carbocyanines and cyanine-dye-3

ATP-binding cassette (ABC) transporters play crucial roles in cellular processes, such as nutrient uptake, drug resistance, cell-volume regulation and others. Despite their importance, all proposed molecular models for transport are based on indirect evidence, i.e. functional interpretation of static crystal structures and ensemble measurements of function and structure. Thus, classical biophysical and biochemical techniques do not readily visualize dynamic structural changes. We recently started to use single-molecule fluorescence techniques to study conformational states and changes of ABC transporters in vitro, in order to observe directly how the different steps during transport are coordinated. This review summarizes our scientific strategy and some of the key experimental advances that allowed the substrate-binding mechanism of prokaryotic ABC importers and the transport cycle to be explored. The conformational states and transitions of ABC-associated substrate-binding domains (SBDs) were visualized with single-molecule FRET, permitting a direct correlation of structural and kinetic information of SBDs. We also delineated the different steps of the transport cycle. Since information in such assays are restricted by proper labelling of proteins with fluorescent dyes, we present a simple approach to increase the amount of protein with FRET information based on non-specific interactions between a dye and the size-exclusion chromatography (SEC) column material used for final purification.

Topics: ATP-Binding Cassette Transporters; Bacterial Proteins; Carbocyanines; Chromatography, Gel; Fluorescence Resonance Energy Transfer; Molecular Dynamics Simulation; Protein Binding; Protein Conformation; Reproducibility of Results

The fluorescent modification of proteins (with genetically encoded low-molecular-mass fluorophores, affinity probes, or other chemically active species) is extraordinarily useful for monitoring and controlling protein functions in vitro, as well as in cell cultures and tissues. The large sizes of some fluorescent tags, such as fluorescent proteins, often perturb normal activity and localization of the protein of interest, as well as other effects. Of the many fluorescent-labeling strategies applied to in vitro and in vivo studies, one is very promising. This requires a very short (6- to 12-residue), appropriately spaced, tetracysteine sequence (-CCXXCC-); this is either placed at a protein terminus, within flexible loops, or incorporated into secondary structure elements. Proteins that contain the tetracysteine motif become highly fluorescent upon labeling with a nonluminescent biarsenical probe, and form very stable covalent complexes. We focus on the development, growth, and multiple applications of this protein research methodology, both in vitro and in vivo. Its application is not limited to intact-cell protein visualization; it has tremendous potential in other protein research disciplines, such as protein purification and activity control, electron microscopy imaging of cells or tissue, protein-protein interaction studies, protein stability, and aggregation studies.

Topics: Arsenic; Carbocyanines; Fluorescein; Fluorescent Dyes; Molecular Structure; Oxazines; Proteins

Microarrays have become an increasingly important tool for biotechnology and molecular diagnostics. Despite many advantages, their sensitivity is still insufficient for such tasks as the analysis of small sample quantities and for the detection of alterations in gene expression of low-abundance genes. Accordingly, amplification strategies are necessary. Approaches to amplify the signal intensity include the increase of the number of dye molecules per target through either particle labels or rolling circle amplification, as used for this study.

Topics: Carbocyanines; DNA; DNA Ligases; DNA, Circular; DNA, Single-Stranded; Fluorescence; Fluorescent Dyes; Models, Chemical; Models, Genetic; Nucleic Acid Amplification Techniques; Nucleic Acid Conformation; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes; Oligonucleotides

DNA microarrays are a powerful tool to investigate differential gene expression for thousands of genes simultaneously. Although DNA microarrays have been widely used to understand the critical events underlying growth, development, homeostasis, behavior and the onset of disease, the management of the resulting data has received little attention. Presently, the fluorescent dyes Cy3 and Cy5 are most often used to prepare labeled cDNA for microarray hybridizations. Raw microarray data are image files that have to be transformed into gene expression formats--a process that requires data manipulation due to systematic variations which may be attributed to differences in the physical and chemical dye applications is to identify differences in transcript levels calculated from fluorescence ratios it is necessary to normalize fluorescence signals to compensate for systematic variations. Here, we will review current normalization strategies applied to cDNA microarrays and discuss their limits. We will show that experimental design determines normalization success.

Topics: Animals; Carbocyanines; DNA, Complementary; Fluorescent Dyes; Humans; Oligonucleotide Array Sequence Analysis

We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the pi-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.

Topics: Carbocyanines; DNA; Energy Transfer; Fluorescence Polarization; Rhodamines; Spectrometry, Fluorescence

The acquisition of simultaneous dual confocal images with red and far-red light has both advantages (e.g. lower autofluorescence) and limitations. An understanding of these requisites is necessary to acquire high-quality images and to avoid the misinterpretation of experimental data. The poor detection of far-red light mandates a high optical transfer efficiency for the system, thus the transmittance of the objective lens and its axial and lateral chromatic aberration in the far-red are important factors for consideration. This technical note is an attempt to 'demystify' the process of filter set design for confocal microscopy by discussing the considerations that went into the construction of a filter set for use with the reagents cyanine 3.18 (Cy3) and cyanine 5.18 (Cy5), and thus to encourage users to look beyond the multi-purpose designs available commercially. The 568-nm laser line exciting Cy3 is at its emission maximum, which limits the collectable Cy3 fluorescence. High-transmission optical filters with sharp band pass cutoffs are thus desirable for maximum light throughout. Light path mirror efficiency rapidly degrades above 700 nm, but the loss of this portion of the Cy5 emission spectrum is acceptable since the fluorophore is very bright, and these very long wavelengths are also likely to introduce aberration. While resolution is decreased with far-red light, there is also greater penetration and less scattering, and it is thus possible to obtain high-quality images from deeper within the specimen.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Carbocyanines; Fluorescent Dyes; Light; Microscopy, Confocal

Single molecule FRET (Forster resonance energy transfer) is very powerful method for studying biomolecular binding dynamics and conformational transitions. Only a few donor - acceptor dye pairs have been characterized for use in single-molecule FRET (smFRET) studies. Hence, introducing and characterizing additional FRET dye pairs is important in order to widen the scope of applications of single-molecule FRET in biomolecular studies. Here we characterize the properties of the Cy3.5 and Cy5.5 dye pair under FRET at the single-molecule level using naked double-stranded DNA (dsDNA) and the nucleosome. We show that this pair of dyes is photostable for ~ 5 min under continuous illumination. We also report Cy3.5-Cy5.5 FRET proximity dependence and stability in the presence of several biochemical buffers and photoprotective reagents in the context of double-stranded DNA. Finally, we demonstrate compatibility of the Cy3.5-Cy5.5 pair for smFRET in vitro studies of nucleosomes.

Topics: Carbocyanines; Coloring Agents; DNA; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Nucleic Acids; Nucleosomes

Nucleic acids, as important substances for biological inheritance, have attracted extensive attention in the biomedical field. More and more cyanine dyes are emerging as one of the probe tools for nucleic acid detection due to their excellent photophysical properties. Here, we discovered that the insertion of the AGRO100 sequence can specifically disrupt the twisted intramolecular charge transfer (TICT) mechanism of the trimethine cyanine dye (

Topics: Carbocyanines; Coloring Agents; Fluorescence; Fluorescent Dyes

Ibrutinib is an inhibitor of Bruton's tyrosine kinase that has been approved for the treatment of patients with chronic lymphocytic leukemia, mantle cell lymphoma and Waldenstrom's macroglobulinemia and is connected with toxicities. To minimize its toxicities, we linked ibrutinib to a cell-targeted, internalizing antibody. To this end, we synthesized a poly-anionic derivate, ibrutinib-Cy3.5, that retains full functionality. This anionic inhibitor is complexed by our anti-CD20-protamine targeting conjugate and free protamine, and thereby spontaneously assembles into an electrostatically stabilized vesicular nanocarrier. The complexation led to an accumulation of the drug driven by the CD20 antigen internalization to the intended cells and an amplification of its pharmacological effectivity. In vivo, we observed a significant enrichment of the drug in xenograft lymphoma tumors in immune-compromised mice and a significantly better response to lower doses compared to the original drug.

Topics: Adenine; Animals; Antibodies, Monoclonal; Antineoplastic Agents; Carbocyanines; Cell Proliferation; Drug Screening Assays, Antitumor; Humans; Lymphoma, Large B-Cell, Diffuse; Mice; Neoplasms, Experimental; Piperidines; Protein Engineering; Protein Kinase Inhibitors; Static Electricity

We developed a one-minute, one-step SARS-CoV-2 antigen assay based on protein-induced fluorescence enhancement of a DNA aptamer. The system showed significant selectivity and sensitivity towards both nucleocapsid protein and SARS-CoV-2 virus lysate, but with marked improvements in speed and manufacturability. We hence propose this platform as a mix-and-read testing strategy for SARS-CoV-2 that can be applied to POC diagnostics in clinical settings, especially in low- and middle-income countries.

Topics: Antigens, Viral; Aptamers, Nucleotide; Biological Assay; Carbocyanines; Coronavirus Nucleocapsid Proteins; COVID-19; COVID-19 Testing; Fluorescence; Fluorescent Dyes; Phosphoproteins; SARS-CoV-2

Dynamic imaging of cell surface pH is extremely challenging due to the slight changes in pH and the fast diffusion of secreted acid to the extracellular environment. In this work, we construct a novel metal-organic framework (MOF)-shell-confined i-motif-based pH probe (MOFC-i) strategy that enables sensitive and dynamic imaging of cell surface pH. The CY3- and CY5-labeled i-motif, which is hybridized via its short complementary chain with two-base mismatches, is optimized for sensing at physiological pH. After efficiently anchoring the optimized pH probes onto the cell membrane with the aid of cholesterol groups, a biocompatible microporous MOF shell is then formed around the cell by cross-linking ZIF-8 nanoparticles via tannic acid. The microporous MOF shell can confine secreted acid without inhibiting the normal physiological activities of cells; thus, the MOFC-i strategy can be used to monitor dynamic changes in the cell surface pH of living cells. Furthermore, this method can not only clearly distinguish the different metabolic behaviors of cancer cells and normal cells but also reveal drug effects on the cell surface pH or metabolism, providing promising prospects in pH-related diagnostics and drug screening.

Topics: Carbocyanines; Cell Membrane; DNA Probes; DNA, Complementary; DNA, Single-Stranded; Estradiol; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Imidazoles; Immobilized Nucleic Acids; MCF-7 Cells; Metal-Organic Frameworks; Nucleotide Motifs; Pyruvates

Dicer-2 cleaves double-stranded RNA into siRNAs in a terminus-dependent manner as part of

Topics: Animals; Carbocyanines; Drosophila melanogaster; Drosophila Proteins; Fluorescent Dyes; Ribonuclease III; RNA Helicases; RNA-Binding Proteins; RNA, Double-Stranded

Cotranscriptional RNA folding is crucial for the timely control of biological processes, but because of its transient nature, its study has remained challenging. While single-molecule Förster resonance energy transfer (smFRET) is unique to investigate transient RNA structures, its application to cotranscriptional studies has been limited to nonnative systems lacking RNA polymerase (RNAP)-dependent features, which are crucial for gene regulation. Here, we present an approach that enables site-specific labeling and smFRET studies of kilobase-length transcripts within native bacterial complexes. By monitoring

Topics: Carbocyanines; Escherichia coli; Escherichia coli Proteins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Riboswitch; Single Molecule Imaging; Transcription Elongation, Genetic

Herein, a highly selective and sensitive "OFF-ON" fluorescent biosensor was designed for intracellular Cu

Topics: Animals; Biocompatible Materials; Biosensing Techniques; Carbocyanines; Catalysis; Cells, Cultured; Click Chemistry; Copper; DNA; Fluorescence; Fluorescent Dyes; Materials Testing; Mice; Particle Size; RAW 264.7 Cells; Surface Properties

Polycaprolactone (PCL) fiber mats with different surface modifications were functionalized with a chitosan nanogel coating to attach the growth factor human bone morphogenetic protein 2 (BMP-2). Three different hydrophilic surface modifications were compared with regard to the binding and in vitro release of BMP-2. The type of surface modification and the specific surface area derived from the fiber thickness had an important influence on the degree of protein loading. Coating the PCL fibers with polydopamine resulted in the binding of the largest BMP-2 quantity per surface area. However, most of the binding was irreversible over the investigated period of time, causing a low release in vitro. PCL fiber mats with a chitosan-graft-PCL coating and an additional alginate layer, as well as PCL fiber mats with an air plasma surface modification boundless BMP-2, but the immobilized protein could almost completely be released. With polydopamine and plasma modifications as well as with unmodified PCL, high amounts of BMP-2 could also be attached directly to the surface. Integration of BMP-2 into the chitosan nanogel functionalization considerably increased binding on all hydrophilized surfaces and resulted in a sustained release with an initial burst release of BMP-2 without detectable loss of bioactivity in vitro.

Topics: Adsorption; Air; Alginates; Animals; Biological Assay; Bone Morphogenetic Protein 2; Carbocyanines; Cell Line; Chitosan; Coated Materials, Biocompatible; Delayed-Action Preparations; Drug Liberation; Humans; Hydrophobic and Hydrophilic Interactions; Indoles; Mice; Nanogels; Polyesters; Polymers; Protein Binding; Protein Refolding; Recombinant Proteins; Surface Properties; Tissue Scaffolds

Dopamine receptors are G protein-coupled receptors that have several essential functions in the central nervous system. A better understanding of the regulatory mechanisms of ligand binding to the receptor may open new possibilities to affect the downstream signal transduction pathways. The majority of the available ligand binding assays use either membrane preparations, cell suspensions, or genetically modified receptors, which may give at least partially incorrect understanding of ligand binding. In this study, we implemented an assay combining fluorescence and bright-field microscopy to measure ligand binding to dopamine D

Topics: Biological Assay; Carbocyanines; Dopamine; Dopamine Antagonists; HEK293 Cells; Humans; Image Processing, Computer-Assisted; Kinetics; Ligands; Machine Learning; Microscopy, Fluorescence; Protein Binding; Receptors, Dopamine; Software; Spiperone

The reported method allows for a simple and rapid monitoring of DNA replication and cell cycle progression in eukaryotic cells in vitro. The DNA of replicating cells is labeled by incorporation of a metabolically-active fluorescent (Cy3) deoxyuridine triphosphate derivative, which is delivered into the cells by a synthetic transporter (SNTT1). The cells are then fixed, stained with DAPI and analyzed by flow cytometry. Thus, this protocol obviates post-labeling steps, which are indispensable in currently used incorporation assays (BrdU, EdU). The applicability of the protocol is demonstrated in analyses of cell cycles of adherent (U-2 OS, HeLa S3, RAW 264.7, J774 A.1, Chem-1, U-87 MG) and suspension (CCRF-CEM, MOLT-4, THP-1, HL-60, JURKAT) cell cultures, including those affected by a DNA polymerase inhibitor (aphidicolin). Owing to a short incorporation time (5-60 min) and reduced number of steps, the protocol can be completed within 1-2 h with a minimal cell loss and with excellent reproducibility.

Topics: Bromodeoxyuridine; Carbocyanines; Cell Cycle; DNA; DNA Replication; Flow Cytometry; Fluorescent Dyes; HeLa Cells; HL-60 Cells; Humans; Jurkat Cells; Reproducibility of Results; Staining and Labeling

Herein, we report a new simple and easy-to-use approach for the characterization of protein oligomerization based on fluorescence resonance energy transfer (FRET) and capillary electrophoresis with LED-induced detection. The FRET pair consisted of quantum dots (QDs) used as an emission tunable donor (emission wavelength of 450 nm) and a cyanine dye (Cy3), providing optimal optical properties as an acceptor. Nonoxidative dimerization of mammalian metallothionein (MT) was investigated using the donor and acceptor covalently conjugated to MT. The main functions of MTs within an organism include the transport and storage of essential metal ions and detoxification of toxic ions. Upon storage under aerobic conditions, MTs form dimers (as well as higher oligomers), which may play an essential role as mediators in oxidoreduction signaling pathways. Due to metal bridging by Cd

Topics: Acetates; Animals; Cadmium; Carbocyanines; Dimerization; Electrophoresis, Capillary; Fluorescence Resonance Energy Transfer; Metallothionein; Models, Molecular; Protein Conformation; Quantum Dots; Rabbits; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Static Electricity

Subcellular stimulation by free radicals is crucial for deeper insight of cell behaviors. However, it remains a tough challenge due to the high spatial precision requirement and short life of radicals. Herein, we report a versatile open microfluidic probe for stable generation of free radical and subcellular stimulation. By optimizing parameters, the chemical reaction can be confined in a microregion with a diameter of several μm, and the real-time produced reactive radicals can attack the desired subcellular region of a single cell. In order to reveal the attacked region, fluorescent cyanine 3 labeled tyramide free radicals are synthesized, and the target microregion on a single cell is successfully stained by the covalent linking reaction between radicals and membrane proteins, which proves the feasibility of our method. We believe this method will open new avenues for short-lived reactive intermediates stimulation at the single-cell/sub-cell level and selective membrane labeling.

Topics: Carbocyanines; Cell Line, Tumor; Fluorescent Dyes; Free Radicals; Humans; Lab-On-A-Chip Devices; Membrane Proteins; Molecular Structure; Single-Cell Analysis; Tyramine

Programmable DNA-based nanostructures (

Topics: Animals; Carbocyanines; DNA; Fluorescent Dyes; Humans; Hypoxia; Light; MCF-7 Cells; Mice; Mitochondria; Nanostructures; Neoplasms; Nitroreductases; Photochemotherapy; Photosensitizing Agents; Porphyrins; Singlet Oxygen

Topics: Carbocyanines; Cations; DNA; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Gene Transfer Techniques; HEK293 Cells; Humans; Hydrophobic and Hydrophilic Interactions; Ligands; Molecular Weight; Organometallic Compounds; Picolines; Plasmids; Polyethyleneimine

During the past decade, fluorescence methods have become valuable tools for characterizing ligand binding to G protein-coupled receptors (GPCRs). However, only a few of the assays enable studying wild-type receptors and monitor the ligand binding in real time. One of the approaches that is inherently suitable for this purpose is the fluorescence anisotropy (FA) assay. In the FA assay, the change of ligand's rotational freedom connected with its binding to the receptor can be monitored with a conventional fluorescence plate reader equipped with suitable optical filters. To achieve the high receptor concentration required for the assay and the low autofluorescence levels essential for reliable results, budded baculoviruses that display GPCRs on their surfaces can be used. The monitoring process generates a substantial amount of kinetic data, which is usually stored as a proprietary file format limiting the flexibility of data analysis. To solve this problem, we propose the use of the data curation software Aparecium ( http://gpcr.ut.ee/aparecium.html ), which integrates experimental data with metadata in a Minimum Information for Data Analysis in Systems Biology (MIDAS) format. Aparecium enables data export to different software packages for fitting to suitable kinetic or equilibrium models. A combination of the FA assay with the novel data analysis strategy is suitable for screening new active compounds, but also for modeling complex systems of ligand binding to GPCRs. We present the proposed approach using different fluorescent probes and assay types to characterize ligand binding to melanocortin 4 (MC

Topics: Animals; Baculoviridae; Binding, Competitive; Biological Assay; Carbocyanines; Fluorescence Polarization; Fluorescent Dyes; Humans; Kinetics; Ligands; Protein Binding; Receptor, Melanocortin, Type 4; Recombinant Proteins; Sf9 Cells

To date, ZnO array-based microfluidic fluorescence assays have been widely investigated and have exhibited excellent performance in the detection of cancer biomarkers. However, the requirements of highly sensitive detection necessitate further improvement of current Zn-based fluorescence detection devices. Here, a rhombus-like Zn(OH)F array-based microfluidic fluorescence detection device is proposed. Construction of Zn(OH)F arrays on the inner wall of a microchannel is carried out via a microfluidic chemical method. A substrate-induced growth strategy for Zn(OH)F arrays is proposed, and various micro/nanostructured Zn(OH)F arrays are successfully obtained. Zn(OH)F nanorod arrays with a high aspect ratio can be constructed on the columnar ZnO nanorod arrays, and the results indicate that the fluorescence enhancement factor (EF) of the Zn(OH)F arrays toward Cy3 is approximately 4-fold that of the ZnO nanorod arrays, which can be attributed to the higher excitation light absorption and evanescent electric field. In human epididymis-specific protein 4 (HE4) detection, the limit of detection (LOD) reaches 9.3 fM, and the dynamic linear range is 10 fM to 100 pM. It has been demonstrated that Zn(OH)F nanorod array-based microfluidic devices are excellent fluorescence assay platforms that also provide a new design and construction strategy for fluorescence enhancement substrates for the detection of biomarkers.

Topics: Antibodies, Immobilized; Biomarkers, Tumor; Carbocyanines; Fluorescence; Fluorescent Dyes; Fluorides; Humans; Hydroxides; Immunoassay; Lab-On-A-Chip Devices; Limit of Detection; Microfluidic Analytical Techniques; Nanotubes; WAP Four-Disulfide Core Domain Protein 2; Zinc Compounds

The use of fluorescence techniques has an enormous impact on various research fields including imaging, biochemical assays, DNA-sequencing and medical technologies. This has been facilitated by the development of numerous commercial dyes with optimized photophysical and chemical properties. Often, however, information about the chemical structures of dyes and the attached linkers used for bioconjugation remain a well-kept secret. This can lead to problems for research applications where knowledge of the dye structure is necessary to predict or understand (unwanted) dye-target interactions, or to establish structural models of the dye-target complex. Using a combination of optical spectroscopy, mass spectrometry, NMR spectroscopy and molecular dynamics simulations, we here investigate the molecular structures and spectroscopic properties of dyes from the Alexa Fluor (Alexa Fluor 555 and 647) and AF series (AF555, AF647, AFD647). Based on available data and published structures of the AF and Cy dyes, we propose a structure for Alexa Fluor 555 and refine that of AF555. We also resolve conflicting reports on the linker composition of Alexa Fluor 647 maleimide. We also conducted a comprehensive comparison between Alexa Fluor and AF dyes by continuous-wave absorption and emission spectroscopy, quantum yield determination, fluorescence lifetime and anisotropy spectroscopy of free and protein-attached dyes. All these data support the idea that Alexa Fluor and AF dyes have a cyanine core and are a derivative of Cy3 and Cy5. In addition, we compared Alexa Fluor 555 and Alexa Fluor 647 to their structural homologs AF555 and AF(D)647 in single-molecule FRET applications. Both pairs showed excellent performance in solution-based smFRET experiments using alternating laser excitation. Minor differences in apparent dye-protein interactions were investigated by molecular dynamics simulations. Our findings clearly demonstrate that the AF-fluorophores are an attractive alternative to Alexa- and Cy-dyes in smFRET studies or other fluorescence applications.

Topics: Carbocyanines; Cysteine; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Magnetic Resonance Spectroscopy; Mass Spectrometry; Molecular Dynamics Simulation; Molecular Structure; Proteins; Rhodamines; Single Molecule Imaging; Sulfonic Acids

Cyanine (Cy) dyes are among the most useful organic fluorophores that have found a wide range of applications in single-molecule and super-resolution imaging as well as in other biophysical studies. However, recent observations that blueshifted derivatives of Cy dyes are formed via photoconversion have raised concerns as to the potential artifacts in multicolor imaging. Here, we report the mechanism for the photoconversion of Cy5 to Cy3 that occurs upon photoexcitation during fluorescent imaging. Our studies show that the formal C

Topics: Carbocyanines; Fluorescent Dyes; HeLa Cells; Humans; Light; Models, Chemical; Oxidation-Reduction; Photochemical Processes; Single Molecule Imaging

Topics: Acrylic Resins; Carbocyanines; DNA; Fluorescence Resonance Energy Transfer; Magnetic Resonance Spectroscopy; Methacrylates; Nylons; Plasmids; Polyethylene Glycols; Polymerization; Polymers; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Temperature

Eukaryotic mRNAs are predominantly translated via the cap-dependent pathway. Initiation is a rate-limiting step in cap-dependent translation and is the main target of translational control mechanisms. There is a lack of high-resolution techniques for characterizing the cap-dependent initiation kinetics. Here, we report an in vitro single-molecule assay that allows characterization of both initiation and peptide chain elongation kinetics for cap-dependent translation. Surprisingly, the histogram of the first-round initiation time is highly asymmetrical and spans a large time range that is several-fold greater than the average peptide synthesis time in translation reactions with a firefly luciferase-encoding mRNA. Both the histogram and single-molecule trajectories reveal an unexpected high-degree of asynchrony in translation activity between mRNA molecules. Furthermore, by inserting a small stem-loop (ΔG = -4.8 kcal/mol) in the middle of the mRNA 5' untranslated region (UTR), our assay robustly detects small changes in budding yeast initiation kinetics, which could not be resolved by bulk luminescence kinetics. Lastly, we demonstrate the general applicability of this assay to distinct cell-free translation systems by using extracts prepared from budding yeast, wheat germ, and rabbit reticulocyte lysates. This assay should facilitate mechanistic studies of eukaryotic cap-dependent translation initiation and translational control.

Topics: Animals; Biological Assay; Carbocyanines; Complex Mixtures; Fluorescent Dyes; Kinetics; Oligopeptides; Peptide Chain Initiation, Translational; Rabbits; Reticulocytes; Ribosomes; RNA Caps; Saccharomyces cerevisiae; Single Molecule Imaging; Triticum

Discriminative identification of homologous miRNAs in miRNA family with high specificity and sensitivity is crucial for accurate classification, diagnosis and prognosis of breast cancer. Herein, we report a reliable, sensitive, and selective assay by coupling fluorescence resonance energy transfer (FRET) with cascade signal amplification. The strategy is developed by designing two programmable DNA probes that can be triggered to shift from "off" to "on" state in a cascade hybridization reaction in the presence of target miRNA let-7a, leading to the generation of an amplified signal. The assay can detect concentrations as low as ∼3.0 pM let-7a and discriminate let-7a from other highly homologous members in the let-7 miRNA family. Moreover, it can also be used to determine let-7a levels at single-cell resolution and evaluate the drug efficacy of let-7a expression among various molecular types of breast cancer cell lines. The advantage of this assay is a combined result of signal generation and amplification triggered by target miRNA, which can satisfy an assay of analogous miRNA in a downregulated manner with high specificity. It has promising potential as a selective assay for homologous miRNAs in precision medicine.

Topics: Antineoplastic Agents; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; DNA Probes; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Inverted Repeat Sequences; MicroRNAs; Microscopy, Confocal; Microscopy, Fluorescence; Nucleic Acid Hybridization; Paclitaxel; Proof of Concept Study

In this work, a hydrazone chemistry assisted DNAzyme has been designed and constructed. The introduction of hydrazone chemistry increases the versatility of DNAzymes. With superior catalytic capability, the hydrazone chemistry assisted DNAzyme has been successfully applied for the analysis of double targets. Taking 5-hydroxymethylfurfural (HMF) and lipopolysaccharide (LPS) as samples, the hydrazone chemistry assisted DNAzyme can be used for the detection of different combinations of targets. Moreover, because hydrazone chemistry is popular in nature, this work may also provide a new insight for the development of DNAzymes and their multifunctionality.

Topics: Carbocyanines; Catalysis; DNA, Catalytic; Fluoresceins; Fluorescent Dyes; Furaldehyde; Hydrazones; Limit of Detection; Lipopolysaccharides; Spectrometry, Fluorescence

RNA-binding proteins with an RNA chaperone activity exert either one or both of the following catalytic activities: (1) RNA annealing, i.e., the protein supports intra- as well as intermolecular RNA-RNA interactions and (2) strand displacement, i.e., the protein mediates the exchange of individual strands of a preexisting RNA duplex. To discriminate and further characterize these activities, it requires defined assay systems. These are based on entirely or partially complementary RNA oligonucleotides that are labeled with fluorescent and/or quencher dyes. The non-catalyzed and the protein-supported associations of the RNA molecules are followed by a real-time fluorescence resonance energy transfer (FRET) system. By site-specific modification of the RNAs or the protein, the substrate- and protein-specific parameters of the RNA chaperone activity can be explored and identified.In this chapter, we present strategies on the design of labeled RNA molecules to be used to characterize the activities of an RNA-binding protein and explain how to monitor progress curves of RNA annealing and strand displacement reactions in single cuvette or well-plate scales. We provide sets of equations and models to determine and analyze different types of reactions, e.g., by calculation of first- and second-order rate constants. Likewise, we demonstrate how to exploit these simple experimental setups to elucidate elementary principles of the reaction mechanisms performed by the protein of interest by applying basic kinetic applications, such as ARRHENIUS and linear free energy relationship analyses. These approaches will be explained by providing example plots and graphs from experiments investigating the RNA chaperone activities of the RNA-binding proteins NF90-NF45 and AUF1 p45.

Topics: Animals; Carbocyanines; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Heterogeneous Nuclear Ribonucleoprotein D0; Humans; Inverted Repeat Sequences; Molecular Chaperones; Nuclear Factor 90 Proteins; RNA Stability; RNA, Small Interfering

Fluorescence resonance energy transfer (FRET) has found widespread uses in biosensing, molecular imaging, and light harvesting. Plasmonic metal nanostructures offer the possibility of engineering photonic environment of specific fluorophores to enhance the FRET efficiency. However, the potential of plasmonic nanostructures to enable tailored FRET enhancement on planar substrates remains largely unrealized, which are of considerable interest for high-performance on-surface bioassays and photovoltaics. The main challenge lies in the necessitated concurrent control over the spectral properties of plasmonic substrates to match that of fluorophores and the fluorophore-substrate spacing. Here, a self-assembled plasmonic substrate based on polydopamine (PDA)-coated plasmonic nanocrystals is developed to effectively address this challenge. The PDA coating not only drives interfacial self-assembly of the nanocrystals to form closely packed arrays with customized optical properties, but also can serve as a tailored nanoscale spacer between the fluorophores and plasmonic nanocrystals, which collectively lead to optimized fluorescence enhancement. The biocompatible plasmonic substrate that allows convenient bioconjugation imparted by PDA has afforded improved FRET efficiency in DNA microarray assay and FRET imaging of live cells. It is envisioned that the self-assembled plasmonic substrates can be readily integrated into fluorescence-based platforms for diverse biomedical and photoconversion applications.

Topics: Biocompatible Materials; Carbocyanines; Cell Line, Tumor; Epidermal Growth Factor; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Gold; Humans; Indoles; Metal Nanoparticles; Microscopy, Confocal; Oligonucleotide Array Sequence Analysis; Polymers; Silver

Up to 20% of solid tumors are characterized by DNA mismatch repair (MMR) deficiency and microsatellite instability that confer resistance to standard of care chemotherapy. MMR-deficient cancers have an increased mutation rate, and DNA mismatches accumulate as part of these cancers. We previously described a class of compounds, rhodium metalloinsertors, that bind DNA mismatches with high specificity and selectivity and have potential as targeted therapy. [Rh(chrysi)(phen)(PPO)]

Topics: Antineoplastic Agents; Carbocyanines; Cell Proliferation; Cell Survival; Coordination Complexes; DNA Damage; DNA Mismatch Repair; Fluorescent Dyes; HCT116 Cells; Humans; Molecular Structure; Optical Imaging; Rhodium

Wound infection is a major challenge in the clinic that greatly hinders the wound healing process. It is highly important to develop smart wound dressings that can sense bacterial infection at early stages and provide on-demand treatment. In this work, a smart hydrogel-based wound dressing capable of monitoring bacterial infection via a pH-responsive fluorescence resonance energy transfer (FRET) transition of Cyanine3 (Cy3) and Cyanine5 (Cy5) in a bacterial environment and providing on-demand treatment of infection via near infrared (NIR) light-triggered antibiotic release was developed. The smart hydrogel was prepared by physical crosslinking of polyvinyl alcohol (PVA) and an ultraviolet (UV)-cleavable polyprodrug (GS-Linker-MPEG), in which Cy3 and Cy5-modified silica nanoparticles (SNP-Cy3/Cy5) were loaded and acted as a pH-responsive fluorescent probe to detect bacterial infection based on the FRET effect between Cy3 and Cy5. Also, up-conversion nanoparticles (UCNP) were loaded into the hydrogels to cleave the UV-responsive GS-Linker-MPEG and achieve NIR-responsive release of GS in the bacterial environment. The in vitro studies proved that the smart hydrogels present good water absorption ability and excellent mechanical properties as well as good biocompatibility, which are necessary for their application in wound dressings. Moreover, the hydrogels showed obvious FRET transitions in both acidic buffer and bacteria solution. Upon irradiating the hydrogels with NIR light, UCNP were able to convert NIR light to UV light to trigger the release of GS from the hydrogels for antibacterial treatment. This research is expected to provide a new strategy for self-reporting and effective treatment of wound infection.

Topics: Animals; Anti-Bacterial Agents; Bacteria; Bandages, Hydrocolloid; Carbocyanines; Colony Count, Microbial; Fluorescence Resonance Energy Transfer; Gentamicins; Hydrogen-Ion Concentration; Infrared Rays; Mice; Nanoparticles; NIH 3T3 Cells; Polyvinyl Alcohol; Prodrugs

Despite extensive studies on transcription mechanisms, it is unknown how termination complexes are disassembled, especially in what order the essential components dissociate. Our single-molecule fluorescence study unveils that RNA transcript release precedes RNA polymerase (RNAP) dissociation from the DNA template much more often than their concurrent dissociations in intrinsic termination of bacterial transcription. As termination is defined by the release of product RNA from the transcription complex, the subsequent retention of RNAP on DNA constitutes a previously unidentified stage, termed here as recycling. During the recycling stage, post-terminational RNAPs one-dimensionally diffuse on DNA in downward and upward directions, and can initiate transcription again at the original and nearby promoters in the case of retaining a sigma factor. The efficiency of this event, termed here as reinitiation, increases with supplement of a sigma factor. In summary, after releasing RNA product at intrinsic termination, recycling RNAP diffuses on the DNA template for reinitiation most of the time.

Topics: Carbocyanines; DNA-Directed RNA Polymerases; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Fluorescent Dyes; Promoter Regions, Genetic; Sigma Factor; Single Molecule Imaging; Terminator Regions, Genetic; Transcription Initiation, Genetic; Transcription Termination, Genetic; Transcription, Genetic

Topics: Adhesins, Bacterial; Antibodies, Bacterial; Bacterial Adhesion; Bacterial Proteins; Bacterial Secretion Systems; Biomechanical Phenomena; Carbocyanines; Flavobacterium; Fluorescent Dyes; Gene Expression; Genes, Reporter; Locomotion; Luminescent Proteins; Protein Binding; Recombinant Fusion Proteins; Rotation

Organelle-specific delivery systems are of significant clinical interest. We demonstrate the use of common cyanine dyes Cy3 and Cy5 as vectors for targeting and delivering cargoes to mitochondria in cancer cells. Specifically, conjugation to the dyes can increase cytotoxicity by up to 1000-fold.

Topics: Antineoplastic Agents; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line; Cell Survival; Fluorescent Dyes; Humans; Mitochondria

FRET (fluorescence resonance energy transfer) between far-upstream (-100) and downstream (+14) cyanine dyes (Cy3, Cy5) showed extensive bending and wrapping of λP

Topics: Carbocyanines; DNA; DNA-Directed RNA Polymerases; Escherichia coli; Escherichia coli Proteins; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Kinetics; Nucleic Acid Conformation; Promoter Regions, Genetic; Protein Binding

Topics: Animals; Antineoplastic Agents; Carbocyanines; Cathepsin B; Fluorescent Dyes; Fluorides; HeLa Cells; Humans; Lanthanoid Series Elements; Light; Metal Nanoparticles; Mice; Neoplasms; NIH 3T3 Cells; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species; Rose Bengal; Xenograft Model Antitumor Assays; Yttrium

The efficacy of fluorescent hybridization assays is often limited by the low signal-to-background ratio of the probes that can be partially overcome by sophisticated signal amplification methods. Deep understanding of the mechanisms of fluorescence quenching and energy transfer in complex DNA probes and the choice of optimal donor/acceptor pairs along with rational design can significantly enhance the performance of DNA probes. Here, we proposed and studied novel Förster resonance energy transfer (FRET) dual DNA probes with the excimer-forming pyrene pair as a donor and sulfo-Cy3 dye as an acceptor, which demonstrated remarkable 75-fold enhancement of sulfo-Cy3 fluorescence upon target capturing. Stokes shift up to 220 nm minimizes fluorescence crosstalk. Time-correlated single-photon counting revealed two excited states of pyrene excimer wherein only one is directly involved in the resonance energy transfer to sulfo-Cy3. Optimized DNA probes demonstrated high sensitivity with excellent signal-to-background ratio, which were applied for visualization of 18S rRNA by fluorescent in situ hybridization in HEK-293T cells.

Topics: Carbocyanines; DNA Probes; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Molecular Structure; Pyrenes; RNA

The drug-target protein interaction is the basis of drug screening and precise therapy in modern clinical medicine. How to acquire the information about the drug-target protein interaction in single living cell is a great challenge due to the shortage of efficient methods. Here we propose a new strategy for in situ studying the drug-target protein interaction in single living cells based on the competition of candidate drugs to the fluorescent probe-target complexes and fluorescence correlation spectroscopy (FCS) with a microfluidic chip. In this study, we used ABL kinase (target) as a model and synthesized a fluorescent probe (Cy3-dasatinib) with an affinity to the target using ABL inhibitor dasatinib as a precursor. We systematically investigated the association of the probe with targets and the dissociation of the drug-target complexes in the presence of candidate drug. We presented a new parameter IC

Topics: Antineoplastic Agents; Carbocyanines; Dasatinib; Fluorescent Dyes; Humans; Molecular Structure; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-abl; Spectrometry, Fluorescence

MicroRNAs (miRNAs) are crucial regulators of gene expression. The abnormal expression of miRNA is often closely related to many diseases. However, the accurate clinical profiling of miRNA expression remains a great challenge due to the high similarity and wide variety of miRNA sequence structures. Here, we report a highly specific and sensitive multiplex miRNA detection scheme with high tension hybridization and dual signal amplification based on the recyclable autocatalytic DNAzyme and a light harvesting conjugated polymer. Multiple signals can be read out simultaneously by single excitation through the efficient multiple fluorescence resonance energy transfer (FRET) between the conjugated polymer and different small molecule dyes. In addition, different types of logic gates can also be operated by observing the emission intensities of the labeling dyes with miRNAs as inputs, thus giving rise to a new way for the specific detection of certain miRNAs according to the logic signals. Furthermore, we successfully applied the strategy for multiple miRNA detection in cell lysates and the results agree well with those of qRT-PCR. Thus, we believe that this platform holds great potential for miRNA detection in biological samples.

Topics: Base Sequence; Carbocyanines; Cell Line, Tumor; DNA Probes; DNA, Catalytic; Fluorenes; Fluoresceins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Magnetic Phenomena; MicroRNAs; Nucleic Acid Amplification Techniques; Nucleic Acid Hybridization; Polymers; Quaternary Ammonium Compounds; Xanthenes

Monitoring RNA synthesis and spatial distribution can help to understand its role in physiology and diseases. However, visualizing newly synthesized RNA in single cells remains a great challenge. Here, we developed a bioorthogonal labeling-primed DNA amplification strategy to visualize newly synthesized RNA in single cells. The new bioorthogonal

Topics: Carbocyanines; DNA Primers; DNA, Neoplasm; Fluorescent Dyes; Humans; In Situ Hybridization, Fluorescence; Molecular Structure; Nucleic Acid Amplification Techniques; Optical Imaging; RNA, Neoplasm; Single-Cell Analysis; Tumor Cells, Cultured

Single-molecule hybridisation of CY3 dye labelled short oligonucleotides to surface immobilised probes was investigated in zero-mode waveguide nanostructures using a modified DNA sequencer. At longer measuring times, we observed changes of the initial hybridisation fluorescence pulse pattern which we attribute to products created by chemical reactions at the nucleobases. The origin is a charge separated state created by a photoinduced electron transfer from nucleobases to the dye followed by secondary reactions with oxygen and water, respectively. The positive charge can migrate through the hybrid resulting in base modifications at distant sites. Static fluorescence spectra were recorded in order to determine the properties of CY3 stacking to different base pairs, and compared to pulse intensities. A characteristic pulse pattern change was assigned to the oxidation of G to 8-oG besides the formation of a number of secondary products that are not yet identified. Further, we present a method to visualise the degree of chemical reactions to gain an overview of ongoing processes. Our study demonstrates that CY3 is able to oxidise nucleobases in ds DNA, and also in ss overhangs. An important finding is the correlation between nucleobase oxidation potential and fluorescence quenching which explains the intensity changes observed in single molecule measurements. The analysis of fluorescence traces provides the opportunity to track complete and coherent reaction sequences enabling to follow the fate of a single molecule over a long period of time, and to observe chemical reactions in real-time. This opens up the opportunity to analyse reaction pathways, to detect new products and short-lived intermediates, and to investigate rare events due to the large number of single molecules observed in parallel.

Topics: Carbocyanines; Fluorescence; Humans; Oligonucleotides; Oxidation-Reduction

Photoexcited fluorescent markers are extensively used in spectroscopy, imaging, and analysis of biological systems. The performance of fluorescent markers depends on high levels of emission, which are limited by competing nonradiative decay pathways. Small-molecule fluorescent dyes have been increasingly used as markers due to their high and stable emission. Despite their prevalence, the nonradiative decay pathways of these dyes have not been determined. Here, we investigate these pathways for a widely used indocarbocyanine dye, Cy3, using transient grating spectroscopy. We identify a nonradiative decay pathway via a previously unknown dark state formed within ∼1 ps of photoexcitation. Our experiments, in combination with electronic structure calculations, suggest that the generation of the dark state is mediated by picosecond vibrational mode coupling, likely via a conical intersection. We further identify the vibrational modes, and thus structural elements, responsible for the formation and dynamics of the dark state, providing insight into suppressing nonradiative decay pathways in fluorescent markers such as Cy3.

Topics: Carbocyanines; Fluorescent Dyes; Light; Spectrum Analysis; Stereoisomerism; Vibration

Gold nanoparticles (AuNPs) conjugated with Cy3-tagged aptamer which can specifically recognize chloramphenicol (CAP) (referred to as AuNPs-AptCAP) are described. CAP can trigger the configuration change of CAP binding aptamer, and thus switching the fluorescence of AuNPs-AptCAP through changing the efficiency of the fluorescence resonance energy transfer (FRET) system with Cy3 as donors and AuNPs as recipients. AuNPs-AptCAP exhibits a linear range of CAP concentrations from 26.0 to 277 μg L

Topics: Aptamers, Nucleotide; Base Sequence; Carbocyanines; Chloramphenicol; DNA; Edetic Acid; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Gold; Immobilized Nucleic Acids; Limit of Detection; Logic; Mercury; Metal Nanoparticles; Nickel

Specific monovalent cation effects on the domain-domain interaction of heterogeneous dimeric protein were investigated using green fluorescent protein (GFP)-glutathione-s-transferase (GST) fusion protein as a model protein. Conjugating N-terminal of GST domain with a fluorescence probe Cyanine3, complementary increase and decrease of fluorescence intensities of Cyanine3 and GFP were recognized on the exclusive excitation of GFP and further the fluorescence decay of GFP was remarkably accelerated to show that an excellent Förster type of resonance excitation energy transfer (FRET) pair was constructed between GFP- and GST-domain. The spectral overlap integral and critical distance of the FRET pair were estimated to be 5.96×10

Topics: Ammonium Chloride; Carbocyanines; Cations; Cesium; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Glutathione Transferase; Green Fluorescent Proteins; Lithium; Potassium; Protein Multimerization; Sodium

Messenger RNA (mRNA) isolated from single cells can generate powerful biological insights, including the discovery of new cell types with unique functions as well as markers potentially predicting a cell's response to various therapeutic agents. We previously introduced an oligonucleotide-based technique for site-selective, photoinduced biotinylation and capture of mRNA within a living cell called transcriptome

Topics: Animals; Biotin; Brain; Carbocyanines; Fluorescent Dyes; Gene Expression Profiling; Light; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Neurons; Nitrobenzenes; RNA Probes; RNA, Messenger; Single-Cell Analysis; Transcriptome

Topics: Base Sequence; Biomedical Enhancement; Biosensing Techniques; Carbocyanines; DNA; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Gold; Metal Nanoparticles; Models, Molecular; Rhodamines; Surface Plasmon Resonance; Surface Properties

Fluorescent nucleoside triphosphates are powerful probes of DNA synthesis, but their potential use in living animals has been previously underexplored. Here, we report the synthesis and characterization of 7-deaza-(1,2,3-triazole)-2'-deoxyadenosine-5'-triphosphate (dATP) derivatives of tetramethyl rhodamine ("TAMRA-dATP"), cyanine ("Cy3-dATP"), and boron-dipyrromethene ("BODIPY-dATP"). Upon microinjection into live zebrafish embryos, all three compounds were incorporated into the DNA of dividing cells; however, their impact on embryonic toxicity was highly variable, depending on the exact structure of the dye. TAMRA-EdATP exhibited superior characteristics in terms of its high brightness, low toxicity, and rapid incorporation and depletion kinetics in both a vertebrate (zebrafish) and a nematode (

Topics: Animals; Boron Compounds; Caenorhabditis elegans; Carbocyanines; Deoxyadenine Nucleotides; DNA; DNA Replication; Fluorescent Dyes; Optical Imaging; Rhodamines; Zebrafish

Accurate quantifications of cellular miRNAs are important not only for accelerating them becoming reliable diagnostics biomarkers but also for deeply understanding their influence on central signaling pathways. Although single-molecule miRNA imaging permits quantifying biomolecules at the single-molecule level, it is limited by the sensitivity and specificity of hybridization-based probes. We report a miRNA single-molecule imaging method by using conjugated polymer nanoparticle (CPN) labeled short DNA probe termed as a nanoflare. The transient hybridization of the nanoflares and target miRNAs yields a featured single-molecule kinetics signal rendering high single-molecule sensitivity and specificity. miRNA can be detected with a remarkable detection limit of 1 fM without using any amplification steps. The discrimination capability of homologous miRNAs was also demonstrated. Taking advantage of the featured single-molecule signal of the nanoflare, we can directly count single miR-21 molecules in single cells by using highly inclined and laminated optical sheet (HILO) microscopy. The statistics of the counting reveals miR-21's cell-to-cell fluctuation and differential expression of tumor cells and normal cells.

Topics: A549 Cells; Carbocyanines; DNA Probes; Fluorenes; Fluorescent Dyes; HEK293 Cells; HeLa Cells; Humans; Limit of Detection; MicroRNAs; Microscopy; Nanoparticles; Nucleic Acid Hybridization; Polymers; Single Molecule Imaging

Sensitive detection of nucleic acids and identification of single nucleotide polymorphism (SNP) is crucial in diagnosis of genetic diseases. Many strategies have been developed for detection and analysis of DNA, including fluorescence, electrical, optical, and mechanical methods. Recent advances in fluorescence resonance energy transfer (FRET)-based sensing have provided a new avenue for sensitive and quantitative detection of various types of biomolecules in simple, rapid, and recyclable platforms. Here, we report single-step FRET-based DNA sensors designed to work via a toehold-mediated strand displacement (TMSD) process, leading to a distinct change in the FRET efficiency upon target binding. Using single-molecule FRET (smFRET), we show that these sensors can be regenerated in situ, and they allow detection of femtomoles DNA without the need for target amplification while still using a dramatically small sample size (fewer than three orders of magnitude compared to the typical sample size of bulk fluorescence). In addition, these single-molecule sensors exhibit a dynamic range of approximately two orders of magnitude. Using one of the sensors, we demonstrate that the single-base mismatch sequence can be discriminated from a fully matched DNA target, showing a high specificity of the method. These sensors with simple and recyclable design, sensitive detection of DNA, and the ability to discriminate single-base mismatch sequences may find applications in quantitative analysis of nucleic acid biomarkers.

Topics: Carbocyanines; DNA; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Limit of Detection; Polymorphism, Single Nucleotide

The strong light-matter interactions between dyes and plasmonic nanoantennas enable the study of fundamental molecular-optical processes. Here, we overcome conventional limitations with high-throughput single-molecule polarization-resolved microscopy to measure dye emission polarization modifications upon near-field coupling to a gold nanorod. We determine that the emission polarization distribution is not only rotated toward the nanorod's dominant localized surface plasmon mode as expected, but it is also unintuitively broadened. With a reduced-order analytical model, we elucidate how this distribution broadening depends upon both far-field interference and off-resonant coupling between the molecular dipole and the nanorod transverse plasmon mode. Experiments and modeling reveal that a nearby plasmonic nanoantenna affects dye emission polarization through a multicolor process, even when the orthogonal plasmon modes are separated by approximately 3 times the dye emission line width. Beyond advancing our understanding of plasmon-coupled emission modifications, this work promises to improve high-sensitivity single-molecule fluorescence imaging, biosensing, and spectral engineering.

Topics: Carbocyanines; Microscopy, Polarization; Nanotubes

RNA interference is a powerful approach to understand gene function both for therapeutic and experimental purposes. Since the lack of knowledge in the gene silencing of various hepatic cell lines, this work was aimed to compare two transfection agents, the liposome-based Lipofectamine™ RNAiMAX and the HepG2-specific, polymer-based GenMute™, in two cellular models of human hepatoma, HepG2 and Huh7.5. In the first part, we assessed transfection efficiency of a fluorescent Cy3-labeled negative control siRNA by cell imaging analysis; we found that cells treated with GenMute present a higher uptake of the fluorescent negative control siRNA when compared to Lipofectamine RNAiMAX-transfected cells, both in HepG2 and in Huh7.5 cells. In the second part, we evaluated GAPDH silencing with the two transfection reagents by RT-PCR similar GAPDH mRNA expression after each transfection treatment. Finally, we measured cell viability by the MTT assay, observing that cells transfected with GenMute have higher viability with respect to Lipofectamine RNAiMAX-administered cells. These results suggest that GenMute reagent might be considered the most suitable transfection agent for hepatic gene silencing.

Topics: Base Sequence; Carbocyanines; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Fluorescent Dyes; Gene Silencing; Gene Transfer Techniques; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Lipid Metabolism; Lipids; Liposomes; Liver Neoplasms; Polymers; RNA Interference; RNA, Small Interfering; Transfection

Current nanomedicine suffers from a big challenge due to the fact that most of the nanocarrier systems lack the desired tumor penetration depth, thereby limiting their clinical translation. Unlike the nanomaterials with a similar size or shape, microgels display excellent softness, fluidity and deformability, as well as stimuli-responsiveness in the tumor microenvironment. Herein, we report the synthesis of temperature-responsive poly(N-vinylcaprolactam)/oligo (ethylene glycol) acrylate/glycidyl methacrylate (PVCL/OEGA/GMA) microgels with different hydrodynamic radii (100-500 nm), crosslinking densities, 2-methoxyethyl acrylate (MEA) contents and OEGA chain lengths using a precipitation polymerization method and the investigation of the microgels in terms of their tumor penetration capability using a multicellular tumor spheroid (MCTS) model. The prepared microgels were well characterized with different techniques. We show that regardless of the size, crosslinking density, MEA content and OEGA chain length, all microgels display the desired cytocompatibility in the given concentration range. In vitro cellular uptake data reveal that similar to 2-dimensional (2-D) adherent cells, microgels with a smaller size display more enhanced cellular uptake than those having a larger size in the 3-D MCTS model. Likewise, 3-D MCTS penetration results indicate that the PVCL/OEGA/GMA microgels with the smallest radius of 100 nm exhibit the deepest penetration length. We then selected the microgels with a radius of 200 nm but with different physicochemical parameters to investigate their cellular uptake and tumor penetration behavior. Our data show that microgels with varying crosslinking densities, MEA contents and OEGA chain lengths do not have any appreciable changes in terms of their cellular uptake and penetration in the 3-D MCTS model. Our study provides new insights for the design of different microgel-based systems for further cancer theranostic applications.

Topics: Antineoplastic Agents; Caprolactam; Carbocyanines; Cell Proliferation; Cell Survival; Cross-Linking Reagents; Drug Screening Assays, Antitumor; HeLa Cells; Humans; Microgels; Microscopy, Fluorescence; Molecular Structure; Nanomedicine; Optical Imaging; Particle Size; Polymers; Spheroids, Cellular; Surface Properties; Temperature

Herein, we construct a structure-switchable gemcitabine (Ge)-containing DNA nanogel that can respond to the intracellular acidic environment, subsequently facilitating the chemodrug release inside the cells. Based on the structural similarity between Ge and deoxycytidine (dC), dC nucleotides in the component DNA strands used for nanogel assembly are fully replaced by Ge during their synthesis. By changing the designed sequences, two Ge-containing Y-shaped motifs with different sticky ends are first assembled and then associated together to form nanogel by sticky-end hybridizations. In particular, one of the sticky-end sequences is arbitrarily designed to be rich of Ge and the other is designed to be partially complementary to the first Ge-rich sticky end. At the neutral or basic condition, the Ge-rich sticky ends hybridize with the partially complementary sticky ends on the second Y motifs, keeping the assembled nanogel stable. Upon being exposed to the acidic condition, Ge-rich sticky ends intend to form intramolecular i-motif-like quadruplex structures, resulting in the disassembly of the nanogel. On the one hand, the nanosized feature enables the Ge-containing nanogel with rapid cellular uptake behavior. On the other hand, the pH-responsive feature endows the rapid disassembly of the nanogel to facilitate the enzymatic drug release inside the cell, resulting in the enhanced anticancer activity of the DNA-based drug delivery system.

Topics: A549 Cells; Antimetabolites, Antineoplastic; Apoptosis; Carbocyanines; Deoxycytidine; DNA; Drug Carriers; Drug Liberation; Endodeoxyribonucleases; Fluorescence Resonance Energy Transfer; Gemcitabine; Humans; Hydrogen-Ion Concentration; Microscopy, Confocal; Nanogels

MicroRNAs (miRNAs), small noncoding endogenous RNA molecules, are emerging as promising biomarkers for early detection of various diseases and cancers. Practical screening tools and strategies to detect these small molecules are urgently needed to facilitate the translation of miRNA biomarkers into clinical practice. In this study, a label-free biosensing technique based on surface-enhanced Raman scattering (SERS), referred to as plasmonic coupling interference (PCI), was applied for the multiplex detection of miRNA biomarkers. The sensing mechanism of the PCI technique relies on the formation of a nanonetwork consisting of nanoparticles with Raman labels located between adjacent nanoparticles that are interconnected by DNA duplexes. Because of the plasmonic coupling effect of adjacent nanoparticles in the nanonetwork, the Raman labels exhibit intense SERS signals. Such effect can be modulated by the addition of miRNA targets of interest that act as inhibitors to interfere with the formation of this nanonetwork, resulting in a diminished SERS signal. In this study, the PCI technique is theoretically analyzed, and the multiplex capability for detection of multiple miRNA cancer biomarkers is demonstrated, establishing the great potential of PCI nanoprobes as a useful diagnostic tool for medical applications.

Topics: Biomarkers, Tumor; Carbocyanines; DNA Probes; Fluorescent Dyes; Humans; Metal Nanoparticles; MicroRNAs; Neoplasms; Rhodamines; RNA, Neoplasm; Sensitivity and Specificity; Silver; Spectrum Analysis, Raman; Surface Plasmon Resonance

The higher-order architecture observed in biological systems, like viruses, is very effective in nucleic acid transport. The replications of this system has been attempted with both synthetic and naturally occurring polymers with mixed results. Here we describe a peptide/siRNA quaternary complex that functions as an siRNA delivery system. The rational design of a peptide assembly is inspired by the viral capsids, but not derived from them. We selected the collagen peptide (COL) to provide the structural stability and the folding framework, and hybridize it with the cell penetrating peptide (CPP) that allows for effective penetration of biological barriers. The peptide/siRNA quaternary complex forms stoichiometric, 10 nm nanoparticles, that show fast cellular uptake (<30 min), effective siRNA release, and gene silencing. The complex provides capsid-like protection for siRNA against nucleases without being immunostimulatory, or cytotoxic. Our data suggests that delivery vehicles based on synthetic quaternary structures that exhibit higher-order architecture may be effective in improving delivery and release of nucleic acid cargo.

Topics: Animals; Biological Transport; Biomimetic Materials; Capsid; Carbocyanines; Cell-Penetrating Peptides; Collagen; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Gene Silencing; Gene Transfer Techniques; Genes, Reporter; Green Fluorescent Proteins; Humans; Hydroxyproline; Mice; Molecular Conformation; Nanoparticles; NIH 3T3 Cells; Polymers; RNA, Small Interfering

SmartFlare technology allows detection of mRNA in single living cells. We studied the possibility of using SmartFlare nanoprobes for detection of small subsets of CD4

Topics: Carbocyanines; Flow Cytometry; Fluorescent Dyes; Gold; Humans; Metal Nanoparticles; RNA, Complementary; RNA, Messenger; T-Lymphocyte Subsets; T-Lymphocytes, Helper-Inducer

Optical imaging is a key modality for observing biological specimen with higher spatial resolution. However, scattering and absorption of light in tissues are inherent barriers in maximizing imaging depth in biological tissues. To achieve this goal, use of light at near-infrared spectrum can improve the present situation. Here, the capability of saturated two-photon saturated excitation (TP-SAX) fluorescence microscopy to image at depths of >2.0 mm, with submicron resolution in transparent mouse brain imaging, is demonstrated. At such depths with scattering-enlarged point spread function (PSF), we find that TP-SAX is capable to provide spatial resolution improvement compared to its corresponding TPFM, which is on the other hand already providing a much improved resolution compared with single-photon confocal fluorescence microscopy. With the capability to further improve spatial resolution at such deep depth with scattering-enlarged PSF, TP-SAX can be used for exquisite visualization of delicate cerebral neural structure in the scattering regime with a submicron spatial resolution inside intact mouse brain.

Topics: Animals; Brain; Carbocyanines; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence, Multiphoton; Nerve Net; Optical Phenomena

An approach combining DNA nanoscaffolds with supramolecular polymers for the efficient and directional propagation of light-harvesting cascades has been developed. A series of photonic wires with different arrangements of fluorophores in DNA-organized nanostructures were linked to light-harvesting supramolecular phenanthrene polymers (SPs) in a self-assembled fashion. Among them, a light-harvesting complex (LHC) composed of SPs and a photonic wire of phenanthrene, Cy3, Cy5, and Cy5.5 chromophores reveals a remarkable energy transfer efficiency of 59 %. Stepwise transfer of the excitation energy collected by the light-harvesting SPs via the intermediate Cy3 and Cy5 chromophores to the final Cy5.5 acceptor proceeds through a Förster resonance energy transfer mechanism. In addition, the light-harvesting properties are documented by antenna effects ranging from 1.4 up to 23 for different LHCs.

Topics: Carbocyanines; DNA; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Models, Molecular; Nanostructures; Phenanthrenes; Photons

While surface-based single-molecule experiments have revolutionized our understanding of biology and biomolecules, the workflow in preparing for such experiments, especially surface cleaning and functionalization, remains labor-intensive and time-consuming. Even worse, meticulously assembled flow channels can be used only once for most experiments. A reusable surface would thus dramatically increase productivity and efficiency of single-molecule experiments. In this paper, we report a novel surface reconditioning strategy termed ERASE (Epitaxial Removal Aided by Strand Exchange) that allows a single flow cell to be used for vast repetition of single-molecule experiments. In this method, biomolecules immobilized to the surface through a nucleic acid duplex are liberated when a competing DNA strand disrupts the duplex via toehold-mediated strand displacement. We demonstrate the wide-range applicability of this method with various common surface preparation techniques, fluorescent dyes, and biomolecules including the bacterial ribosome. Beyond time and cost savings, we also show ERASE can assort molecules based on a nucleic acid barcode sequence, thus allowing experiments on different molecules in parallel. Our method increases the utility of prepared surfaces and is a significant improvement to the current single-use paradigm.

Topics: Carbocyanines; Fluorescent Dyes; Nucleic Acid Hybridization; Oligodeoxyribonucleotides; Single Molecule Imaging

Advances in nanoparticle design have led to the development of nanoparticulate systems that can sense intracellular molecules, alter cellular processes, and release drugs to specific targets in vitro. In this work, we demonstrate that oligonucleotide-coated gold nanoparticles are suitable for the detection of mRNA in live Hydra vulgaris, a model organism, without affecting the animal's integrity. We specifically focus on the detection of Hymyc1 mRNA, which is responsible for the regulation of the balance between stem cell self-renewal and differentiation. Myc deregulation is found in more than half of human cancers, thus the ability to detect in vivo related mRNAs through innovative fluorescent systems is of outmost interest.

Topics: Animals; Carbocyanines; DNA; Gold; Hydra; Metal Nanoparticles; Microscopy, Fluorescence; Oligonucleotides; Proto-Oncogene Proteins c-myc; RNA, Messenger

Small interfering RNA (siRNA)-based therapies are proving to be efficient for treating liver-associated disorders. However, extra-hepatic delivery remains challenging, limiting therapeutic siRNA utility. We synthesized a panel of fifteen lipid-conjugated siRNAs and systematically evaluated the impact of conjugate on siRNA tissue distribution and efficacy. Generally, conjugate hydrophobicity defines the degree of clearance and the liver-to-kidney distribution profile. In addition to primary clearance tissues, several conjugates achieve significant siRNA accumulation in muscle, lung, heart, adrenal glands and fat. Oligonucleotide distribution to extra-hepatic tissues with some conjugates was significantly higher than with cholesterol, a well studied conjugate, suggesting that altering conjugate structure can enhance extra-hepatic delivery. These conjugated siRNAs enable functional gene silencing in lung, muscle, fat, heart and adrenal gland. Required levels for productive silencing vary (5-200 μg/g) per tissue, suggesting that the chemical nature of conjugates impacts tissue-dependent cellular/intracellular trafficking mechanisms. The collection of conjugated siRNA described here enables functional gene modulation in vivo in several extra-hepatic tissues opening these tissues for gene expression modulation. A systemic evaluation of a panel of conjugated siRNA, as reported here, has not previously been investigated and shows that chemical engineering of lipid siRNAs is essential to advance the RNA therapeutic field.

Topics: Animals; Carbocyanines; Cholesterol; Fatty Acids; Female; Fluorescent Dyes; Kidney; Lipids; Liver; Mice; Phosphorylcholine; RNA Interference; RNA, Small Interfering; Tissue Distribution

Molecular crowding creates a unique environment in cells and imposes physical constraints such as the excluded volume effect, water activity, and dielectric constant that can affect the structure and function of biomolecules. It is therefore important to develop a method for quantifying the effects of molecular crowding in cells. In this study, we developed a Förster resonance energy transfer (FRET) probe based on a guanine-quadruplex (G4) DNA motif that shows distinct FRET signals in response to crowding conditions in the presence of salt and poly(ethylene glycol). FRET efficiencies varied in different solutions, reflecting the dependence of G4 stability and topology on salt concentration and water activity. In living cells, FRET signals in the nucleus were higher than those in the cytosol; the signals in membraneless nuclear compartments (i.e., nucleolus) were especially high, suggesting that a decrease in water activity is important for the crowding effect in the nucleus. Thus, the use of DNA sensors with variable structures can elucidate the local effects of molecular crowding in cells.

Topics: Animals; Carbocyanines; Cattle; Cell Nucleus; Cytoplasm; DNA; DNA Probes; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; G-Quadruplexes; HeLa Cells; Humans; Intracellular Space; Oligodeoxyribonucleotides; Polyethylene Glycols; Potassium; Serum Albumin, Bovine; Sodium

Abundant post-translational modification through methylation alters the function, stability, and/or localization of a protein. Malfunctions in post-translational modification are associated with severe diseases. To unravel protein methylation sites and their biological functions, chemical methylation reporters have been developed. However, until now, their usage was limited to cell lysates. Herein, we present the first generally applicable approach for imaging methylation of individual proteins in human cells, which is based on a combination of chemical reporter strategies, bioorthogonal ligation reactions, and FRET detected by means of fluorescence lifetime imaging microscopy. Through this approach, methylation of histone 4 and the non-histone proteins tumor suppressor p53, kinase Akt1, and transcription factor Foxo1 in two human cell lines has been successfully imaged. To further demonstrate its potential, the localization-dependent methylation state of Foxo1 in the cellular context has been visualized.

Topics: Alkynes; Azides; Carbocyanines; Fluorescent Dyes; Forkhead Box Protein O1; HEK293 Cells; HeLa Cells; Histones; Humans; Methylation; Methyltransferases; Microscopy, Confocal; Microscopy, Fluorescence; Proto-Oncogene Proteins c-akt; Selenomethionine; Tumor Suppressor Protein p53

In this work, we report a component-minimal spherical nucleic acid (SNA) from monodisperse DNA-polymer conjugates that can load and release nucleic acid therapeutics in a stimuli-responsive manner. We show that this vehicle assembles from only four strands, and conditional release of its antisense therapeutic cargo can be induced upon recognition of specific oligonucleotide triggers via strand displacement. The latter (triggers) may be a microRNA that offers additional synergistic therapy, in addition to the previously shown ability of the SNA to load hydrophobic drugs. The SNA is easy to prepare, has dynamic character, releases its cargo only upon the presence of both triggers, and can survive biological conditions while protecting its cargo. The gene silencing potency of the cargo was tested in live cells and shown to be suppressed when loaded in the SNA, and its activity was restored only upon release with the two triggers. This vehicle has the essential characteristics of versatility, ease of synthesis, low cost, highly responsive behavior, and ability to support combination therapies, making it a promising candidate for cell-selective drug delivery and clinical transition.

Topics: Carbocyanines; Drug Carriers; Fluorescence Resonance Energy Transfer; Gene Silencing; HeLa Cells; Humans; Hydrophobic and Hydrophilic Interactions; Nanotechnology; Nucleic Acids; Oligonucleotides, Antisense; Polymers; RNA, Small Interfering

Heterochromatin protein 1α (HP1α) undergoes liquid-liquid phase separation (LLPS) and forms liquid droplets and gels in vitro, properties that also appear to be central to its biological function in heterochromatin compaction and regulation. Here we use solid-state NMR spectroscopy to track the conformational dynamics of phosphorylated HP1α during its transformation from the liquid to the gel state. Using experiments designed to probe distinct dynamic modes, we identify regions with varying mobilities within HP1α molecules and show that specific serine residues uniquely contribute to gel formation. The addition of chromatin disturbs the gelation process while preserving the conformational dynamics within individual bulk HP1α molecules. Our study provides a glimpse into the dynamic architecture of dense HP1α phases and showcases the potential of solid-state NMR to detect an elusive biophysical regime of phase separating biomolecules.

Topics: Amino Acid Sequence; Carbocyanines; Chromatin; Chromobox Protein Homolog 5; Chromosomal Proteins, Non-Histone; Fluorescence Recovery After Photobleaching; Gels; Humans; Nuclear Magnetic Resonance, Biomolecular

Imaging and detecting microRNAs (miRNAs) is of central importance in tumor cell analysis. It stays challenging to establish simple, accurate, and sensitive analytical assays for imaging and detection of miRNA in a single living cell, because of intracellular complex environment and miRNA sequence similarity. Herein, we designed a dual-signal twinkling probe (DSTP) with triplex-stem structure which employed a fluorescence-SERS signal reciprocal switch. The spatiotemporal dynamics of the miRNA molecular and intracellular uptake of the probe are monitored by fluorescence-SERS signal switch of the DSTP. Meanwhile, using the surface-enhanced Raman scattering (SERS) signals of DSTP, the measure of absolute value coupling of reciprocal signals is first used to real-time detection of miRNA. Through simultaneous enhancing the target response signal value and reducing blank value, this work deducted the background effect, and showed high sensitivity and reproducibility. Moreover, the probe shows excellent reversibility and specificity in real quantitative detection of intracellular miRNA. miR-203 was successfully monitored in MCF-7, in accord with the results in vitro as well as in cell lysates. We anticipate that this new dual-signal twinkling and dual-spectrum switch method will be generally useful to image and detect various types of biomolecules in single living cell.

Topics: Carbocyanines; Fluorescent Dyes; Gold; Humans; Immobilized Nucleic Acids; Inverted Repeat Sequences; MCF-7 Cells; Metal Nanoparticles; MicroRNAs; Microscopy, Fluorescence; Nucleic Acid Hybridization; Oligodeoxyribonucleotides; Reproducibility of Results; Single-Cell Analysis; Spectrum Analysis, Raman

The development of new nanoparticle formulations that are capable of high transfection efficiency without toxicity is essential to provide new tools for gene therapy. However, the issues of complex, poorly reproducible manufacturing methods, and low efficiencies during in vivo testing have prevented translation to the clinic. We have previously reported the use of cholesteryl oleate as a novel excipient for solid lipid nanoparticles (SLNs) for the development of highly efficient and nontoxic nucleic acid delivery carriers. Here, we performed an extensive characterization of this novel formulation to make the scale up under Good Manufacturing Practice (GMP) possible. We also describe the complete physicochemical and biological characterization of cholesteryl oleate-loaded SLNs to ensure the reproducibility of this formula and the preservation of its characteristics before and after the lyophilization process. We defined the best manufacturing method and studied the influence of some parameters on the obtained nanoparticles using the Quality by Design (ICH Q8) guideline to obtain cholesteryl oleate-loaded SLNs that remain stable during storage and guarantee in vitro nucleic acid delivery efficacy. Our results indicate that this improved formulation is suitable for gene therapy with the possibility of scale-up the manufacturing of nanoparticles under GMP conditions.

Topics: Amines; Carbocyanines; Cations; Cholesterol Esters; Factor Analysis, Statistical; Fluorescent Dyes; Gene Transfer Techniques; HeLa Cells; Humans; Microscopy, Fluorescence; Nanoparticles; Particle Size; Plasmids; Poloxamer; RNA, Small Interfering; Stearic Acids; Transfection

Clinical trials involving genome-edited cells are growing in popularity, where CAR-T immunotherapy and CRISPR/Cas9 editing are more recognized strategies. Genetic reporters are needed to localize the molecular events inside these cells in patients. Specifically, a nonimmunogenic genetic reporter is urgently needed as current reporters are immunogenic due to derivation from nonhuman sources. Prostate-specific membrane antigen (PSMA) is potentially nonimmunogenic due to its natural, low-level expression in select tissues (self-MHC display). PSMA overexpression on human prostate adenocarcinoma is also visible with excellent contrast. We exploit these properties in a transduced, two-component,

Topics: Animals; Antigens, Surface; Carbocyanines; Cell Line, Tumor; Cell Tracking; Fluorescent Dyes; Genes, Reporter; Glutamate Carboxypeptidase II; Humans; Male; Mice; Models, Molecular; Optical Imaging; Positron-Emission Tomography; Prostatic Neoplasms

Fluorescence imaging is a powerful tool for studying biologically relevant macromolecules, but its applicability is often limited by the fluorescent probe, which must demonstrate both high site-specificity and emission efficiency. In this regard, M13 virus, a versatile biological scaffold, has previously been used to both assemble fluorophores on its viral capsid with molecular precision and to also target a variety of cells. Although M13-fluorophore systems are highly selective, these complexes typically suffer from poor molecular detection limits due to low absorption cross-sections and moderate quantum yields. To overcome these challenges, a coassembly of the M13 virus, cyanine 3 dye, and silver nanoparticles is developed to create a fluorescent tag capable of binding with molecular precision with high emissivity. Enhanced emission of cyanine 3 of up to 24-fold is achieved by varying nanoparticle size and particle-fluorophore separation. In addition, it is found that the fluorescence enhancement increases with increasing dye surface density on the viral capsid. Finally, this highly fluorescent probe is applied for in vitro staining of E. coli. These results demonstrate an inexpensive framework for achieving tuned fluorescence enhancements. The methodology developed in this work is potentially amendable to fluorescent detection of a wide range of M13/cell combinations.

Topics: Bacteriophage M13; Carbocyanines; Fluorescence; Metal Nanoparticles; Particle Size; Polyethylene Glycols; Silver

Reactive oxygen species (ROS) are potent signaling molecules with critical roles in cellular pathology and homeostasis. They are produced in all cell types via a diverse array of cellular machinery, giving rise to an equally diverse repertoire of molecular effects. These range from cytotoxic killing of microbes to alteration of the cellular transcriptional response to stress. Despite their importance, research into ROS has been difficult given their inherent instability and transient signaling properties. Herein we describe methods for the use of the redox-sensitive probe hydro-Cy3 for the detection and quantification of ROS both in vitro and in vivo.

Topics: Animals; Carbocyanines; Cell Membrane Permeability; Fluorescent Dyes; Mice; Microscopy, Confocal; Molecular Imaging; Molecular Structure; NADPH Oxidases; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species

Here we report the endocytosis and excretion pathways of two different dye-conjugated cucurbit[7]urils, (cyanine 3-conjugated CB[7] and rhodamine X-conjugated CB[7]), which have great potential as molecular probes for live cell imaging. The dye-CB[7]s are translocated into live cells (human breast carcinoma cells, MCF-7) via multiple pathways, predominantly by clathrin-mediated endocytosis, and excreted from cells via lysosome-associated exocytosis. Interestingly, the CB[7] moiety has a substantial influence on the uptake and excretion pathways. These findings may widen the applications of the dyes conjugated to CB[7] and assist in the design of new molecular probes for live cell imaging.

Topics: Bridged-Ring Compounds; Carbocyanines; Endocytosis; Exocytosis; Fluorescence; Fluorescent Dyes; Humans; Imidazoles; Lysosomes; MCF-7 Cells; Rhodamines

The cyanine dye Cy3 is a popular fluorophore used to probe the binding of proteins to nucleic acids as well as their conformational transitions. Nucleic acids labeled only with Cy3 can often be used to monitor interactions with unlabeled proteins because of an enhancement of Cy3 fluorescence intensity that results when the protein contacts Cy3, a property sometimes referred to as protein-induced fluorescence enhancement (PIFE). Although Cy3 fluorescence is enhanced upon contacting most proteins, we show here in studies of human replication protein A and Escherichia coli single-stranded DNA binding protein that the magnitude of the Cy3 enhancement is dependent on both the protein as well as the orientation of the protein with respect to the Cy3 label on the DNA. This difference in PIFE is due entirely to differences in the final protein-DNA complex. We also show that the origin of PIFE is the longer fluorescence lifetime induced by the local protein environment. These results indicate that PIFE is not a through space distance-dependent phenomenon but requires a direct interaction of Cy3 with the protein, and the magnitude of the effect is influenced by the region of the protein contacting Cy3. Hence, use of the Cy3 PIFE effect for quantitative studies may require careful calibration.

Topics: Carbocyanines; DNA; DNA-Binding Proteins; Escherichia coli Proteins; Fluorescent Dyes; Fluorometry; Humans; Protein Binding; Replication Protein A

We describe a molecular sensor that reports, using fluorescence resonance energy transfer (FRET), on the degree of macromolecular crowding in different cellular compartments. The oligonucleotide-based sensor is sensitive to changes in the volume fraction of macromolecules over a wide range in vitro and, when introduced in cells, rapidly distributes and shows a striking contrast between the cytosol and the nucleus. This contrast can be modulated by osmotic stress or by using a number of drugs that alter chromatin organization within the nucleus. These findings suggest that the sensor can be used as a tool to probe chromosome organization. Further, our finding that the cell maintains different degrees of macromolecular crowding in the cytoplasm and nucleoplasm has implications on molecular mechanisms since crowding can alter protein conformations, binding rates, reaction kinetics, and therefore protein function.

Topics: Animals; Carbocyanines; Cell Nucleus; Chromatin; Cytoplasm; Deoxyribonucleotides; Fibroblasts; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Mice; Osmotic Pressure

Surface-enhanced Raman scattering (SERS)-based sensing is promising in that it has potential to allow for highly sensitive, selective, and multiplexed detection and imaging. However, the controlled assembly and gap formation between plasmonic particles for generating strong SERS signals in a quantitative manner is highly challenging, especially on biodetection platforms, and particle-to-particle variation in the signal enhancement can vary by several orders of magnitude in a single batch, largely limiting the reliable use of SERS for practical sensing applications. Here, a hierarchic-nanocube-assembly based SERS (H-Cube-SERS) bioassay to controllably amplify the electromagnetic field between gold nanocubes (AuNCs) is developed. Based on this strategy, H-Cube-SERS assay allows for detecting target DNA with a wide dynamic range from 100 aM to 10 pM concentrations in a stable and reproducible manner. It is also found that the uniformly formed AuNCs with flat surfaces are much more suitable for highly sensitive, reliable, and quantitative biodetection assays due to faster DNA binding kinetics, sharper DNA melting transition, wider hot spot regions, and less dependence on light polarization direction than spherical Au nanoparticles with curved interfaces. This work paves the pathways to the quantitative and sensitive biodetection on a SERS platform and can be extended to other particle assembly systems.

Topics: Biological Assay; Carbocyanines; DNA; Fluorescent Dyes; Gold; Limit of Detection; Metal Nanoparticles; Nanoparticles; Nucleic Acid Denaturation; Reproducibility of Results; Spectrum Analysis, Raman

Sialic acid (SA), usually located at the termini of glycan chains, is one of the most important monosaccharide blocks for glycosylation of proteins. The expression level of sialoglycoconjugates (SiaGCs) in cellular secretome is of great significance in diagnosis of tumor malignancy. This work developed a fluorescent visual method for the detection of SiaGCs secreted from living cells by a boronic acid modified chip based chemoselective recognition and hybridization chain reaction. The cell-secreted SiaGCs, which were labeled with the azide group through a metabolic labeling technique during cell culture, were captured by the chip through chemoselective recognition of boronic acid toward SA. After further conjugating the azide group with an alkyne modified DNA probe, the captured SiaGCs could be conveniently endowed with the amplified fluorescent signal through a hybridization chain reaction of a pair of dye-labeled DNA hairpins, which led to a quantitative imaging method for detection of SiaGCs. The average amount of metabolically labeled SiaGCs secreted from a single HeLa cell and MCF-7 cell was 2.18 × 10

Topics: Alkynes; Azides; Boronic Acids; Carbocyanines; Cell Line, Tumor; DNA; DNA Probes; Fluorescent Dyes; Fluorometry; Glycoconjugates; Hexosamines; Humans; N-Acetylneuraminic Acid; Nucleic Acid Amplification Techniques; Nucleic Acid Hybridization; Sulfhydryl Compounds

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is increasingly used for detection of various macromolecules and metabolites in biological samples. Here, we present a detailed analysis of the CuAAC reaction conditions in cells and tissue sections. Using the optimized CuAAC conditions, we have devised a highly sensitive immunostaining technique, based on the tyramide signal amplification/catalyzed reporter deposition (TSA/CARD) method with a novel alkyne tyramide substrate. The described method offers improved detection threshold compared to conventional immunofluorescent staining and produces significantly lower non-specific background than TSA/CARD with fluorescent tyramides.

Topics: Animals; Azides; Boron Compounds; Brain Chemistry; Bromodeoxyuridine; Carbocyanines; Cells, Cultured; Click Chemistry; Copper; Deoxyuridine; DNA; Female; Fluorescent Antibody Technique; Fluorescent Dyes; Horseradish Peroxidase; Humans; Male; Mice; Pluripotent Stem Cells; Sensitivity and Specificity; Tyramine

DNA nanomachines have received great interest due to their potential to mimic various natural biomolecular machines. Intracellular pH sensing and imaging are of great significance to understand cellular behaviors and disease diagnostics. In this work, we report the novel molecular switching of a self-assembled 3D DNA triangular prism nanomachine (TPN) for pH sensing and imaging in living cells. The TPN was self-assembled in quantitative yields by hybridization with two DNA triangles and three I-strands (containing i-motif sequences). At acidic conditions, the TPN was compressed due to the I-strand that formed an intramolecular i-tetraplex, which was in between the fluorophores Cy3 and Cy5, resulting in a significant fluorescence resonance energy transfer (FRET) signal. At neutral or weakly alkaline conditions, the TPN adopted an extended state due to the random coil form of the I-strand, leading to spatial separation of the two fluorophores and the FRET being blocked. The TPN was fully reversible and could rapidly respond to the pH changes, entered into living cells automatically via an endocytic pathway, monitored spatiotemporal pH changes during endocytosis, maintained its structural integrity after escape from lysosomes, still had the ability for pH sensing, and also visualized pH fluctuations under varying stimuli in living cells. We foresee that this TPN can become a generic platform for a pH-related cell biology study and in disease diagnostics.

Topics: Biosensing Techniques; Carbocyanines; DNA; Endocytosis; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; HeLa Cells; Humans; Hydrogen-Ion Concentration; Lysosomes; Nanostructures; Nucleic Acid Conformation; Nucleic Acid Hybridization

One of the universal mechanisms for the response of Escherichia coli to stress is the increase of the synthesis of specific histone-like proteins that bind the DNA, Dps. As a result, two-and three-dimensional crystalline arrays may be observed in the cytoplasm of starving cells. Here, we determined the conditions to obtain very thin two-dimensional DNA-Dps co-crystals in vitro, and studied their projection structures, using electron microscopy. Analysis of the projection maps of the free Dps crystals revealed two lattice types: hexagonal and rectangular. We used the fluorescently labeled DNA to prove that the DNA is present within the co-crystals with Dps in vitro, and visualized its position using transmission electron microscopy. Molecular modeling confirmed the DNA position within the crystal. We have also suggested a structural model for the DNA-Dps co-crystal dissolving in the presence of Mg

Topics: Bacterial Outer Membrane Proteins; Carbocyanines; Crystallization; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Fluorescent Dyes; Gene Expression; Magnesium Chloride; Microscopy, Electron, Transmission; Models, Molecular; Protein Binding; Staining and Labeling

Direct cellular imaging of the localization and dynamics of biomolecules helps to understand their function and reveals novel mechanisms at the single-cell resolution. In contrast to routine fluorescent-protein-based protein imaging, technology for RNA imaging remains less well explored because of the lack of enabling technology. Herein, we report the development of an aptamer-initiated fluorescence complementation (AiFC) method for RNA imaging by engineering a green fluorescence protein (GFP)-mimicking turn-on RNA aptamer, Broccoli, into two split fragments that could tandemly bind to target mRNA. When genetically encoded in cells, endogenous mRNA molecules recruited Split-Broccoli and brought the two fragments into spatial proximity, which formed a fluorophore-binding site in situ and turned on fluorescence. Significantly, we demonstrated the use of AiFC for high-contrast and real-time imaging of endogenous RNA molecules in living mammalian cells. We envision wide application and practical utility of this enabling technology to in vivo single-cell visualization and mechanistic analysis of macromolecular interactions.

Topics: Actins; Aptamers, Nucleotide; Carbocyanines; Cytoplasm; DNA Probes; Green Fluorescent Proteins; HeLa Cells; Humans; Microscopy, Confocal; Plasmids; RNA, Messenger; Time-Lapse Imaging; Transcription, Genetic

A supramolecular FRET pair based on the ultrahigh binding affinity between cyanine 3 conjugated cucurbit[7]uril (CB[7]-Cy3) and cyanine 5 conjugated adamantylamine (AdA-Cy5) was exploited as a new synthetic tool for imaging cellular processes in live cells. Confocal laser scanning microscopy revealed that CB[7]-Cy3 and AdA-Cy5 were intracellularly translocated and accumulated in lysosomes and mitochondria, respectively. CB[7]-Cy3 and AdA-Cy5 then formed a host-guest complex, reported by a FRET signal, as a result of the fusion of lysosomes and mitochondria. This observation not only indicated that CB[7] forms a stable complex with AdA in a live cell, but also suggested that this FRET pair can visualize dynamic organelle fusion processes, such as those involved in the degradation of mitochondria through autophagy (mitophagy), by virtue of its small size, chemical stability, and ease of use.

Topics: Amantadine; Autophagosomes; Autophagy; Bridged-Ring Compounds; Carbocyanines; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Imidazoles; Lysosomes; MCF-7 Cells; Membrane Fusion; Microscopy, Confocal

Designer DNA nanodevices have attracted extensive interest for detection of specific targets in living cells. However, it still remains a great challenge to construct DNA sensing devices that can be activated at desired time with a remotely applied stimulus. Here we report a rationally designed, synthetic DNA nanodevice that can detect ATP in living cells in an upconversion luminescence-activatable manner. The nanodevice consists of a UV light-activatable aptamer probe and lanthanide-doped upconversion nanoparticles which acts as the nanotransducers to operate the device in response to NIR light. We demonstrate that the nanodevice not only enables efficient cellular delivery of the aptamer probe into live cells, but also allows the temporal control over its fluorescent sensing activity for ATP by NIR light irradiation in vitro and in vivo. Ultimately, with the availability of diverse aptamers selected in vitro, the DNA nanodevice platform will allow NIR-triggered sensing of various targets as well as modulation of biological functions in living systems.

Topics: Adenosine Triphosphate; Carbocyanines; DNA; HeLa Cells; Humans; Luminescence; Microscopy, Electron, Transmission; Nanoparticles; Ultraviolet Rays

Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged β2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.

Topics: Carbocyanines; Coloring Agents; Endocytosis; Endosomes; Exocytosis; Fluorescent Dyes; HEK293 Cells; Humans; Hydrogen-Ion Concentration; Indicators and Reagents; Microscopy, Confocal; Protein Transport; Receptors, Adrenergic, beta-2; Rosaniline Dyes; Single-Chain Antibodies

Accumulation of amyloid-beta (Aβ), which is associated with Alzheimer's disease, can be caused by excess production or insufficient clearance. Because of its β-sheet structure, fibrillar Aβ is resistant to proteolysis, which would contribute to slow degradation of Aβ plaques in vivo. Fibrillar Aβ can be internalized by microglia, which are the scavenger cells of the brain, but the fibrils are degraded only slowly in microglial lysosomes. Cathepsin B is a lysosomal protease that has been shown to proteolyze fibrillar Aβ. Tripeptidyl peptidase 1 (TPP1), a lysosomal serine protease, possesses endopeptidase activity and has been shown to cleave peptides between hydrophobic residues. Herein, we demonstrate that TPP1 is able to proteolyze fibrillar Aβ efficiently. Mass spectrometry analysis of peptides released from fibrillar Aβ digested with TPP1 reveals several endoproteolytic cleavages including some within β-sheet regions that are important for fibril formation. Using molecular dynamics simulations, we demonstrate that these cleavages destabilize fibrillar β-sheet structure. The demonstration that TPP1 can degrade fibrillar forms of Aβ provides insight into the turnover of fibrillar Aβ and may lead to new therapeutic methods to increase degradation of Aβ plaques.

Topics: Aminopeptidases; Amyloid; Amyloid beta-Peptides; Carbocyanines; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Lysosomes; Mass Spectrometry; Models, Molecular; Molecular Dynamics Simulation; Peptide Fragments; Protein Conformation, beta-Strand; Protein Domains; Protein Stability; Serine Proteases; Time Factors; Tripeptidyl-Peptidase 1

Ultrasound application combined with microbubbles has shown great potential for intracellular gene delivery. However, the fundamental mechanistic question of how plasmid DNA enters the intracellular space mediated by ultrasound and microbubble has not been fully explored and understood. The goal of this study is to unveil the detailed intracellular uptake process of plasmid DNA stimulated by ultrasound and microbubbles, uniquely highlighting the role of microbubbles play in this process. The usage of targeted microbubbles pinpointed the subcellular membrane site, where ultrasound exerted acoustic force onto the cell membrane. With the combination of high-speed video microscopy and 3D confocal fluorescence microscopy, we show the spatiotemporal correlation between the microbubble dynamics and intracellular plasmid DNA distribution. Two ultrasound modes (high pressure short pulse and low pressure long pulse) were chosen to trigger different plasmid DNA uptake routes. We found that reversible cell membrane disruption, induced by high pressure short pulse ultrasound, permitted plasmid DNA passage across cell membrane, but not in an exclusive way. Under both ultrasound modes, with or without cell membrane disruption, global plasmid DNA internalization, even nuclear-localization, was observed immediately post ultrasound application. Our results show that plasmid DNA uptake evoked by localized acoustically excited microbubbles is a fast (<2min), global (not limited to the site where microbubbles were attached), and multi-mechanisms involved process.

Topics: Biological Transport; Carbocyanines; Cell Survival; DNA; Fluorescent Dyes; Gene Transfer Techniques; Green Fluorescent Proteins; HeLa Cells; Humans; Microbubbles; Plasmids; Ultrasonic Waves

Exciton delocalization in dye aggregate systems is a phenomenon that is revealed by spectral features, such as Davydov splitting, J- and H-aggregate behavior, and fluorescence suppression. Using DNA as an architectural template to assemble dye aggregates enables specific control of the aggregate size and dye type, proximal and precise positioning of the dyes within the aggregates, and a method for constructing large, modular two- and three-dimensional arrays. Here, we report on dye aggregates, organized via an immobile Holliday junction DNA template, that exhibit large Davydov splitting of the absorbance spectrum (125 nm, 397.5 meV), J- and H-aggregate behavior, and near-complete suppression of the fluorescence emission (∼97.6% suppression). Because of the unique optical properties of the aggregates, we have demonstrated that our dye aggregate system is a viable candidate as a sensitive absorbance and fluorescence optical reporter. DNA-templated aggregates exhibiting exciton delocalization may find application in optical detection and imaging, light-harvesting, photovoltaics, optical information processing, and quantum computing.

Topics: Carbocyanines; DNA, Cruciform; Fluorescence; Fluorescent Dyes; Models, Molecular; Templates, Genetic

Despite the differences of the host, parasitic nematodes may share commonalities in their parasitizing genes. Setaria digitata novel protein (SDNP) is such an entity which is parasitic nematode-specific and having sequence similarities with those of W. bancrofti, B. malayi, Loa loa and Onchocerca volvulus. Post-transcriptional gene silencing by siRNA mediated RNA interference (RNAi) is a widely used technique in functional genomics. Though the technique has been used in several free-living, plant and animal parasitic nematodes, it has not yet been tried out for the filarial worm S. digitata. In this study, we developed an effective siRNA delivery method by microinjection and utilized the siRNAi tool to knockdown SDNP to study the phenotypic and cellular changes associated with the interference. qPCR analysis revealed, a significant reduction of SDNP transcript levels following siRNA microinjection into S. digitata adult worms. Similarly, immunohistochemical staining indicated a reduction of SDNP protein expression. Furthermore, worms treated with siRNA showed a significant reduction of microfilariae release together with embryonic lethality by arresting an early developmental stage compared to non-treated worms. A distinct motility reduction was also observed in treated worms compared to non-treated counterparts. This is the first report of the amenability of S. digitata to the siRNA induced RNAi. The presence of inter-domain linkers of muscle-specific twitchin kinase and calcium-dependent protein kinase isoform CDPK1 together with what our results revealed suggest that SDNP is most likely a protein involved in muscle movement and growth and development of the nematode. Hence SDNP has the characteristics of a potential drug target.

Topics: Animals; Carbocyanines; Cattle; Female; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Gene Knockdown Techniques; Gene Silencing; Helminth Proteins; Immunohistochemistry; Microinjections; Movement; Polymerase Chain Reaction; Reverse Transcription; RNA Interference; RNA, Small Interfering; Setaria Nematode

Light-activated ("caged") antisense oligonucleotides are powerful molecules for regulating gene expression at submicron spatial resolution through the focal modulation of endogenous cellular processes. Cyclized caged oligos are particularly promising structures because of their inherent stability and similarity to naturally occurring circular DNA and RNA molecules. Here, we introduce an efficient route for cyclizing an antisense oligodeoxynucleotide incorporating a photocleavable linker. Oligo cyclization was achieved for several sequences in nearly quantitative yields through intramolecular copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Caging stability and light activation were characterized by FRET efficiency, denaturing gel assay, and melting temperature measurements. Finally, a cyclized caged oligo was designed to target gfap, and it gave a tenfold reduction in glial fibrillary acidic protein upon photoactivation in astrocytes.

Topics: Alkynes; Animals; Astrocytes; Azides; Base Sequence; Carbocyanines; Catalysis; Click Chemistry; Copper; Cyclization; Cycloaddition Reaction; Gene Expression; Glial Fibrillary Acidic Protein; Humans; Oligonucleotides, Antisense; Optogenetics

A modular route to prepare functional self-assembling dendritic peptide amphiphiles decorated with mannosides, to effectively target antigen-presenting cells, such as macrophages, is reported. The monomeric building blocks were equipped with tetra(ethylene glycol)s (TEGs) or labeled with a Cy3 fluorescent probe. Experiments on the uptake of the multifunctional supramolecular particles into murine macrophages (Mφs) were monitored by confocal microscopy and fluorescence-activated cell sorting. Mannose-decorated supramolecular polymers trigger a significantly higher cellular uptake and distribution, relative to TEG carrying bare polymers. No cytotoxicity or negative impact on cytokine production of the treated Mφs was observed, which emphasized their biocompatibility. The modular nature of the multicomponent supramolecular polymer coassembly protocol is a promising platform to develop fully synthetic multifunctional vaccines, for example, in cancer immunotherapy.

Topics: Animals; Antigen-Presenting Cells; Biological Transport; Carbocyanines; Cells, Cultured; Dendrimers; Fluorescent Dyes; Macrophages; Mannosides; Mice; Microscopy, Confocal; Models, Molecular; Peptides; Polyethylene Glycols; Surface-Active Agents

To develop structural modifications of dNTPs that are compatible with Taq DNA polymerase activity, we synthesized eight dUTP derivatives conjugated with Cy3 or Cy5 dye analogues that differed in charge and charge distribution throughout the fluorophore. These dUTP derivatives and commercial Cy3- and Cy5-dUTP were studied in Taq polymerase-dependent polymerase chain reactions (PCRs) and in primer extension reactions using model templates containing one, two and three adjacent adenine nucleotides. The relative amounts of amplified DNA and the kinetic parameters Km and Vmax characterizing the incorporation of labelled dUMPs have been estimated using fluorescence measurements and analysed. The dUTPs labelled with electroneutral zwitterionic analogues of Cy3 or Cy5 fluorophores were used by Taq polymerase approximately one order of magnitude more effectively than the dUTPs labelled with negatively charged analogues of Cy3 or Cy5. The nucleotidyl transferase activity of Taq polymerase was also observed and resulted in the addition of dUMPs labelled with electroneutral or positively charged fluorophores to the 3' ends of DNA. The introduction of mutually compensating charges into fluorophores or other functional groups conjugated to dNTPs can be considered a basis for the creation of PCR-compatible modified nucleoside triphosphates.

Topics: Carbocyanines; Deoxyuracil Nucleotides; DNA-Directed DNA Polymerase; Fluorescent Dyes; Kinetics; Nucleotides; Nucleotidyltransferases; Polymerase Chain Reaction; Taq Polymerase; Templates, Genetic

Site-specific incorporation of labeled nucleotides is an extremely useful synthetic tool for many structural studies (e.g., NMR, electron paramagnetic resonance (EPR), fluorescence resonance energy transfer (FRET), and X-ray crystallography) of RNA. However, specific-position-labeled RNAs >60 nt are not commercially available on a milligram scale. Position-selective labeling of RNA (PLOR) has been applied to prepare large RNAs labeled at desired positions, and all the required reagents are commercially available. Here, we present a step-by-step protocol for the solid-liquid hybrid phase method PLOR to synthesize 71-nt RNA samples with three different modification applications, containing (i) a

Topics: Biotin; Carbocyanines; Chemistry Techniques, Synthetic; Isotopes; Nucleotides; RNA; Staining and Labeling; Uracil

Here we report ultrastable synthetic binding pairs between cucurbit[7]uril (CB[7]) and adamantyl- (AdA) or ferrocenyl-ammonium (FcA) as a supramolecular latching system for protein imaging, overcoming the limitations of protein-based binding pairs. Cyanine 3-conjugated CB[7] (Cy3-CB[7]) can visualize AdA- or FcA-labeled proteins to provide clear fluorescence images for accurate and precise analysis of proteins. Furthermore, controllability of the system is demonstrated by treating with a stronger competitor guest. At low temperature, this allows us to selectively detach Cy3-CB[7] from guest-labeled proteins on the cell surface, while leaving Cy3-CB[7] latched to the cytosolic proteins for spatially conditional visualization of target proteins. This work represents a non-protein-based bioimaging tool which has inherent advantages over the widely used protein-based techniques, thereby demonstrating the great potential of this synthetic system.

Topics: Animals; Bridged-Ring Compounds; Caenorhabditis elegans; Carbocyanines; Chlorocebus aethiops; COS Cells; Fluorescent Antibody Technique; Imidazoles; Intravital Microscopy; Microscopy, Confocal; Molecular Imaging; Protein Binding; Staining and Labeling

Wave-based optical elastography is rapidly emerging as a powerful technique for quantifying tissue biomechanical properties due to its noninvasive nature and high displacement sensitivity. However, current approaches are limited in their ability to produce high-frequency waves and highly localized mechanical stress. In this Letter, we demonstrate that the rapid liquid-to-gas phase transition of dye-loaded perfluorocarbon nanodroplets ("nanobombs") initiated by a pulsed laser can produce highly localized, high-frequency, and broadband elastic waves. The waves were detected by an ultra-fast line-field low-coherence holography system. For comparison, we also excited waves using a focused micro-air-pulse. Results from tissue-mimicking phantoms showed that the nanobombs produced elastic waves with frequencies up to ∼9  kHz, which was much greater than the ∼2  kHz waves excited by the air-pulse. Consequently, the nanobombs enabled more accurate quantification of sample viscoelasticity. Combined with their potential for functionalization, the nanobombs show promise for accurate and highly specific noncontact all-optical elastography.

Topics: Biomechanical Phenomena; Carbocyanines; Elastic Modulus; Elasticity Imaging Techniques; Fluorocarbons; Lasers, Solid-State; Microspheres; Phantoms, Imaging; Stress, Mechanical; Tomography, Optical Coherence

Cytochrome P450 oxidoreductase (POR) is the primary electron donor in eukaryotic cytochrome P450 (CYP) containing systems. A wealth of ensemble biophysical studies of Cytochrome P450 oxidoreductase (POR) has reported a binary model of the conformational equilibrium directing its catalytic efficiency and biomolecular recognition. In this study, full length POR from the crop plant Sorghum bicolor was site-specifically labeled with Cy3 (donor) and Cy5 (acceptor) fluorophores and reconstituted in nanodiscs. Our single molecule fluorescence resonance energy transfer (smFRET) burst analyses of POR allowed the direct observation and quantification of at least three dominant conformational sub-populations, their distribution and occupancies. Moreover, the state occupancies were remodeled significantly by ionic strength and the nature of reconstitution environment, i.e. phospholipid bilayers (nanodiscs) composed of different lipid head group charges vs. detergent micelles. The existence of conformational heterogeneity in POR may mediate selective activation of multiple downstream electron acceptors and association in complexes in the ER membrane.

Topics: Carbocyanines; Catalysis; Cytochrome P-450 Enzyme System; Detergents; Electrons; Fluorescence Resonance Energy Transfer; Lipid Bilayers; Membranes; Micelles; Osmolar Concentration; Oxidoreductases Acting on CH-CH Group Donors; Phospholipids; Protein Conformation; Sorghum

Dynamic DNA nanotechnology has yielded nontrivial autonomous behaviours such as stimulus-guided locomotion, computation and programmable molecular assembly. Despite these successes, DNA-based nanomachines suffer from slow kinetics, requiring several minutes or longer to carry out a handful of operations. Here, we pursue the speed limit of an important class of reactions in DNA nanotechnology-toehold exchange-through the single-molecule optimization of a novel class of DNA walker that undergoes cartwheeling movements over a field of complementary oligonucleotides. After optimizing this DNA 'acrobat' for rapid movement, we measure a stepping rate constant approaching 1 s

Topics: Carbocyanines; DNA, Single-Stranded; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Kinetics; Models, Molecular; Motion; Nanostructures; Nanotechnology; Oligonucleotides

Fluorescence lifetime correlation analysis is becoming a powerful tool to understand the conformational heterogeneity of biomolecules and their dynamics with an unprecedented detection sensitivity and time resolution. However, its application to the study of biomembranes is very limited. Here, we report on two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) in combination with total internal reflection (TIR) microscopy (TIR 2D-FLCS). High depth resolution in TIR microscopy and species-specific correlation analysis in 2D FLCS give us the opportunity to selectively analyze molecules in or on a supported lipid bilayer, a model biomembrane formed on the glass surface. Feasibility experiments performed in this study clearly demonstrated that TIR 2D-FLCS has a potential to selectively analyze the diffusion and the conformational dynamics of proteins peripherally bound on the membrane in the presence of substantial amounts of unbound molecules in the bulk phase.

Topics: Carbocyanines; Diffusion; DNA; Glass; Lipid Bilayers; Microscopy, Interference; Molecular Conformation; Spectrometry, Fluorescence

Silver nanoparticles (AgNPs) have a high affinity for sulfhydryl (thiol) groups, which can be exploited for functionalization with various tracking and targeting moieties. Here, we describe how to reliably and reproducibly functionalize AgNPs with the fluorescent moiety cyanine3-polyethelyne glycol (5000 molecular weight)-thiol (Cy3-PEG

Topics: Breast Neoplasms; Carbocyanines; Cell Culture Techniques; Female; Fluorescent Dyes; Humans; Metal Nanoparticles; Molecular Imaging; Silver; Spectrophotometry, Ultraviolet; Tumor Cells, Cultured

Release factors RF1 and RF2 promote hydrolysis of peptidyl-tRNA during translation termination. The GTPase RF3 promotes recycling of RF1 and RF2. Using single molecule FRET and biochemical assays, we show that ribosome termination complexes that carry two factors, RF1-RF3 or RF2-RF3, are dynamic and fluctuate between non-rotated and rotated states, whereas each factor alone has its distinct signature on ribosome dynamics and conformation. Dissociation of RF1 depends on peptide release and the presence of RF3, whereas RF2 can dissociate spontaneously. RF3 binds in the GTP-bound state and can rapidly dissociate without GTP hydrolysis from termination complex carrying RF1. In the absence of RF1, RF3 is stalled on ribosomes if GTP hydrolysis is blocked. Our data suggest how the assembly of the ribosome-RF1-RF3-GTP complex, peptide release, and ribosome fluctuations promote termination of protein synthesis and recycling of the release factors.

Topics: Carbocyanines; Escherichia coli; Escherichia coli Proteins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Guanosine Triphosphate; Hydrolysis; Kinetics; Peptide Chain Termination, Translational; Peptide Termination Factors; Protein Binding; Protein Conformation; Ribosomes; RNA, Transfer, Amino Acyl; Single Molecule Imaging; Thermodynamics

Nucleic acid (NA)-based therapy is proposed to address serious diseases such as cardiovascular diseases (CVDs). Powerful NA delivery vehicles are essential for effective gene therapy. Herein, a novel type of delivery vehicle, an unlockable core-shell nanocomplex (Hep@PGEA) with self-accelerating NA release, is structurally designed. Hep@PGEA is composed of disulfide-bridged heparin nanoparticle (HepNP) core and low-toxicity PGEA cationic shell. In comparison with NA, heparin, a negatively charged polysaccharide macromolecule, exhibits stronger interactions with cationic species. Upon the breakdown of redox-responsive HepNP cores, unlocked heparin would interact with the outer cationic shells and replace the condensed NA to facilitate NA release. Such unique Hep@PGEA is successfully explored for effective miRNA-pDNA staged gene therapy of myocardial infarction (MI), one of the most serious CVDs. With the progression of MI, glutathione amounts in heart tissues increase. MiR-499 (for the inhibition of cardiomyocyte apoptosis) and plasmid encoding vascular endothelial growth factor (for the promotion of angiogenesis) are sequentially delivered for systemic treatment of MI. Such treatment produces impressive results in restoring heart function and suppressing cardiac hypertrophy. Due to the wide existence of redox agents in cells, the proposed unlockable delivery nanovehicle and staged therapy strategy can provide new methods to effectively treat different serious diseases.

Topics: Animals; Carbocyanines; DNA; Genetic Therapy; Glutathione; Heparin; Mice; MicroRNAs; Microscopy, Atomic Force; Microscopy, Confocal; Myocardial Infarction; Nanoparticles; Particle Size; Polymethacrylic Acids; Vascular Endothelial Growth Factor A

Supramolecular one-dimensional (1D) architectures are of high interest in drug delivery and templation of complex linear arrays due to their high aspect ratio and rigidity. A particular desire is the access of 1D nanostructures with high functionality and biorelevance, which opens the door to  their applications in materials science and nanomedicine. Here we report the discovery that the site-specific introduction of a cyanine (Cy3) dye unit in sequence-defined DNA amphiphiles causes a complete shift of the overall structure from spheres to 1D DNA nanofibers in aqueous media. We show that the generation of DNA nanofibers is dependent on the presence of cyanine units and their position within the DNA-polymer hybrid. We further demonstrate an example of stimuli-responsive shape-shifting DNA nanofibers to highlight the role of the dye in the overall assembly. Notably, we show the preparation of fibers with controlled length by seeded-growth mechanism. Additionally, the DNA nanofibers exhibit a change in Cy3 dye optical properties upon assembly, typical of cyanine dye aggregation, which can be used to monitor the fiber growth process. To demonstrate the functionality of these structures, we show the templation of gold nanoparticles (AuNP) along the fiber length and demonstrate the directional templation of DNA nanofibers on rectangular DNA origami. Our findings provide a method for generating functional nanomaterials and hierarchical complex architectures and show promise as a platform for biosensing and targeted drug delivery.

Topics: Carbocyanines; DNA; Fluorescence; Fluorescent Dyes; Gold; Metal Nanoparticles; Molecular Structure; Nanofibers; Nucleic Acid Conformation

A core-shell nanostructure is fabricated with a pH-sensitive metal-organic framework shell and a peptide functionalized gold nanoparticle core via a mild synthetic route. The nanostructure can be applied as a dual-recognition switch in response to an acidic environment and enzyme activity, sequentially, leading to a stepwise-responsive strategy for imaging lysosomal cathepsin B.

Topics: Carbocyanines; Cathepsin B; Fluorescence; Fluorescent Dyes; Gold; HeLa Cells; Humans; Hydrogen-Ion Concentration; Imidazoles; Lysosomes; Metal Nanoparticles; Metal-Organic Frameworks; Microscopy, Confocal; Microscopy, Fluorescence; Particle Size; Peptides; Zeolites

Leucine-rich alpha-2-glycoprotein-1 (Lrg1) is an emerging biomarker for angiogenesis. Its expression in ocular tissues is up-regulated in both human patients with proliferative diabetic retinopathy and rodent models of pathological angiogenesis. However, there is no existing sensor that allows visualization and monitoring of Lrg1 expression noninvasively and in real time. Herein, we report a nucleic acid-gold nanorod-based nanosensor for the noninvasive monitoring of cellular Lrg1 expression in angiogenesis. Specifically, this platform is constructed by covalently conjugating molecular beacons onto gold nanorods, which prequench the fluorophores on the molecular beacons. Upon intracellular entry and endosomal escape, the complexes interact with cellular Lrg1 mRNA through hybridization of the loop area of the molecular beacons. This complexation distances the fluorophores from nanorod and restores the prequenched fluorescence. The reliability of this platform is confirmed by examining the increased Lrg1 expression in migrating keratinocytes and the Lrg1 gene changes in different postnatal stages of mouse retinal vasculature growth in the mouse retina model.

Topics: Animals; Carbocyanines; Cell Line; Fluorescence; Fluorescent Dyes; Glycoproteins; Gold; Humans; Inverted Repeat Sequences; Mice, Inbred C57BL; Nanotubes; Neovascularization, Pathologic; Nucleic Acid Hybridization; Retina; RNA, Messenger; Up-Regulation

Rapid detection and quantification of pharmaceutical drugs directly in human plasma is of major importance for the development of relevant point-of-care testing devices. Here, we report a method for detection and quantification of small molecules in human plasma. An assay employing a small molecule-linked hybridization chain reaction (HCR) has been devised for the detection of the pharmaceutically relevant drugs digoxin (Dig) and methotrexate (MTX). Double modification by small molecule ligands on the initiator strand act as sites to control the rate of the HCR. Upon protein binding to the modified initiator strand, the HCR is greatly inhibited. If the protein is preincubated with a sample containing the small molecule analyte, the protein binding site is occupied by the analyte and the initiator strand will initiate the HCR. This enables efficient detection and quantification of small-molecule analytes in nanomolar concentration even in 50% human plasma within 4 min. Thus, the rapidity and simplicity of this assay has potential for point-of-care testing.

Topics: Antibodies; Biotin; Carbocyanines; Digoxigenin; Digoxin; DNA, Complementary; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Limit of Detection; Methotrexate; Nucleic Acid Amplification Techniques; Nucleic Acid Hybridization; Point-of-Care Testing; Streptavidin

Fluorescence-activated cell sorting (FACS) offers a powerful approach to high-throughput library screening in directed evolution experiments. However, FACS is rarely used in the evolution of stereoselective enzymes, due to the difficulty of designing fluorescence-based assays for measuring enantiopurity. Here, we describe a new FACS-based enantiopurity analysis approach that overcomes these limitations by using enantiomeric DNA biosensors labeled with orthogonal fluorophores. By co-encapsulating the biosensors with a mixture of target enantiomers in microfluidic droplets, we could demonstrate the use of FACS to differentiate between droplets having various levels of target enantiopurity. We envision the utility of this method for high-throughput screening of enantiopurity in the directed evolution of stereoselective enzymes, thereby facilitating the discovery of new asymmetric biocatalysts for the synthesis of pharmaceuticals and other high-value chemicals.

Topics: Alkanesulfonates; Aptamers, Nucleotide; Azo Compounds; Biosensing Techniques; Carbocyanines; DNA; Flow Cytometry; Fluorescence; Fluorescent Dyes; High-Throughput Screening Assays; Lab-On-A-Chip Devices; Stereoisomerism; Tyrosine

Optical manipulation appears to be a powerful tool for spatiotemporally controlling a variety of cellular functions. Herein, a photocontrolled DNA assembly approach is described which enables light-induced activation of cellular signal transduction by triggering protein dimerization (c-Met signalling in this case). Three kinds of DNA probes are designed, including a pair of receptor recognition probes with adaptors and a blocker probe with a photocleavable linker (PC-linker). By implementing PC-linkers in blocker probes, the designed DNA probes response to light irradiation, which then induces the assembly of receptor recognition probes through adaptor complementing. Consequently, light-mediated DNA assembly promotes the dimerization of c-Met receptors, resulting in activation of c-Met signalling. It is demonstrated that the proposed photocontrolled DNA assembly approach is effective for regulating c-Met signalling and modulating cellular behaviours, such as cell proliferation and migration. Therefore, this simple approach may offer a promising strategy to manipulate cell signalling pathways precisely in living cells.

Topics: Aptamers, Nucleotide; Carbocyanines; Cell Line, Tumor; Cell Membrane; Cell Movement; Cell Proliferation; Cell Survival; DNA Probes; Fluorescent Dyes; Humans; Light; Photochemical Processes; Protein Multimerization; Proto-Oncogene Proteins c-met; Signal Transduction

From gene expression to nanotechnology, understanding and controlling DNA requires a detailed knowledge of its higher order structure and dynamics. Here we take advantage of the environment-sensitive photoisomerization of cyanine dyes to probe local and global changes in DNA structure. We report that a covalently attached Cy3 dye undergoes strong enhancement of fluorescence intensity and lifetime when stacked in a nick, gap or overhang region in duplex DNA. This is used to probe hybridization dynamics of a DNA hairpin down to the single-molecule level. We also show that varying the position of a single abasic site up to 20 base pairs away modulates the dye-DNA interaction, indicative of through-backbone allosteric interactions. The phenomenon of stacking-induced fluorescence increase (SIFI) should find widespread use in the study of the structure, dynamics and reactivity of nucleic acids.

Topics: Allosteric Regulation; Carbocyanines; DNA; Fluorescence; Fluorescent Dyes; Nucleic Acid Conformation; Single Molecule Imaging; Spectrometry, Fluorescence

Aptamers are short nucleic-acid biopolymers selected to have high affinity and specificity for protein or small-molecule target analytes. Aptamers can be engineered into split-aptamer biosensors comprising two nucleic acid strands that coassemble as they bind to a target, resulting in a large signal change from attached molecular probes (e.g., molecular beacons). The kinetics of split-aptamer assembly and their dependence on target recognition are largely unknown; knowledge of these kinetics could help in design and optimization of split-aptamer biosensors. In this work, we measure assembly kinetics of cocaine-dependent split-aptamer molecules using single-molecule fluorescence imaging. Assembly is monitored between a DNA strand tethered to a glass substrate and solutions containing the other strand tagged with a fluorescent label, with varying concentrations of the cocaine analyte. Dissociation rates are measured by tracking individual molecules and measuring their bound lifetimes. Dissociation-time distributions are biexponential, possibly indicating different folded states of the aptamer. The dissociation rate of only the longer-lived complex decreases with cocaine concentration, suggesting that cocaine stabilizes the long-lived aptamer complex. The variation in the slow dissociation rate with cocaine concentration is well described with an equilibrium-binding model, where the dissociation rate approaches a saturation limit consistent with the dissociation-equilibrium constant for cocaine-binding to the split aptamer. This single-molecule methodology provides a sensitive readout of cocaine-binding based on the dissociation kinetics of the split aptamer, allowing one to distinguish target-dependent aptamer assembly from background assembly. This methodology could be used to study other systems where target association affects the stability of aptamer duplexes.

Topics: Aptamers, Nucleotide; Carbocyanines; Cocaine; DNA Probes; Fluorescence; Fluorescent Dyes; Kinetics; Nucleic Acid Conformation; Nucleic Acid Hybridization; Single Molecule Imaging

Two 2'-deoxyuridines as new building blocks for automated DNA synthesis carry a small aryltetrazole as "photoclickable" group at their 5-positions. The postsynthetic "photoclick" labeling of such presynthesized DNA using a maleimide-modified Cy3 dye shows an up to 17-fold fluorogenicity due to an energy transfer between the pyrazoline moiety and the Cy3 fluorophore in the DNA products. This concept is also applicable to other maleimide-modified dyes.

Topics: Base Sequence; Carbocyanines; Cycloaddition Reaction; Deoxyuridine; DNA; Fluorescent Dyes; Maleimides; Photochemical Processes; Staining and Labeling

Exosomes, as novel noninvasive biomarkers for disease prediction and diagnosis, have shown fascinating prospects in monitoring cancer-linked public health issues. Herein, a unique Cy3 labeled CD63 aptamer (Cy3-CD63 aptamer)/Ti

Topics: Aptamers, Nucleotide; Biosensing Techniques; Carbocyanines; Cell Line, Tumor; Exosomes; Fluorescence; Fluorescence Resonance Energy Transfer; Humans; Limit of Detection; Nanostructures; Tetraspanin 30; Titanium

The sortase-catalyzed coupling reaction is an efficient strategy to incorporate chemically defined modifications into proteins of interest. Despite its widespread applications in protein chemistry, the conventional bulk fluorescence measurement is not sufficient to characterize sortase due to the fluorescence inner filter effect-induced self-quenching. Herein, we develop a new method to visualize and quantify sortase A (SrtA) activity at the single-molecule level by using transpeptidation-directed intramolecular Förster resonance energy transfer (FRET). This assay utilizes two cyanine dye-peptide conjugates, in which one carries an LPXTG motif and a donor fluorophore while the other harbors an oligoglycine nucleophile and an acceptor fluorophore as the substrate of SrtA. The presence of SrtA catalyzes the fusion of two conjugates and allows for the occurrence of intramolecular FRET. The FRET signal is recorded at the single-molecule level via total internal reflection fluorescence (TIRF)-based imaging. The proposed assay not only can accurately determine the kinetic parameters of SrtA but also can characterize the inhibition of SrtA activity by berberine chloride both in vitro and in Staphylococcus aureus ( S. aureus) cells. Moreover, the assay is very specific, and it can sensitively measure SrtA down to 7.08 pM, which is much lower than most of the reported methods. This strategy may provide a valuable tool for an in-depth study of sortases and for the discovery of anti-infective agents.

Topics: Aminoacyltransferases; Bacterial Proteins; Carbocyanines; Cysteine Endopeptidases; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Kinetics; Single Molecule Imaging; Staphylococcus aureus

Neural stem cells (NSCs) have been attractive donor sources for cell therapy in traumatic brain injuries (TBI). Monitoring the fate of transplanted cells, including the survival and differentiation, will provide vital information to assess the outcome during the therapy time course. However, the current labeling methods are based on the principles of cell endocytosis, demanding relatively high fluorescent probes concentration and long incubation time, which may affect the proliferation and differentiation of transplanted cells. In our study, an efficient and relatively fast labeling strategy for NSCs with Cy3 based on DNA hybridization was proposed for monitoring the fate of transplanted cells. The oligo[dA]

Topics: Animals; Brain Injuries, Traumatic; Carbocyanines; Cell Differentiation; Cell Survival; Disease Models, Animal; DNA; Mice; Neural Stem Cells; Nucleic Acid Hybridization; Stem Cell Transplantation

The visualization of glycosylation states of specific proteins in vivo is of great importance for uncovering their roles in disease development. However, the ubiquity of glycosylation makes probing the glycans on a certain protein as difficult as looking for a needle in a haystack. Herein, we demonstrate a proximity-induced hybridization chain reaction (HCR) strategy for amplified visualization of protein-specific glycosylation. The strategy relies on designing two kinds of DNA probes, glycan conversion probes and protein recognition probes, which are attached to glycans and target proteins, respectively. Upon sequential binding to the targets, the proximity-induced hybridization between two probes occurs, which leads to the structure-switching of protein recognition probes, followed by triggering of HCR assembly. This strategy has been used to visualize tyrosine-protein kinase 7-specific sialic acid in living CEM cells and zebrafish and to monitor its variation during drug treatment. It provides a potential tool for investigating protein-specific glycosylation and researching the relation between dynamic glycans state and disease process.

Topics: Animals; Azides; Carbocyanines; Cell Line, Tumor; DNA; DNA Probes; Fluorescence; Fluorescent Dyes; Glycoproteins; Glycosylation; Hexosamines; Humans; Microscopy, Confocal; Microscopy, Fluorescence; Nucleic Acid Hybridization; Polysaccharides; Tunicamycin; Zebrafish

RNA interference (RNAi)-mediated gene silencing offers a novel treatment and prevention strategy for human immunodeficiency virus (HIV) infection. HIV was found to infect and replicate in human brain cells and can cause neuroinfections and neurological deterioration. We designed dual-antibody-modified chitosan/small interfering RNA (siRNA) nanoparticles to deliver siRNA across the blood-brain barrier (BBB) targeting HIV-infected brain astrocytes as a strategy for inhibiting HIV replication. We hypothesized that transferrin antibody and bradykinin B2 antibody could specifically bind to the transferrin receptor (TfR) and bradykinin B2 receptor (B2R), respectively, and deliver siRNA across the BBB into astrocytes as potential targeting ligands. In this study, chitosan nanoparticles (CS-NPs) were prepared by a complex coacervation method in the presence of siRNA, and antibody was chemically conjugated to the nanoparticles. The antibody-modified chitosan nanoparticles (Ab-CS-NPs) were spherical in shape, with an average particle size of 235.7 ± 10.2 nm and a zeta potential of 22.88 ± 1.78 mV. The therapeutic potential of the nanoparticles was evaluated based on their cellular uptake and gene silencing efficiency. Cellular accumulation and gene silencing efficiency of Ab-CS-NPs in astrocytes were significantly improved compared to non-modified CS-NPs and single-antibody-modified CS-NPs. These results suggest that the combination of anti-Tf antibody and anti-B2 antibody significantly increased the knockdown effect of siRNA-loaded nanoparticles. Thus, antibody-mediated dual-targeting nanoparticles are an efficient and promising delivery strategy for inhibiting HIV replication in astrocytes. Graphical abstract Graphic representation of dual-antibody-conjugated chitosan nanoparticles for the targeted delivery of siRNA across the blood-brain barrier (BBB) for inhibiting HIV replication in astrocytes. a Nanoparticle delivery to the BBB and penetration. b TfR-mediated transcytosis of nanoparticles across the epithelial cells. c B2R-mediated endocytosis of nanoparticles in astrocytes. d The molecular interactions between HIV-1 Tat protein and Cyclin T1 and Tip110 cellular proteins. e A schematic representation of chitosan nanoparticles with its components. RNAPII RNA polymerase II, TAR transactivation response RNA element, LTR long terminal repeat, Ab antibody, CS chitosan, TPP tripolyphosphate.

Topics: Antibodies; Antigens, Neoplasm; Astrocytes; Blood-Brain Barrier; Carbocyanines; Cell Line; Cell Survival; Chitosan; Cyclin T; Fluorescent Dyes; HIV-1; Humans; Nanoparticles; Receptor, Bradykinin B2; RNA-Binding Proteins; RNA, Small Interfering; Transferrin; Virus Replication

The development of functional DNA-based nanosensors in living cells has experienced some design challenges, including, for example, poor cellular uptake, rapid nuclease degradation, and high false positives. Herein, we designed selectively permeable poly(methacrylic acid) (PMA) nanocapsules to encapsulate functional DNAs for metal ions and small-molecules sensing in living cells. Since functional DNAs are concentrated in the nanocapsules, an increasing reaction rate is obtained in vitro. During endocytosis, polymeric capsules simultaneously improve cellular uptake of functional DNAs and preserve their structural integrity inside the confined capsule space. More importantly, selective shell permeability allows for the free diffusion of small molecular targets through capsule shells but limits the diffusion of large biomolecules, such as nuclease and nonspecific protein. Compared to the free DNAzyme, PMA nanocapsules could reduce false positives and enhance detection accuracy. Furthermore, PMA nanocapsules are biocompatible and biodegradable. Through the controllability of wall thickness, permeability, and size distribution, these nanocapsules could be expanded easily to other targets, such as microRNAs, small peptides, and metabolites. These nanocapsules will pave the way for in situ monitoring of various biological processes in living cells and in vivo.

Topics: Bioreactors; Biosensing Techniques; Carbocyanines; DNA; DNA, Catalytic; Humans; Lead; Lipid Bilayers; MCF-7 Cells; Microscopy, Confocal; Nanocapsules; Particle Size; Polymethacrylic Acids; Silicon Dioxide; Spectrometry, Fluorescence; Zinc

Hydrophobins are surface-active proteins produced by filamentous fungi. They have amphiphilic structures and form multimers in aqueous solution to shield their hydrophobic regions. The proteins rearrange at interfaces and self-assemble into films that can show a very high degree of structural order. Little is known on dynamics of multimer interactions in solution and how this is affected by other components. In this work we examine the multimer dynamics by stopped-flow fluorescence measurements and Förster Resonance Energy Transfer (FRET) using the class II hydrophobin HFBII. The half-life of exchange in the multimer state was 0.9s at 22°C with an activation energy of 92kJ/mol. The multimer exchange process of HFBII was shown to be significantly affected by the closely related HFBI hydrophobin, lowering both activation energy and half-life for exchange. Lower molecular weight surfactants interacted in very selective ways, but other surface active proteins did not influence the rates of exchange. The results indicate that the multimer formation is driven by specific molecular interactions that distinguish different hydrophobins from each other.

Topics: Carbocyanines; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Fungal Proteins; Half-Life; Hydrophobic and Hydrophilic Interactions; Kinetics; Models, Molecular; Mycelium; Protein Isoforms; Protein Multimerization; Rheology; Surface-Active Agents; Temperature; Thermodynamics; Trichoderma

Fifty-five mono- and disaccharide analogues were prepared and used for the construction of microarrays to uncover lectin-selective ligands. The microarray study showed that two disaccharide analogues, 28' and 44', selectively bind to Solanum tuberosum lectin (STL) and wheat germ agglutinin (WGA), respectively. Cell studies indicated that 28' and 44' selectively block the binding of STL and WGA to mammalian cells, unlike the natural ligand LacNAc, which suppresses binding of both STL and WGA to cells.

Topics: Carbocyanines; Carbohydrate Conformation; Disaccharides; Dose-Response Relationship, Drug; Fluorescent Dyes; HeLa Cells; Humans; Ligands; Microarray Analysis; Monosaccharides; Plant Lectins; Protein Binding; Solanum tuberosum; Staining and Labeling; Triticum; Wheat Germ Agglutinins

Here, we demonstrate a phosphorodiamidate morpholino oligos (PMO)-functionalized nanochannel biosensor for label-free detection of microRNAs (miRNAs) with ultrasensitivity and high sequence specificity. PMO, as a capture probe, was covalently anchored on the nanochannel surface. Because of the neutral character and high sequence-specific affinity of PMO, hybridization efficiency between PMO and miRNAs was enhanced, thus largely decreasing background signals and highly improving the detection specificity and sensitivity. The miRNAs detection was realized through observing the change of surface charge density when PMO/miRNAs hybridization occurred. Not only could the developed biosensor specifically discriminate complementary miRNAs (Let-7b) from noncomplementary miRNAs (miR-21) and one-base mismatched miRNAs (Let-7c), but also it could detect target miRNAs in serum samples. In addition, this nanochannel-based biosensor attained a reliable limit of detection down to 1 fM in PBS and 10 fM in serum sample, respectively. It is expected that such a new method will benefit miRNA detection in clinical diagnosis.

Topics: Animals; Biosensing Techniques; Carbocyanines; Cattle; Limit of Detection; MicroRNAs; Microscopy, Confocal; Morpholinos; Nucleic Acid Hybridization; Reproducibility of Results

The use of synthetic messenger ribonucleic acid (mRNA) to express specific proteins is a highly promising therapeutic and vaccine approach that avoids many safety issues associated with viral or DNA-based systems. However, in order to optimize mRNA designs and delivery, technology advancements are required to study fundamental mechanisms of mRNA uptake and localization at the single-cell and tissue level. Here, we present a single RNA sensitive fluorescent labeling method which allows us to label and visualize synthetic mRNA without significantly affecting function. This approach enabled single cell characterization of mRNA uptake and release kinetics from endocytic compartments, the measurement of mRNA/protein correlations, and motivated the investigation of mRNA induced cellular stress, all important mechanisms influencing protein production. In addition, we demonstrated this approach can facilitate near-infrared imaging of mRNA localization in vivo and in ex-vivo tissue sections, which will facilitate mRNA trafficking studies in pre-clinical models. Overall, we demonstrate the ability to study fundamental mechanisms necessary to optimize delivery and therapeutic strategies, in order to design the next generation of novel mRNA therapeutics and vaccines.

Topics: Animals; Biological Transport; Carbocyanines; Cell Line; Cytoplasmic Granules; Endosomes; Fibroblasts; Fluorescent Dyes; Green Fluorescent Proteins; HeLa Cells; Humans; Infrared Rays; Injections, Intramuscular; Mice; Molecular Probes; Nucleic Acid Hybridization; Optical Imaging; RNA, Messenger; Single-Cell Analysis; Staining and Labeling

We investigated the efficacy and safety of ultrasound (US)-targeted microbubble (MB) destruction (UTMD)-enhanced delivery of monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly-L-lysine (mPEG-PLGA-PLL) nanoparticles (NPs) loading Cy3-labelled platelet-derived growth factor BB (PDGF-BB) siRNA to rat retina in vivo. Eighty Wistar rats were divided into five groups (G). The right eyes, respectively, received an intravitreal injection as follows: normal saline (NS) (G1), NPs and NS (G2), NPs and MBs (G3), NPs and NS (G4) and NPs and MBs (G5). In G4 and G5, the eyes were exposed to US for 5 mins. Twenty-four hours after transfection, the uptake and distribution of Cy3-labelled siRNA in rat retina were observed by fluorescent microscope. The percentage of Cy3- labelled siRNA-positive cells was evaluated by flow cytometer. The levels of PDGF-BB mRNA in retinal pigment epithelium (RPE) cells and secreted PDGF-BB proteins were also measured. Hematoxylin and eosin staining and frozen sections were used to observe tissue damage. Our results showed that the number of Cy3-labelled siRNApositive cells in G5 was significantly higher than those of the other groups (P less than 0.05 for all comparisons). The maximum efficiency of siRNA uptake in neural retina was 18.22 +/_ 1.67%. In G4 and G5, a small number of Cy3- labelled siRNA-positive cells were also detected in the pigmented cell layer of the retina. NPs loading siRNA delivered with UTMD could more effectively down-regulate the mRNA and protein expression of PDGF-BB than NPs plus US (P=0.014 and P=0.007, respectively). Histology showed no evident tissue damage after UTMDmediated NPs loading siRNA transfection. UTMD could be used safely to enhance the delivery of mPEG-PLGAPLL NPs loading siRNA into rat retina.

Topics: Animals; Becaplermin; Biocompatible Materials; Carbocyanines; Drug Delivery Systems; Lactic Acid; Male; Microbubbles; Nanoparticles; Polyethylene Glycols; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polylysine; Proto-Oncogene Proteins c-sis; Rats; Rats, Wistar; Retina; RNA, Small Interfering; Ultrasonics

Changes in microscopic viscosity and macromolecular crowding accompany the transition of proteins from their monomeric forms into highly organised fibrillar states. Previously, we have demonstrated that viscosity sensitive fluorophores termed 'molecular rotors', when freely mixed with monomers of interest, are able to report on changes in microrheology accompanying amyloid formation, and measured an increase in rigidity of approximately three orders of magnitude during aggregation of lysozyme and insulin. Here we extend this strategy by covalently attaching molecular rotors to several proteins capable of assembly into fibrils, namely lysozyme, fibrinogen and amyloid-β peptide (Aβ(1-42)). We demonstrate that upon covalent attachment the molecular rotors can successfully probe supramolecular assembly in vitro. Importantly, our new strategy has wider applications in cellulo and in vivo, since covalently attached molecular rotors can be successfully delivered in situ and will colocalise with the aggregating protein, for example inside live cells. This important advantage allowed us to follow the microscopic viscosity changes accompanying blood clotting and during Aβ(1-42) aggregation in live SH-SY5Y cells. Our results demonstrate that covalently attached molecular rotors are a widely applicable tool to study supramolecular protein assembly and can reveal microrheological features of aggregating protein systems both in vitro and in cellulo not observable through classical fluorescent probes operating in light switch mode.

Topics: Amyloid beta-Peptides; Boron Compounds; Carbocyanines; Cell Line; Fibrinogen; Fluorescent Dyes; Humans; Insulin; Microscopy, Electron, Transmission; Molecular Probes; Muramidase; Nanoconjugates; Optical Imaging; Peptide Fragments; Protein Aggregates; Viscosity

Quantitative measurement of mRNA levels in single cells is necessary to understand phenotypic variability within an otherwise isogenic population of cells. Single-molecule mRNA Fluorescence In Situ Hybridization (FISH) has been established as the standard method for this purpose, but current protocols require a long region of mRNA to be targeted by multiple DNA probes. Here, we introduce a new single-probe FISH protocol termed sFISH for budding yeast, Saccharomyces cerevisiae using a single DNA probe labeled with a single fluorophore. In sFISH, we markedly improved probe specificity and signal-to-background ratio by using methanol fixation and inclined laser illumination. We show that sFISH reports mRNA changes that correspond to protein levels and gene copy number. Using this new FISH protocol, we can detect >50% of the total target mRNA. We also demonstrate the versatility of sFISH using FRET detection and mRNA isoform profiling as examples. Our FISH protocol with single-fluorophore sensitivity significantly reduces cost and time compared to the conventional FISH protocols and opens up new opportunities to investigate small changes in RNA at the single cell level.

Topics: Carbocyanines; DNA Probes; Fluorescent Dyes; Gene Expression Regulation, Fungal; In Situ Hybridization, Fluorescence; RNA, Messenger; Saccharomyces cerevisiae; Sensitivity and Specificity; Single Molecule Imaging

Cyanine (Cy) dyes show a general propensity to localize in polarized mitochondria. This mitochondriotropism was used to perform a copper-free click reaction in the mitochondria of living cells. The in organello reaction of dyes Cy3 and Cy5 led to a product that was easily traceable by Förster resonance energy transfer (FRET). As determined by confocal laser scanning microscopy, the Cy3-Cy5 conjugate showed enhanced retention in mitochondria, relative to that of the starting compounds. This enhancement of a favorable property can be achieved by synthesis in organello, but not outside mitochondria.

Topics: Animals; Carbocyanines; Cell Line; Click Chemistry; Copper; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Microscopy, Confocal; Mitochondria; Rats

Structural DNA nanotechnology finds applications in numerous areas, but the construction of objects, 2D and 3D crystalline lattices and devices is prominent among them. Each of these components has been developed individually, and most of them have been combined in pairs. However, to date there are no reports of independent devices contained within 3D crystals. Here we report a three-state 3D device whereby we change the colour of the crystals by diffusing strands that contain dyes in or out of the crystals through the mother-liquor component of the system. Each colouring strand is designed to pair with an extended triangle strand by Watson-Crick base pairing. The arm that contains the dyes is quite flexible, but it is possible to establish the presence of the duplex proximal to the triangle by X-ray crystallography. We modelled the transition between the red and blue states through a simple kinetic model.

Topics: Carbocyanines; Color; DNA; Nanotechnology; Nucleic Acid Hybridization; Phase Transition

The use of therapeutic antibodies, delivered by intravenous (IV) instillation, is a rapidly expanding area of biomedical treatment for a variety of conditions. However, little is known about how the antibodies are anatomically distributed following infusion and the underlying mechanism mediating therapeutic antibody distribution to specific anatomical sites remains to be elucidated. Current efforts utilize low resolution and sensitivity methods such as ELISA and indirect labeling imaging techniques, which often leads to high background and difficulty in assessing biodistribution. Here, using the in vivo non-human primate model, we demonstrate that it is possible to utilize the fluorophores Cy5 and Cy3 directly conjugated to antibodies for direct visualization and quantification of passively transferred antibodies in plasma, tissue, and in mucosal secretions. Antibodies were formulated with 1-2 fluorophores per antibody to minimally influence antibody function. Fluorophore conjugated Gamunex-C (pooled human IgG) were tested for binding to protein A, via surface plasmon resonance, and showed similar levels of binding when compared to unlabeled Gamunex-C. In order to assess the effect fluorophore labeling has on turnover and localization, rhesus macaques were IV infused with either labeled or unlabeled Gamunex-C. Plasma, vaginal Weck-Cel fluid, cervicovaginal mucus, and vaginal/rectal tissue biopsies were collected up to 8weeks. Similar turnover and biodistribution was observed between labeled and unlabeled antibodies, showing that the labeling process did not have an obvious deleterious effect on localization or turnover. Cy5 and Cy3 labeled antibodies were readily detected in the same pattern regardless of fluorophore. Tissue distribution was measured in macaque vaginal and rectal biopsies. The labeled antibody in macaque biopsies was found to have similar biodistribution pattern to endogenous antibodies in macaque and human tissues. In the vaginal and rectal mucosa, endogenous and infused antibodies were found primarily within the lamina propria. In the mucosal squamous epithelium of the vaginal vault, significant antibody was also observed in a striated pattern in the superficial, nonviable, stratum corneum. Endogenous antibody distribution in both human and macaque squamous tissues exhibited a similar pattern as seen with the labeled and unlabeled antibodies. This proof-of-principle study reveals that the labeled antibody is stable and physiologically s

Topics: Animals; Carbocyanines; Cervix Mucus; Drug Stability; Female; Fluorescent Antibody Technique; Fluorescent Dyes; Humans; Immunoglobulins, Intravenous; Infusions, Intravenous; Macaca mulatta; Microscopy, Fluorescence; Models, Animal; Mucous Membrane; Plasma; Protein Stability; Rectum; Surface Plasmon Resonance; Tissue Distribution; Vagina

Telomere length has been correlated with various diseases, including cardiovascular disease and cancer. The use of currently available telomere-length measurement techniques is often restricted by the requirement of a large amount of cells (Southern-based techniques) or the lack of information on individual cells or telomeres (PCR-based methods). Although several methods have been used to measure telomere length in tissues as a whole, the assessment of cell-type-specific telomere length provides valuable information on individual cell types. The development of fluorescence in situ hybridization (FISH) technologies enables the quantification of telomeres in individual chromosomes, but the use of these methods is dependent on the availability of isolated cells, which prevents their use with fixed archival samples. Here we describe an optimized quantitative FISH (Q-FISH) protocol for measuring telomere length that bypasses the previous limitations by avoiding contributions from undesired cell types. We have used this protocol on small paraffin-embedded cardiac-tissue samples. This protocol describes step-by-step procedures for tissue preparation, permeabilization, cardiac-tissue pretreatment and hybridization with a Cy3-labeled telomeric repeat complementing (CCCTAA)

Topics: Carbocyanines; Humans; In Situ Hybridization, Fluorescence; Myocardium; Peptide Nucleic Acids; Telomere

Short oligonucleotide sequences are now being widely investigated for their potential therapeutic properties. The modification of oligonucleotide termini with short fluorinated residues is capable of drastically altering their behavior in complex

Topics: Carbocyanines; Cell Line, Tumor; Electrophoretic Mobility Shift Assay; Fatty Acids; Fluorocarbons; Humans; Intracellular Space; Molecular Dynamics Simulation; Molecular Imaging; Molecular Probes; Oligodeoxyribonucleotides; Polylysine; Proton Magnetic Resonance Spectroscopy; STAT3 Transcription Factor

Cy3 and Cy5 dyes linked to the 5' end of a double stranded DNA molecule are known to attach to both strands in a way that is very similar to an additional base pair and has a stabilizing effect on the oligonucleotide. Here we adapt the Peyrard-Bishop mesoscopic model to incorporate cyanine dyes and use the technique of thermal equivalence to obtain the appropriate parameters from existing melting temperatures. We have found that the stacking parameters are in the same range of ordinary AT and CG base pairs, in particular Cy3-A was found to be most rigidly stacked. While the cyanines stabilize the AT hydrogen bonds quite strongly the CG bonds are mostly unaffected.

Topics: Base Pairing; Carbocyanines; DNA; Hydrogen Bonding; Models, Molecular; Nucleic Acid Denaturation; Temperature

A multicolour protein labelling technique using a protein tag and fluorogenic probes is a powerful approach for spatio-temporal analyses of proteins in living cells. Since cyanine fluorophores have attractive properties for multicolour imaging of proteins, there is a huge demand to develop fluorogenic cyanine probes for specific protein labelling in living cells. Herein, we develop fluorogenic cyanine probes for labelling a protein tag by using a dinitrobenzene fluorescence quencher. The probes enhanced fluorescence intensity upon labelling reactions and emitted orange or far-red fluorescence. Intramolecular interactions between the cyanine fluorophores and the dinitrobenzene quencher led not only to fluorescence quenching of the probes in the free state but also to promotion of labelling reactions. Furthermore, the probes successfully imaged cell-surface proteins without a washing process. These findings offer valuable information on the design of fluorogenic cyanine probes and indicate that the probes are useful as novel live-cell imaging tools.This article is part of the themed issue 'Challenges for chemistry in molecular imaging'.

Topics: Carbocyanines; Dinitrobenzenes; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; HEK293 Cells; Humans; Kinetics; Microscopy, Fluorescence, Multiphoton; Molecular Imaging; Proteins; Spatio-Temporal Analysis

Elaborating efficient strategies and deepening the understanding of light transport at the nanoscale is of great importance for future designs of artificial light-harvesting assemblies and dye-based photonic circuits. In this work, we focus on studying the phenomenon of Förster resonance energy transfer (FRET) among fluorophores of the same kind (homo-FRET) and its implications for energy cascades containing two or three different dye molecules. Utilizing the spatial programmability of DNA origami, we arranged a chain of cyanine 3 (Cy3) dyes flanked at one end with a dye of lower excitation energy, cyanine 5 (Cy5), with or without an additional dye of higher excitation energy, Alexa488, at the other end. We characterized the response of our fluorophore assemblies with bulk and single-molecule spectroscopy and support our measurements by Monte Carlo modeling of energy transfer within the system. We find that, depending on the arrangement of the fluorophores, homo-FRET between the Cy3 dyes can lead to an overall enhanced energy transfer to the acceptor fluorophore. Furthermore, we systematically analyzed the homo-FRET system by addressing the fluorescence lifetime and anisotropy. Finally, we built a homo-FRET-mediated photonic wire capable of transferring energy through the homo-FRET system from the blue donor dye (Alexa488) to the red acceptor fluorophore (Cy5) across a total distance of 16 nm.

Topics: Carbocyanines; Computer Simulation; DNA; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Monte Carlo Method; Nanostructures; Photons; Single Molecule Imaging; Spectrometry, Fluorescence

Topics: Binding Sites; Carbocyanines; DNA Damage; DNA Helicases; DNA Repair; DNA Replication; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Gene Expression; Kinetics; Microscopy, Fluorescence; Models, Molecular; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Multimerization; Single Molecule Imaging; Thermodynamics

Herein, a novel upconversion@polydopamine core@shell nanoparticle (termed as UCNP@PDA NP) -based aptameric biosensor has been fabricated for the quantitative analysis of cytochrome c (Cyt c) inside living cells, which comprises an UCNP@PDA NP, acting as an internal reference and fluorescence quenching agent, and Cy3 modified aptamer enabling ratiometric quantitative Cyt c measurement. After the hybridization of Cy3 labeled aptamer with amino-terminated single DNA on the UCNP@PDA NP surface (termed as UCNP@PDA@AP), the fluorescence of Cy3 can be efficiently quenched by the PDA shell. With the spontaneous cellular uptake of UCNP@PDA@AP, the Cyt c aptamer dissociates from UCNP@PDA NP surface through formation of aptamer-Cyt c complex, resulting in concomitant activation of the Cy3 fluorescence. High amount of Cyt c leads to high fluorescence emission, enabling direct visualization/measurement of the Cyt c by fluorescence microscopy/spectroscopy. The steady upconversion luminescent (UCL) signals can be employed not only for intracellular imaging, but also as an internal reference for evaluating intracellular Cyt c amount using the ratio of fluorescence intensity of Cy3 with the UCL intensity of UCNP. The UCNP@PDA@AP shows a reasonable detection limit (20nM) and large dynamic range (50nM to 10μM, which covers the literature reported values (1-10μM) for cytosolic Cyt c in apoptotic cells) for detecting Cyt c in buffer with excellent selectivity. In addition, the UCNP@PDA@AP has been successfully used to monitor etoposide induced intracellular releasing of Cyt c, providing the possibility for cell-based screening of apoptosis-inducing drugs.

Topics: Aptamers, Nucleotide; Biosensing Techniques; Carbocyanines; Cytochromes c; Fluorescent Dyes; Hep G2 Cells; Humans; Indoles; Lanthanoid Series Elements; Microscopy, Fluorescence; Models, Molecular; Nanoparticles; Optical Imaging; Polymers

Hydrocyanines are widely used as fluorogenic probes to monitor reactive oxygen species (ROS) generation in cells. Their brightness, stability to autoxidation and photobleaching, large signal change upon oxidation, pH independence and red/near infrared emission are particularly attractive for imaging ROS in live tissue.. Using confocal fluorescence microscopy we have examined an interference of mitochondrial membrane potential (ΔΨm) with fluorescence intensity and localisation of a commercial hydro-Cy3 probe in respiring and non-respiring colon carcinoma HCT116 cells.. We found that the oxidised (fluorescent) form of hydro-Cy3 is highly homologous to the common ΔΨm-sensitive probe JC-1, which accumulates and aggregates only in 'energised' negatively charged mitochondrial matrix. Therefore, hydro-Cy3 oxidised by hydroxyl and superoxide radicals tends to accumulate in mitochondrial matrix, but dissipates and loses brightness as soon as ΔΨm is compromised. Experiments with mitochondrial inhibitor oligomycin and uncoupler FCCP, as well as a common ROS producer paraquat demonstrated that signals of the oxidised hydro-Cy3 probe rapidly and strongly decrease upon mitochondrial depolarisation, regardless of the rate of cellular ROS production.. While analysing ROS-derived fluorescence of commercial hydrocyanine probes, an accurate control of ΔΨm is required.. If not accounted for, non-specific effect of mitochondrial polarisation state on the behaviour of oxidised hydrocyanines can cause artefacts and data misinterpretation in ROS studies.

Topics: Animals; Carbocyanines; Cell Line, Tumor; Fluorescence; HCT116 Cells; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mitochondria; Oligomycins; Oxidation-Reduction; Reactive Oxygen Species; Superoxides

Podosomes are adhesive structures formed on the plasma membrane abutting the extracellular matrix of macrophages, osteoclasts, and dendritic cells. They consist of an f-actin core and a ring structure composed of integrins and integrin-associated proteins. The podosome ring plays a major role in adhesion to the underlying extracellular matrix, but its detailed structure is poorly understood. Recently, it has become possible to study the nano-scale structure of podosome rings using localization microscopy. Unlike traditional microscopy images, localization microscopy images are reconstructed using discrete points, meaning that standard image analysis methods cannot be applied. Here, we present a pipeline for podosome identification, protein position calculation, and creating a podosome ring model for use with localization microscopy data.

Topics: Actin Cytoskeleton; Carbocyanines; Cell Movement; Cells, Cultured; Dendritic Cells; Extracellular Matrix; Fibroblasts; Fluorescent Dyes; Gene Expression; Genes, Reporter; Humans; Luminescent Proteins; Macrophages; Microscopy, Fluorescence; Osteoclasts; Paxillin; Podosomes; Red Fluorescent Protein; Staining and Labeling; Talin; Vinculin

Despite recent advances in scientific knowledge and clinical practice, cardiovascular disease management and treatment remain a major burden. While several treatment strategies using drugs and surgeries are being developed for cardiovascular manifestations, gene-based therapies hold significant promise. Recent findings from our laboratory unveiled a novel mechanism that exosomes, secreted nanovesicles from stem cells, mediate cardiac repair via transferring their unique repertoire of microRNAs (miRNA) to recipient cells in the heart. Exosomes, unlike other vectors for gene delivery, present unique advantages such that exosomes are a cell-free natural system for ferrying RNA between cells, robust exosomal membrane can protect the RNA/gene of interest from digestion, and exosomes are rapidly taken up by target cells making them a more efficient vehicle for gene delivery. Here, we describe a stepwise protocol developed in our laboratory for generating exosomes from human CD34

Topics: Carbocyanines; Cells, Cultured; Exosomes; Flow Cytometry; Gene Transfer Techniques; Genetic Therapy; Humans; MicroRNAs; Stem Cells; Transfection

Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation of pulmonary artery smooth muscle cells and endothelial cells associated with obliteration of small pulmonary arterioles and formation of plexiform lesions. To date, no curative treatments have been identified for pulmonary arterial hypertension. There are various therapeutic options, including conventional medical therapies and oral, subcutaneous, intravenous, and inhalation delivery. We have previously shown that miR-143/145 knockout can prevent the development of chronic hypoxia-induced pulmonary hypertension (PH) in mice. Here, we use chronic hypoxia-induced PH as a disease model to evaluate miR-143/145 inhibition after delivery of antimiRNAs via the subcutaneous or intranasal routes. We use qRT-PCR and immunofluorescence to confirm that both delivery strategies efficiently inhibit miR-143/145 in lung tissue from mice with chronic hypoxia-induced PH.

Topics: Administration, Intranasal; Animals; Antagomirs; Carbocyanines; Chronic Disease; Disease Models, Animal; Female; Fluorescent Antibody Technique; Gene Transfer Techniques; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Lung; Mice, Inbred C57BL; MicroRNAs; Real-Time Polymerase Chain Reaction

This study presents an innovative method for the synthesis of polymeric nanoparticles (NPs) for central nervous system (CNS) targeting. The method is based on Ultraviolet light (UV)-induced crosslinking of diacrylamide-terminated oligomers of poly(amidoamine)s (PAAs), a widely used class of synthetic polymers in biomedical field research, especially in drug delivery thanks to their excellent biocompatibility and controlled biodegradability. Previous attempts aiming at preparing PAA-based NPs by self-assembly were challenged by lack of structural stability and consequently their early degradation and premature drug release. Here, the UV-induced crosslinked PAA NPs demonstrated to overcome main disadvantages of the self-assembled ones, as they showed improved stability and controlled release properties. Besides the remarkable efficiency to produce monodisperse and stable PAA NPs, the UV-induced crosslinking method is featured by great versatility and low environmental impact, since it does not require use of organic solvents and multiple purification steps. The capability of PAA NPs to encapsulate a fluorescently labelled model protein was experimentally demonstrated in this study. Cell culture experiments showed that PAA NPs were biocompatible and highly permeable across an in vitro blood-brain barrier model, thus highlighting their great potential as drug delivery vectors for CNS delivery.

Topics: Animals; Biocompatible Materials; Blood-Brain Barrier; Brain; Carbocyanines; Central Nervous System; Cross-Linking Reagents; Drug Carriers; Drug Delivery Systems; Human Umbilical Vein Endothelial Cells; Humans; Immunoglobulin G; Light; Mice; Microscopy, Fluorescence; Nanoparticles; Permeability; Photochemistry; Polyamines; Polymers; Scattering, Radiation; Serum Albumin; Solvents; Ultraviolet Rays

Lanthanide-doped upconversion nanoparticles with a suitable surface coating are appealing for biomedical applications. Because high-quality upconversion nanoparticles are typically prepared in an organic solvent and passivated by hydrophobic oleate ligands, a convenient and reliable method for the surface modification of upconversion nanoparticles is thus highly desired to satisfy downstream biological investigations. In this work, we describe a facile and versatile strategy for displacing native oleate ligands on upconversion nanoparticles with a diversity of hydrophilic molecules. The ligand-exchange procedure involves the removal of original oleate ligands followed by the attachment of new ligands in a separate step. The successful coating of relevant ligands was confirmed by Fourier transform infrared spectroscopy, thermogravimetry analysis, and ζ-potential measurement. The surface-modified nanoparticles display high stability and good biocompatibility, as revealed by electron microscopy, photoluminescence spectroscopy, and cytotoxicity assessment. Our study demonstrates that functional biomolecules such as biotin can be directly immobilized on the nanoparticle surface using this approach for the quick and effective detection of streptavidin.

Topics: A549 Cells; Acrylic Resins; Carbocyanines; Fluorides; Humans; Ligands; Nanoparticles; Oleic Acids; Streptavidin; Yttrium

Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope.

Topics: Anti-Bacterial Agents; beta-Lactamases; Biosensing Techniques; Carbapenems; Carbocyanines; Dimethylpolysiloxanes; DNA Probes; DNA, Circular; Drug Resistance, Microbial; Escherichia coli; Escherichia coli Proteins; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Gene Expression; Humans; Lab-On-A-Chip Devices; Methicillin; Microscopy, Fluorescence; Mutation; Oligonucleotides; Polymerase Chain Reaction; Pseudomonas aeruginosa; Staphylococcus aureus

Bioorthogonal chemistry is an effective tool for elucidating metabolic pathways and measuring cellular activity, yet its use is currently limited by the difficulty of getting probes past the cell membrane and into the cytoplasm, especially if more complex probes are desired. Here we present a simple and minimally perturbative technique to deliver functional probes of glycosylation into cells by using a nanostructured "nanostraw" delivery system. Nanostraws provide direct intracellular access to cells through fluid conduits that remain small enough to minimize cell perturbation. First, we demonstrate that our platform can deliver an unmodified azidosugar, N-azidoacetylmannosamine, into cells with similar effectiveness to a chemical modification strategy (peracetylation). We then show that the nanostraw platform enables direct delivery of an azidosugar modified with a charged uridine diphosphate group (UDP) that prevents intracellular penetration, thereby bypassing multiple enzymatic processing steps. By effectively removing the requirement for cell permeability from the probe, the nanostraws expand the toolbox of bioorthogonal probes that can be used to study biological processes on a single, easy-to-use platform.

Topics: Aluminum Oxide; Animals; Azides; Carbocyanines; Cell Membrane Permeability; CHO Cells; Cricetulus; Drug Delivery Systems; Fluorescent Dyes; Glycosylation; Hexosamines; Molecular Probes; Nanostructures; Protein Processing, Post-Translational; Rhodamines; Uridine Diphosphate N-Acetylgalactosamine

The replicative polymerases cannot accommodate distortions to the native DNA sequence such as modifications (lesions) to the native template bases from exposure to reactive metabolites and environmental mutagens. Consequently, DNA synthesis on an afflicted template abruptly stops upon encountering these lesions, but the replication fork progresses onward, exposing long stretches of the damaged template before eventually stalling. Such arrests may be overcome by translesion DNA synthesis (TLS) in which specialized TLS polymerases bind to the resident proliferating cell nuclear antigen (PCNA) and replicate the damaged DNA. Hence, a critical aspect of TLS is maintaining PCNA at or near a blocked primer/template (P/T) junction upon uncoupling of fork progression from DNA synthesis by the replicative polymerases. The single-stranded DNA (ssDNA) binding protein, replication protein A (RPA), coats the exposed template and might prohibit diffusion of PCNA along the single-stranded DNA adjacent to a blocked P/T junction. However, this idea had yet to be directly tested. We recently developed a unique Cy3-Cy5 Forster resonance energy transfer (FRET) pair that directly reports on the occupancy of DNA by PCNA. In this study, we utilized this FRET pair to directly and continuously monitor the retention of human PCNA at a blocked P/T junction. Results from extensive steady state and pre-steady state FRET assays indicate that RPA binds tightly to the ssDNA adjacent to a blocked P/T junction and restricts PCNA to the upstream duplex region by physically blocking diffusion of PCNA along ssDNA.

Topics: Base Sequence; Biotin; Carbocyanines; Cell Line; DNA Damage; DNA Repair; DNA Replication; DNA, Single-Stranded; Escherichia coli; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Gene Expression; Humans; Nucleic Acid Conformation; Oligonucleotides; Proliferating Cell Nuclear Antigen; Protein Binding; Recombinant Proteins; Replication Protein A

Many transgenes are silenced in mammalian cells (donor cells used for somatic cell nuclear transfer [SCNT]). Silencing correlated with a repressed chromatin structure or suppressed promoter, and it impeded the production of transgenic animals. Gene transcription studies in live cells are challenging because of the drawbacks of reverse-transcription polymerase chain reaction and fluorescence in situ hybridization. Nano-flare probes provide an effective approach to detect RNA in living cells. We used 18S RNA, a housekeeping gene, as a reference gene. This study aimed to establish a platform to detect RNA in single living donor cells using a Nano-flare probe prior to SCNT and to verify the safety and validity of the Nano-flare probe in order to provide a technical foundation for rescuing silenced transgenes in transgenic cloned embryos. We investigated cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts, characterized the distribution of the 18S RNA-Nano-flare probe in living cells and investigated the effect of the 18S RNA-Nano-flare probe on the development of cloned embryos after SCNT. The cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts was dose-dependent, and 18S RNA was detected using the 18S RNA-Nano-flare probe. In addition, treating donor cells with 500 pM 18S RNA-Nano-flare probe did not have adverse effects on the development of SCNT embryos at the pre-implantation stage. In conclusion, we established a preliminary platform to detect RNA in live donor cells using a Nano-flare probe prior to SCNT.

Topics: Animals; Animals, Genetically Modified; Carbocyanines; Cloning, Organism; Female; Fibroblasts; Fluorescent Dyes; Gene Expression; Gene Silencing; Genetic Engineering; In Situ Hybridization, Fluorescence; Metal Nanoparticles; Nuclear Transfer Techniques; Nucleic Acid Hybridization; Oocytes; RNA, Ribosomal, 18S; Swine; Tissue Donors; Transgenes

We present a non-invasive fluorescence method for imaging of scaffold degradation in vivo by quantifying the degradation of porcine cartilage-derived extracellular matrix powder (PCP).Three-dimensional porous scaffolds should be biocompatible and bioresorbable, with a controllable degradation and resorption rate to match tissue growth. However, in vivo scaffold degradation and tissue ingrowth processes are not yet fully understood. Unfortunately, current analysis methods require animal sacrifice and scaffold destruction for the quantification of scaffold degradation and cannot monitor the situation in real time. In this study, Cy3, a fluorescent dye, was used for visualizing PCP and a real-time degradation profile was obtained quantitatively by a non-invasive method using an imaging system in which the reduction in fluorescence intensity depended on PCP scaffold degradation. Real-time PCP scaffold degradation was confirmed through changes in the volume and morphology of the scaffold using micro-computed tomography and microscopy. Our results suggest that extracellular matrix degradation was induced by collagen degradation because of the binding between Cy3 and collagen. This non-invasive real-time monitoring system for scaffold degradation will increase our understanding of in vivo matrix and/or scaffold degradation.

Topics: Animals; Carbocyanines; Cartilage; Esters; Extracellular Matrix; Mice; Optical Imaging; Powders; Swine; Tissue Scaffolds; X-Ray Microtomography

The regulation of RNA transcription is central to cellular function. Changes in gene expression drive differentiation and cellular responses to events such as injury. RNA trafficking can also have a large impact on protein expression and its localization. Thus, the ability to image RNA transcription and trafficking in real time and in living cells is a worthwhile goal that has been difficult to achieve. The availability of "light-up" aptamers that cause an increase in fluorescence of their ligands when bound by the aptamer have shown promise for reporting on RNA production and localization in vivo. Here we have investigated two light-up aptamers (the malachite green aptamer and the Spinach aptamers) for their suitabilities as reporters of RNA expression in vivo using two eukaryotic cell types, yeast and mammalian. Our analysis focused on the aptamer ligands, their contributions to background noise, and the impact of tandem aptamer strings on signal strength and ligand affinity. Whereas the background fluorescence is very low in vitro, this is not always true for cell imaging. Our results suggest the need for caution in using light-up aptamers as reporters for imaging RNA. In particular, images should be collected and analyzed by operators blinded to the sample identities. The appropriate control condition of ligand with the cells in the absence of aptamer expression must be included in each experiment. This control condition establishes that the specific interaction of ligand with aptamer, rather than nonspecific interactions with unknown cell elements, is responsible for the observed fluorescent signals. High background signals due to nonspecific interactions of aptamer ligands with cell components can be minimized by using IMAGEtags (Intracellular Multiaptamer GEnetic tags), which signal by FRET and are promising RNA reporters for imaging transcription.

Topics: Animals; Aptamers, Nucleotide; Benzyl Compounds; Carbocyanines; Cell Line; CHO Cells; Cricetulus; Epithelial Cells; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Imidazolines; Kinetics; Ligands; Lung; Mink; Molecular Probes; Optical Imaging; RNA; Rosaniline Dyes; Saccharomyces cerevisiae; Signal-To-Noise Ratio; Transcription, Genetic

We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87 μM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H(+) homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles.

Topics: Acridine Orange; Amino Acid Sequence; Animals; Antimalarials; Biological Transport; Carbocyanines; Cell-Penetrating Peptides; Cells, Cultured; Chloroquine; Crotalid Venoms; Crotalus; Dose-Response Relationship, Drug; Erythrocytes; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Inhibitory Concentration 50; Plasmodium falciparum; Snake Venoms; Staining and Labeling; Vacuoles

Nanoparticle delivery is becoming an increasingly more valuable technique in cancer drug treatments. The use of fluorescent probes, in particular, can provide noninvasive strategies to interrogate the internalization mechanisms of cancer cells and aid in drug design. Here we describe the delivery of fluorescently labeled polysaccharide-based nanoparticles to breast cancer cells in vitro and their subsequent immunofluorescence microscopy examination. The description of the synthesis, preparation, and delivery of the nanoparticles can be widely applicable to other in vitro drug delivery studies.

Topics: Animals; Biological Transport; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cryopreservation; Fluorescent Dyes; Intracellular Space; Mice; Microscopy, Fluorescence; Nanoparticles; Particle Size; Polysaccharides; Sterilization

DNA origami nanostructures are a versatile tool to arrange metal nanostructures and other chemical entities with nanometer precision. In this way gold nanoparticle dimers with defined distance can be constructed, which can be exploited as novel substrates for surface enhanced Raman scattering (SERS). We have optimized the size, composition and arrangement of Au/Ag nanoparticles to create intense SERS hot spots, with Raman enhancement up to 10(10), which is sufficient to detect single molecules by Raman scattering. This is demonstrated using single dye molecules (TAMRA and Cy3) placed into the center of the nanoparticle dimers. In conjunction with the DNA origami nanostructures novel SERS substrates are created, which can in the future be applied to the SERS analysis of more complex biomolecular targets, whose position and conformation within the SERS hot spot can be precisely controlled.

Topics: Carbocyanines; Dimerization; DNA; DNA, Single-Stranded; Gold; Metal Nanoparticles; Microscopy, Atomic Force; Microscopy, Electron, Scanning; Nanotechnology; Nucleic Acid Hybridization; Rhodamines; Scattering, Radiation; Silicon; Silver; Spectrum Analysis, Raman

The fluorescent intensity of Cy3 and Cy5 dyes is strongly dependent on the nucleobase sequence of the labeled oligonucleotides. Sequence-dependent fluorescence may significantly influence the data obtained from many common experimental methods based on fluorescence detection of nucleic acids, such as sequencing, PCR, FRET, and FISH. To quantify sequence dependent fluorescence, we have measured the fluorescence intensity of Cy3 and Cy5 bound to the 5' end of all 1024 possible double-stranded DNA 5mers. The fluorescence intensity was also determined for these dyes bound to the 5' end of fixed-sequence double-stranded DNA with a variable sequence 3' overhang adjacent to the dye. The labeled DNA oligonucleotides were made using light-directed, in situ microarray synthesis. The results indicate that the fluorescence intensity of both dyes is sensitive to all five bases or base pairs, that the sequence dependence is stronger for double- (vs single-) stranded DNA, and that the dyes are sensitive to both the adjacent dsDNA sequence and the 3'-ssDNA overhang. Purine-rich sequences result in higher fluorescence. The results can be used to estimate measurement error in experiments with fluorescent-labeled DNA, as well as to optimize the fluorescent signal by considering the nucleobase environment of the labeling cyanine dye.

Topics: Carbocyanines; DNA; Fluorescence

The remarkable performance and quantum efficiency of biological light-harvesting complexes has prompted a multidisciplinary interest in engineering biologically inspired antenna systems as a possible route to novel solar cell technologies. Key to the effectiveness of biological "nanomachines" in light capture and energy transport is their highly ordered nanoscale architecture of photoactive molecules. Recently, DNA origami has emerged as a powerful tool for organizing multiple chromophores with base-pair accuracy and full geometric freedom. Here, we present a programmable antenna array on a DNA origami platform that enables the implementation of rationally designed antenna structures. We systematically analyze the light-harvesting efficiency with respect to number of donors and interdye distances of a ring-like antenna using ensemble and single-molecule fluorescence spectroscopy and detailed Förster modeling. This comprehensive study demonstrates exquisite and reliable structural control over multichromophoric geometries and points to DNA origami as highly versatile platform for testing design concepts in artificial light-harvesting networks.

Topics: Carbocyanines; DNA; Light; Photochemical Processes; Spectrometry, Fluorescence

We describe a supramolecular surface competition assay for quantifying glutamine uptake from single cells. Cy3-labeled cyclodextrins were immobilized on a glass surface as a supramolecular host/FRET donor, and adamantane-BHQ2 conjugates were employed as the guest/quencher. An adamantane-labeled glutamine analog was selected through screening a library of compounds and validated by cell uptake experiments. When integrated onto a single cell barcode chip with a multiplex panel of 15 other metabolites, associated metabolic enzymes, and phosphoproteins, the resultant data provided input for a steady-state model that describes energy potential in single cells and correlates that potential with receptor tyrosine kinase signaling. We utilize this integrated assay to interrogate a dose-dependent response of model brain cancer cells to EGFR inhibition. We find that low-dose (1 μM erlotinib) drugging actually increases cellular energy potential even as glucose uptake and phosphoprotein signaling is repressed. We also identify new interactions between phosphoprotein signaling and cellular energy processes that may help explain the facile resistance exhibited by certain cancer patients to EGFR inhibitors.

Topics: Brain Neoplasms; Carbocyanines; Cell Line, Tumor; Dose-Response Relationship, Drug; ErbB Receptors; Erlotinib Hydrochloride; Fluorescence Resonance Energy Transfer; Glioblastoma; Glutamine; Humans; Molecular Probes

We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA.

Topics: Binding Sites; Carbocyanines; DNA; DNA Polymerase I; DNA Replication; DNA, Single-Stranded; Escherichia coli; Fluorescence Resonance Energy Transfer; Microscopy, Atomic Force; Nanoparticles; Nanotechnology

Ribosome assembly has been studied intensively using Förster resonance energy transfer (FRET) with fluorophore-labeled fragments of RNA produced by chemical synthesis. However, these studies are limited by the size of the accessible RNA fragments. We have developed a replicable unnatural base pair (UBP) formed between (d)5SICS and (d)MMO2 or (d)NaM, which efficiently directs the transcription of RNA containing unnatural nucleotides. We now report the synthesis and evaluation of several of the corresponding ribotriphosphates bearing linkers that enable the chemoselective attachment of different functionalities. We found that the RNA polymerase from T7 bacteriophage does not incorporate NaM derivatives but does efficiently incorporate 5SICS(CO), whose linker enables functional group conjugation via Click chemistry, and when combined with the previously identified MMO2(A), whose amine side chains permits conjugation via NHS coupling chemistry, enables site-specific double labeling of transcribed RNA. To study ribosome assembly, we transcribed RNA corresponding to a 243-nt fragment of the central domain of Thermus thermophilus 16S rRNA containing 5SICS(CO) and MMO2(A) at defined locations and then site-specifically attached the fluorophores Cy3 and Cy5. FRET was characterized using single-molecule total internal reflection fluorescence (smTIRF) microscopy in the presence of various combinations of added ribosomal proteins. We demonstrate that each of the fragment's two three-helix junctions exist in open and closed states, with the latter favored by sequential protein binding. These results elucidate early and previously uncharacterized folding events underlying ribosome assembly and demonstrate the applicability of UBPs for biochemical, structural, and functional studies of RNAs.

Topics: Base Pairing; Base Sequence; Carbocyanines; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; RNA, Bacterial; RNA, Ribosomal, 16S; Thermus thermophilus

The present study was designed to visualize the cellular trafficking of adeno-associated virus (AAV) vectors in general and AAV5 serotype vectors in particular. We fluorescently labeled AAV5 wild-type and a mutant (S652A) virus and studied their infection process by live cell imaging and confocal microscopy. Our data demonstrate considerable difference in the ability of these vectors to reach the nuclear compartment within the first 6 hr after infection.

Topics: Carbocyanines; Cytoplasm; Dependovirus; Fluorescent Dyes; Genetic Vectors; HeLa Cells; Humans; Transcytosis

High-performing hybridization platforms fabricated by reactive microcontact printing of DNA probes are presented. Multishaped PDMS molds are used to covalently bind oligonucleotides over a functional copolymer (DMA-NAS-MAPS) surface. Printed structures with minimum width of about 1.5 μm, spaced by 10 μm, are demonstrated, with edge corrugation lower than 300 nm. The quantification of the immobilized surface probes via fluorescence imaging gives a remarkable concentration of 3.3 × 10(3) oligonucleotides/μm(2), almost totally active when used as probes in DNA-DNA hybridization assays. Indeed, fluorescence and atomic force microscopy show a 95% efficiency in target binding and uniform DNA hybridization over printed areas.

Topics: Carbocyanines; DNA Probes; DNA, Single-Stranded; Fluorescent Dyes; Methacrylates; Nucleic Acid Hybridization; Oligodeoxyribonucleotides; Succinimides

miR-144 has potential benefits in protecting against myocardial ischemia and suppression of tumor growth. We have previously shown that a single intravenous injection of miR-144 provides potent cardioprotection, but its kinetics and distribution are not known.. Single stranded mature miR-144 or Cy3-labelled-miR-144 was delivered into C57/B6 mice by tail vein injection.. After intravenous injection, the signal of Cy3-labelled-miR-144 in the kidney, brain, heart and liver peaks at 60 minutes, and is predominantly localised to the endothelium at that stage. In the kidney and heart, Cy3-labelled-miR-144 signal is detectable within the parenchymal tissues for at least 3 days, after which it starts to decrease, but brain Cy3-miR-144 signal rapidly decreases after 1 hour, and is lost at day 1, with no parenchymal uptake detected. Cy3-miR-144 signal can be detected until day 28 in the liver. Stem loop RTPCR confirmed the temporal pattern shown by miR-144 in kidney, brain and heart, but in liver there was a continuous rise following the initial injection until day 28 with no signs of decrease, suggesting de-novo synthesis.. There is early endothelial uptake of injected miR-144 followed by organ-specific distribution and kinetics. In the liver, there appears to be a positive feedback process that leads to continued accumulation of miR-144 that persists for at least 28 days. These observations should be taken into account when designing experiments utilizing parenteral miR-144 and assessing the biology of its actions.

Topics: Administration, Intravenous; Animals; Argonaute Proteins; Brain; Carbocyanines; Kidney; Liver; Mice; Mice, Inbred C57BL; MicroRNAs; Myocardium; Staining and Labeling

Two novel cyanine-based 1-amino-1-deoxy-β-glucose conjugates (Glu-1N-Cy3 and Glu-1N-Cy5) were designed, synthesized and their fluorescence characteristics were studied. Both Glu-1N-Cy3 and Glu-1N-Cy5 accumulate in living HT29 human colon cancer cells, which overexpress glucose transporters (GLUTs). The cellular uptake of the bioprobes was inhibited by natural GLUT substrate d-glucose and 2-deoxy-d-glucose. The GLUT specificity of the probes was validated with quercetin, which is both a permeant substrate via GLUTs and a high-affinity inhibitor of GLUT-mediated glucose transport. Competitive fluorometric assay for GLUT substrate cell uptake revealed that Glu-1N-Cy3 and Glu-1N-Cy5 are 5 and 10 times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. This study provides fundamental data supporting the potential of these two conjugates as new powerful tools for GLUT-mediated theranostics, in vitro and in vivo molecular bioimaging and drug R&D.

Topics: Carbocyanines; Fluorescent Dyes; Glucose; Glucose Transport Proteins, Facilitative; Image Enhancement; Microscopy, Fluorescence; Molecular Imaging; Reproducibility of Results; Sensitivity and Specificity; Subcellular Fractions

Steroid ligands can easily diffuse through the cell membrane and this property makes it feasible to be used for in-situ staining of the nuclear receptors. However, nonspecific binding of the internalized ligand probe with the cellular components has caused serious interferences for the detection of receptor-expressing cells. We report a novel gold nanocluster (AuNC)-conjugated estrogen probe that can eliminate nonspecific internalization and accelerate nuclear localization to achieve selective and rapid detection of estrogen receptors (ERs) in live cells. The AuNC, protected by bovine serum albumin (BSA), BSA-AuNCs, was prepared by the synthesis and confirmed to be 1.9 nm in core size and 18 nm in diameter. Ethinyl estradiol was used as the precursor of 17β-estradial (E2) to conjugate with BSA-protected AuNCs via polyethylene glycol linker (E2-PEG/BSA-AuNCs) or to conjugate with Cy3 dyes (E2-Cy3). The conjugated probe was determined to contain five E2 molecules per BSA-AuNC by mass spectrometry and exhibit an emission maximum of around 640 nm, which was not altered by E2 conjugation indicating that the structural integrity of BSA-AuNCs was conserved. E2-PEG/BSA-AuNCs probes were quickly internalized by MCF-7 (ER+) cells and localized to the nuclei in 2 h. Such internalization was sensitive to competition by free E2 and was rarely detected in the controls using either non-conjugated BSA-AuNCs in MCF-7 (ER+) cells or E2-PEG/BSA-AuNCs in MDA-MB-231 (ER-) cells. In contrast to the high specificity of E2-PEG/BSA-AuNCs probe, the uptake of E2-Cy3 probe could not differentiate between MCF-7(ER+) and MDA-MB-231(ER-) cells during the early phases of the treatment. Moreover, nuclear targeting by E2-Cy3 was three times slower than that by the E2-PEG/BSA-AuNC probe. Such accelerated nuclei targeting was consistent with the enhanced cell viability by conjugating E2 with BSA-AuNC. In conclusion, the E2-PEG/BSA-AuNC probes are promising candidates that can be used for the detection of ER+ tumor tissues and the same strategy can be applied to fabricate other steroid probes.

Topics: Alkynes; Animals; Biological Transport; Carbocyanines; Cattle; Cell Survival; Click Chemistry; Estradiol; Ethinyl Estradiol; Gold; Humans; MCF-7 Cells; Metal Nanoparticles; Optical Imaging; Receptors, Estrogen; Serum Albumin, Bovine

Single-molecule, protein-induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Förster-type resonance energy transfer (FRET) or photoinduced electron transfer. In order to provide two simultaneous measurements of two distances on different molecular length scales for the analysis of macromolecular complexes, we and others recently combined measurements of PIFE and FRET (PIFE-FRET) on the single molecule level. PIFE relies on steric hindrance of the fluorophore Cy3, which is covalently attached to a biomolecule of interest, to rotate out of an excited-state trans isomer to the cis isomer through a 90° intermediate. In this work, we provide a theoretical framework that accounts for relevant photophysical and kinetic parameters of PIFE-FRET, show how this framework allows the extraction of the fold-decrease in isomerization mobility from experimental data, and show how these results provide information on changes in the accessible volume of Cy3. The utility of this model is then demonstrated for experimental results on PIFE-FRET measurement of different protein-DNA interactions. The proposed model and extracted parameters could serve as a benchmark to allow quantitative comparison of PIFE effects in different biological systems.

Topics: Binding Sites; Carbocyanines; Computer Simulation; DNA; DNA Restriction Enzymes; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Isomerism; Kinetics; Models, Theoretical; Photochemical Processes

Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3'-Cy3-labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5' to 3' ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5' to 3') pushing of the SSB toward the 3' ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3' to 5') direction, are observed with EcRep and EcUvrD (both 3' to 5' ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.

Topics: Adenosine Triphosphate; Carbocyanines; DNA Replication; DNA-Binding Proteins; DNA, Single-Stranded; Escherichia coli; Escherichia coli Proteins; Fluorescence Resonance Energy Transfer; Kinetics; Protein Binding; Saccharomyces cerevisiae; Spectrometry, Fluorescence

In this Communication, a molecular beacon-based DNA switch (LMB) is developed as an efficient and reversible pH sensing probe. Remarkably, LMB exhibited reversible structural transition between the closed (molecular beacon) and open (A-motif) states very efficiently in synthetic vesicles and live cells without the need for any transfection agents.

Topics: Carbocyanines; Cell Survival; DNA; Fluorescence Resonance Energy Transfer; HeLa Cells; Humans; Hydrogen-Ion Concentration; Molecular Probes

The reactivation of stalled DNA replication via fork regression invokes Holliday junction formation, branch migration, and the recovery of the replication fork after DNA repair or error-free DNA synthesis. The coordination mechanism for these DNA structural transitions by molecular motors, however, remains unclear. Here we perform single-molecule fluorescence experiments with Werner syndrome protein (WRN) and model replication forks. The Holliday junction is readily formed once the lagging arm is unwound, and migrated unidirectionally with 3.2 ± 0.03 bases/s velocity. The recovery of the replication fork was controlled by branch migration reversal of WRN, resulting in repetitive fork regression. The Holliday junction formation, branch migration, and migration direction reversal are all ATP dependent, revealing that WRN uses the energy of ATP hydrolysis to actively coordinate the structural transitions of DNA.

Topics: Adenosine Triphosphate; Animals; Base Pairing; Carbocyanines; DNA Replication; DNA, Cruciform; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Gene Expression; Humans; Recombinant Fusion Proteins; Sf9 Cells; Single Molecule Imaging; Spodoptera; Werner Syndrome Helicase

Clostridium difficile is the bacterium responsible for most antibiotic-associated diarrhea in North America and Europe. This bacterium, which colonizes the gut of humans and animals, produces toxins that are known to contribute directly to damage of the gut. It is known that bacterial flagella are involved in intestinal lesions through the inflammatory host response. The C. difficile flagellin recognizes TLR5 and consequently activates the NF-κB and the MAPK signaling pathways which elicit the synthesis of pro-inflammatory cytokines. Increasing interest on the role of C. difficile flagella in eliciting this cell response was recently developed and the development of tools to study cell response triggered by C. difficile flagella will improve our understanding of the pathogenesis of C. difficile.

Topics: Bacterial Proteins; Caco-2 Cells; Carbocyanines; Cell Survival; Clostridioides difficile; Cytokines; Flagella; Flagellin; Gene Expression Regulation; Histidine; Host-Pathogen Interactions; Humans; Immunity, Innate; Mitogen-Activated Protein Kinases; NF-kappa B; Oligopeptides; Protein Array Analysis; Recombinant Fusion Proteins; Signal Transduction; Streptavidin; Toll-Like Receptor 5

Peroxynitrite (OONO(-)) is profoundly implicated in health and disease. The physiological and pathological outcome of OONO(-) is related to its local concentration, and hence, a reliable OONO(-) assay is highly desired. We have developed a FRET-based small-molecule fluorescent probe (PNCy3Cy5), harnessing the differential reactivity of Cy3 and Cy5 toward OONO(-) by fine-tuning. It exhibits high detection sensitivity and yields a ratiometric fluorescent signal. We have exemplified that it can be applied in semiquantitative determination of OONO(-) in living cells. Notably, it specifically localizes in mitochondria, where endogenous OONO(-) is predominantly generated. Thus, PNCy3Cy5 is a promising molecular tool for peroxynitrite biology.

Topics: Animals; Carbocyanines; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Mice; Mitochondria; Optical Imaging; Peroxynitrous Acid; RAW 264.7 Cells

Two novel fluorescent bioprobes, namely, 6N-Gly-Cy3 and 6N-Gly-Cy5, were designed and synthesized for real-time glucose transport imaging as well as potentially useful tracer for galactokinase metabolism. The structure of the bioprobes was fully characterized by

Topics: Carbocyanines; Fluorescent Dyes; Glucose Transport Proteins, Facilitative; Glycoconjugates; HT29 Cells; Humans; Microscopy, Fluorescence; Molecular Imaging; Spectrometry, Fluorescence

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Binding Sites; Carbocyanines; Elasticity; Fluorescent Dyes; Hydrolysis; Ion Transport; Kinetics; Models, Chemical; Protein Binding; Proton-Translocating ATPases; Rotation; Single Molecule Imaging

The key challenge in the field of fluorescent nanoparticles (NPs) for biological applications is to achieve superior brightness for sizes equivalent to single proteins (3-7 nm). We propose a concept of shell-cross-linked fluorescent micelles, in which PEGylated cyanine 3 and 5 bis-azides form a covalently attached corona on micelles of amphiphilic calixarene bearing four alkyne groups. The fluorescence quantum yield of the obtained monodisperse NPs, with a size of 7 nm, is a function of viscosity and reached up to 15 % in glycerol. In the on-state they are circa 2-fold brighter than quantum dots (QD-585), which makes them the smallest PEGylated organic NPs of this high brightness. FRET between cyanine 3 and 5 cross-linkers at the surface of NPs suggests their integrity in physiological media, organic solvents, and living cells, in which the NPs rapidly internalize, showing excellent imaging contrast. Calixarene micelles with a cyanine corona constitute a new platform for the development of protein-sized ultrabright fluorescent NPs.

Topics: Azides; Calixarenes; Carbocyanines; Click Chemistry; Cross-Linking Reagents; Fluorescent Dyes; HeLa Cells; Humans; Micelles; Nanoparticles; Optical Imaging; Particle Size; Polyethylene Glycols; Proteins

During type I-E CRISPR-Cas immunity, the Cascade surveillance complex utilizes CRISPR-derived RNAs to target complementary invasive DNA for destruction. When invader mutation blocks this interference activity, Cascade instead triggers rapid primed adaptation against the invader. The molecular basis for this dual Cascade activity is poorly understood. Here we show that the conformation of the Cse1 subunit controls Cascade activity. Using FRET, we find that Cse1 exists in a dynamic equilibrium between "open" and "closed" conformations, and the extent to which the open conformation is favored directly correlates with the attenuation of interference and relative increase in priming activity upon target mutation. Additionally, the Cse1 L1 motif modulates Cascade activity by stabilizing the closed conformation. L1 mutations promote the open conformation and switch immune response from interference to priming. Our results demonstrate that Cascade conformation controls the functional outcome of target recognition, enabling tunable CRISPR immune response to combat invader evolution.

Topics: Binding Sites; Carbocyanines; CRISPR-Associated Proteins; CRISPR-Cas Systems; DNA Helicases; Escherichia coli K12; Escherichia coli Proteins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Gene Expression Regulation, Bacterial; Mutation; Plasmids; Protein Binding; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Staining and Labeling

Objective To develop a simple, effective, time-saving and low-cost fluorescence protein microarray method for detecting serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC). Method Non-contact piezoelectric print techniques were applied to fluorescence protein microarray to reduce the cost of prey antibody. Serum samples from patients with HCC and healthy control subjects were collected and evaluated for the presence of AFP using a novel fluorescence protein microarray. To validate the fluorescence protein microarray, serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA). Results A total of 110 serum samples from patients with HCC ( n = 65) and healthy control subjects ( n = 45) were analysed. When the AFP cut-off value was set at 20 ng/ml, the fluorescence protein microarray had a sensitivity of 91.67% and a specificity of 93.24% for detecting serum AFP. Serum AFP quantified via fluorescence protein microarray had a similar diagnostic performance compared with ELISA in distinguishing patients with HCC from healthy control subjects (area under receiver operating characteristic curve: 0.906 for fluorescence protein microarray; 0.880 for ELISA). Conclusion A fluorescence protein microarray method was developed for detecting serum AFP in patients with HCC.

Topics: Aged; alpha-Fetoproteins; Antibodies; Area Under Curve; Biomarkers, Tumor; Carbocyanines; Carcinoma, Hepatocellular; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Female; Fluorescence; Fluorescent Dyes; Gene Expression; Humans; Liver Neoplasms; Male; Middle Aged; Protein Array Analysis; ROC Curve; Streptavidin

A wide variety of organic dyes form, under certain conditions, clusters know as J- and H-aggregates. Cyanine dyes are such a class of molecules where the spatial proximity of several dyes leads to overlapping electron orbitals and thus to the creation of a new energy landscape compared to that of the individual units. In this work, we create artificial H-aggregates of exactly two Cyanine 3 (Cy3) dyes by covalently linking them to a DNA molecule with controlled subnanometer distances. The absorption spectra of these coupled systems exhibit a blue-shifted peak, whose intensity varies depending on the distance between the dyes and the rigidity of the DNA template. Simulated vibrational resolved spectra, based on molecular orbital theory, excellently reproduce the experimentally observed features. Circular dichroism spectroscopy additionally reveals distinct signals, which indicates a chiral arrangement of the dye molecules. Molecular dynamic simulations of a Cy3-Cy3 construct including a 14-base pair DNA sequence verified chiral stacking of the dye molecules.

Topics: Carbocyanines; Circular Dichroism; DNA; Molecular Dynamics Simulation

Fluorescence correlation spectroscopy (FCS) allows determining diffusion and relaxation properties of fluorescent molecules. It requires only extremely small amounts of sample, down to picomolar concentrations, in an effective analysis volume of a few femtoliters. In essence, FCS determines the autocorrelation of fluorescence fluctuations caused by single labeled molecules passing through the confocal volume of a microscope equipped with a suitable detector; it permits investigating interactions of (macro)molecules, even in single cells. We present an FCS protocol for exploring, under in vitro conditions, the dynamic processes that take place during the early steps of virus infection. We cover two important issues of rhinovirus research, the kinetics of directional RNA release, and virus-receptor interactions exemplified by using human rhinovirus type A2 (HRV-A2) as a model.

Topics: Capsid; Carbocyanines; Fluorescent Dyes; Host-Pathogen Interactions; Humans; Receptors, Virus; Rhinovirus; RNA, Viral; Spectrometry, Fluorescence

The generation of induced pluripotent stem (iPS) cells has successfully been achieved in many species. However, the identification of truly reprogrammed iPS cells still remains laborious and the detection of pluripotency markers requires fixation of cells in most cases. Here, we report an approach with nanoparticles carrying Cy3-labeled sense oligonucleotide reporter strands coupled to gold-particles. These molecules are directly added to cultured cells without any manipulation and gene expression is evaluated microscopically after overnight incubation. To simultaneously detect gene expression in different species, probe sequences were chosen according to interspecies homology. With a common target-specific probe we could successfully demonstrate expression of the GAPDH house-keeping gene in somatic cells and expression of the pluripotency markers NANOG and GDF3 in embryonic stem cells and iPS cells of murine, human, and porcine origin. The population of target gene positive cells could be purified by fluorescence-activated cell sorting. After lentiviral transduction of murine tail-tip fibroblasts Nanog-specific probes identified truly reprogrammed murine iPS cells in situ during development based on their Cy3-fluorescence. The intensity of Nanog-specific fluorescence correlated positively with an increased capacity of individual clones to differentiate into cells of all three germ layers. Our approach offers a universal tool to detect intracellular gene expression directly in live cells of any desired origin without the need for manipulation, thus allowing conservation of the genetic background of the target cell. Furthermore, it represents an easy, scalable method for efficient screening of pluripotency which is highly desirable during high-throughput cell reprogramming and after genomic editing of pluripotent stem cells.

Topics: Animals; Carbocyanines; Cellular Reprogramming; Embryonic Stem Cells; Gene Expression Regulation; Humans; Induced Pluripotent Stem Cells; Mice; Mice, Transgenic; Oligonucleotides; Swine

We present an all-thermoplastic integrated sample-to-answer centrifugal microfluidic Lab-on-Disc system (LoD) for nucleic acid analysis. The proposed CD system and engineered platform were employed for analysis of Bacillus atrophaeus subsp. globigii spores. The complete assay comprised cellular lysis, polymerase chain reaction (PCR) amplification, amplicon digestion, and microarray hybridization on a plastic support. The fluidic robustness and operating efficiency of the assay were ensured through analytical optimization of microfluidic tools enabling beneficial implementation of capillary valves and accurate control of all flow timing procedures. The assay reliability was further improved through the development of two novel microfluidic strategies for reagents mixing and flow delay on the CD platform. In order to bridge the gap between the proof-of-concept LoD and production prototype demonstration, low-cost thermoplastic elastomer (TPE) was selected as the material for CD fabrication and assembly, allowing the use of both, high quality hot-embossing and injection molding processes. Additionally, the low-temperature and pressure-free assembly and bonding properties of TPE material offer a pertinent solution for simple and efficient loading and storage of reagents and other on-board components. This feature was demonstrated through integration and conditioning of microbeads, magnetic discs, dried DNA buffer reagents and spotted DNA array inserts. Furthermore, all microfluidic functions and plastic parts were designed according to the current injection mold-making knowledge for industrialization purposes. Therefore, the current work highlights a seamless strategy that promotes a feasible path for the transfer from prototype toward realistic industrialization. This work aims to establish the full potential for TPE-based centrifugal system as a mainstream microfluidic diagnostic platform for clinical diagnosis, water and food safety, and other molecular diagnostic applications.

Topics: Bacillus subtilis; Carbocyanines; DNA, Bacterial; Elastomers; Microfluidic Analytical Techniques; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction

The search for viral entry inhibitors that selectively target viral envelope glycoproteins has attracted increasing interest in recent years. Amongst the handful of molecules reported to show activity as hepatitis C virus (HCV) entry inhibitors are a variety of glycan-binding proteins including the lectins, cyanovirin-N (CV-N) and griffithsin. We recently demonstrated that boronic acid-modified nanoparticles are able to reduce HCV entry through a similar mechanism to that of lectins. A major obstacle to any further development of these nanostructures as viral entry inhibitors is their only moderate maximal inhibition potential. In the present study, we report that lipid nanocapsules (LNCs), surface-functionalized with amphiphilic boronic acid (BA) through their post-insertion into the semi-rigid shell of the LNCs, are indeed far superior as HCV entry inhibitors when compared with previously reported nanostructures. These 2(nd) generation particles (BA-LNCs) are shown to prevent HCV infection in the micromolar range (IC50 = 5.4 μM of BA moieties), whereas the corresponding BA monomers show no significant effects even at the highest analyzed concentration (20 μM). The new BA-LNCs are the most promising boronolectin-based HCV entry inhibitors reported to date and are thus observed to show great promise in the development of a pseudolectin-based therapeutic agent.

Topics: Antibodies; Boronic Acids; Carbocyanines; Cell Line; Cell Survival; Hepacivirus; Humans; Microscopy, Fluorescence; Nanocapsules; Particle Size; Polyethylene Glycols; Triglycerides; Viral Envelope Proteins; Virus Internalization

The G protein-coupled receptors (GPCRs) form one of the largest membrane receptor families. The nature of the ligands that interact with these receptors is highly diverse; they include light, peptides and hormones, neurotransmitters, and small molecular weight compounds. The GPCRs are involved in a wide variety of physiological processes and thus hold considerable therapeutic potential.GPCR function is usually determined in cell-based assays, whose complexity nonetheless limits their use. The use of alternative, cell-free assays is hampered by the difficulties in purifying these seven-transmembrane domain receptors without altering their functional properties. Several methods have been proposed to immobilize GPCR on biosensor surfaces which use antibodies or avidin-/biotin-based capture procedures, alone or with reconstitution of the GPCR physiological microenvironment. Here we propose a method for GPCR immobilization in their native membrane microenvironment that requires no manipulation of the target receptor and maintains the many conformations GPCR can adopt in the cell membrane.

Topics: Carbocyanines; Fluorescent Dyes; Gene Expression; Genetic Vectors; Green Fluorescent Proteins; HEK293 Cells; Humans; Immobilized Proteins; Lentivirus; Plasmids; Receptors, CXCR4; Recombinant Proteins; Surface Plasmon Resonance; Transfection; Vesiculovirus; Virion; Virus Assembly

The photostability of reporter fluorophores in single-molecule fluorescence imaging is of paramount importance, as it dictates the amount of relevant information that may be acquired before photobleaching occurs. Quenchers of triplet excited states are thus required to minimize blinking and sensitization of singlet oxygen. Through a combination of single-molecule studies and ensemble mechanistic studies including laser flash photolysis and time-resolved fluorescence, we demonstrate herein that Ni(2+) provides a much desired physical route (chemically inert) to quench the triplet excited state of Cy3, the most ubiquitous green emissive dye utilized in single-molecule studies.

Topics: Carbocyanines; Fluorescence; Molecular Imaging; Molecular Structure; Nickel; Photochemical Processes; Singlet Oxygen

CoFe nanowires have been synthesized by the electrodeposition technique into the pores of a polycarbonate membrane with a nominal pore diameter of 50 nm, and the composition of CoFe nanowires varying by changing the source concentration of iron. The synthesized nanowire surfaces were functionalized with amine groups by treatment with aminopropyltriethoxysilane (APTES) linker, and then conjugated with streptavidin-Cy3 protein via ethyl (dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide coupling chemistry. The oxide surface of CoFe nanowire is easily modified with aminopropyltriethoxysilane to form an amine terminating group, which is covalently bonded to streptavidin-Cy3 protein. The physicochemical properties of the nanowires were analyzed through different characterization techniques such as scanning electron microscope, energy dispersive spectroscopy, and vibrating sample magnetometer. Fluorescence microscopic studies and Fourier transform infrared studies confirmed the immobilization of protein on the nanowire surface. In addition, the transmission electron microscope analysis reveals the thin protein layer which is around 12-15 nm on the nanowire surfaces.

Topics: Carbocyanines; Cobalt; Electroplating; Ferrous Compounds; Immobilized Proteins; Nanowires; Streptavidin

Commercial platforms consisting of ready-to-use microarrays printed with target-specific DNA probes, a microarray scanner, and software for data analysis are available for different applications in medical diagnostics and food analysis, detecting, e.g., viral and bacteriological DNA sequences. The transfer of these tools from basic research to routine analysis, their broad acceptance in regulated areas, and their use in medical practice requires suitable calibration tools for regular control of instrument performance in addition to internal assay controls. Here, we present the development of a novel assay-adapted calibration slide for a commercialized DNA-based assay platform, consisting of precisely arranged fluorescent areas of various intensities obtained by incorporating different concentrations of a "green" dye and a "red" dye in a polymer matrix. These dyes present "Cy3" and "Cy5" analogues with improved photostability, chosen based upon their spectroscopic properties closely matching those of common labels for the green and red channel of microarray scanners. This simple tool allows to efficiently and regularly assess and control the performance of the microarray scanner provided with the biochip platform and to compare different scanners. It will be eventually used as fluorescence intensity scale for referencing of assays results and to enhance the overall comparability of diagnostic tests.

Topics: Calibration; Carbocyanines; Equipment Design; Fluorescent Dyes; Food Analysis; Food Contamination; Food Safety; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; Spectrometry, Fluorescence

Cyanine dyes are important chemical modifications of oligonucleotides exhibiting intensive and stable fluorescence at visible light wavelengths. When Cy3 or Cy5 dye is attached to 5' end of a DNA duplex, the dye stacks on the terminal base pair and stabilizes the duplex. Using optical melting experiments, we have determined thermodynamic parameters that can predict the effects of the dyes on duplex stability quantitatively (ΔG°, Tm). Both Cy dyes enhance duplex formation by 1.2 kcal/mol on average, however, this Gibbs energy contribution is sequence-dependent. If the Cy5 is attached to a pyrimidine nucleotide of pyrimidine-purine base pair, the stabilization is larger compared to the attachment to a purine nucleotide. This is likely due to increased stacking interactions of the dye to the purine of the complementary strand. Dangling (unpaired) nucleotides at duplex terminus are also known to enhance duplex stability. Stabilization originated from the Cy dyes is significantly larger than the stabilization due to the presence of dangling nucleotides. If both the dangling base and Cy3 are present, their thermodynamic contributions are approximately additive. New thermodynamic parameters improve predictions of duplex folding, which will help design oligonucleotide sequences for biophysical, biological, engineering, and nanotechnology applications.

Topics: Carbocyanines; DNA; Models, Chemical; Oligonucleotides; Thermodynamics; Ultraviolet Rays

Excessive or misplaced production of ClO(-) in living systems is usually associated with many human diseases. Therefore, it is of great importance to develop an effective and sensitive method to detect ClO(-) in living systems. Herein, we designed an 808 nm excited upconversion luminescence nanosystem, composed of the Nd(3+)-sensitized core-shell upconversion nanophosphor NaYF4:30%Yb,1%Nd,0.5%Er@NaYF4:20%Nd, which serves as an energy donor, and the ClO(-)-responsive cyanine dye hCy3, which acts as an energy acceptor, for ratiometric upconversion luminescence (UCL) monitoring of ClO(-). The detection limit of ClO(-) for this nanoprobe in aqueous solution is 27 ppb and the nanoprobe was successfully used to detect the ClO(-) in the living cells by ratiometric upconversion luminescence. Importantly, the nanoprobe realized the detection of ClO(-) in a mouse model of arthritis, which produced an excess of ROS, under 808 nm irradiation in vivo. The excitation laser efficiently reduced the heating effect, compared to the commonly used 980 nm laser for upconversion systems.

Topics: Animals; Arthritis; Carbocyanines; Cell Survival; HeLa Cells; Humans; Hypochlorous Acid; Luminescent Measurements; Metal Nanoparticles; Mice; Molecular Imaging; Neodymium

We demonstrated the surface functionalization of a highly three-dimensional, superhydrophilic wicking substrate using light to immobilize functional biomolecules for sensor or microarray applications. We showed here that the three-dimensional substrate was compatible with photo-attachment and the performance of functionalization was greatly improved due to both increased surface capacity and reduced substrate reflectivity. In addition, photo-attachment circumvents the problems induced by wicking effect that was typically encountered on superhydrophilic three-dimensional substrates, thus reducing the difficulty of producing miniaturized sites on such substrate. We have investigated various aspects of photo-attachment process on the nanowire substrate, including the role of different buffers, the effect of wavelength as well as how changing probe structure may affect the functionalization process. We demonstrated that substrate fabrication and functionalization can be achieved with processes compatible with microelectronics processes, hence reducing the cost of array fabrication. Such functionalization method coupled with the high capacity surface makes the substrate an ideal candidate for sensor or microarray for sensitive detection of target analytes.

Topics: Carbocyanines; DNA Probes; Immobilized Nucleic Acids; Microarray Analysis; MicroRNAs; Miniaturization; Nanowires; Nucleic Acid Hybridization; Nucleic Acids; Silicon; Surface Properties; Ultraviolet Rays

We report that Cy3 undergoes triple helix conformation-specific blinking in DNA. Blinking patterns were affected by the stabilization of the Hoogsteen base-pair, suggesting that not only the presence but also the fluctuating behaviour of the triple helix can be monitored by the changes in the Cy3 blinking patterns.

Topics: Base Pairing; Base Sequence; Carbocyanines; DNA; Fluorescent Dyes; Nucleic Acid Conformation

HSV is a large double stranded DNA virus, capable of causing a variety of diseases from the common cold sore to devastating encephalitis. Although DNA within the HSV virion does not contain any histone protein, within 1 h of infecting a cell and entering its nucleus the viral genome acquires some histone protein (nucleosomes). During lytic infection, partial micrococcal nuclease (MNase) digestion does not give the classic ladder band pattern, seen on digestion of cell DNA or latent viral DNA. However, complete digestion does give a mono-nucleosome band, strongly suggesting that there are some nucleosomes present on the viral genome during the lytic infection, but that they are not evenly positioned, with a 200 bp repeat pattern, like cell DNA. Where then are the nucleosomes positioned? Here we perform HSV-1 genome wide nucleosome mapping, at a time when viral replication is in full swing (6 hr PI), using a microarray consisting of 50mer oligonucleotides, covering the whole viral genome (152 kb). Arrays were probed with MNase-protected fragments of DNA from infected cells. Cells were not treated with crosslinking agents, thus we are only mapping tightly bound nucleosomes. The data show that nucleosome deposition is not random. The distribution of signal on the arrays suggest that nucleosomes are located at preferred positions on the genome, and that there are some positions that are not occupied (nucleosome free regions -NFR or Nucleosome depleted regions -NDR), or occupied at frequency below our limit of detection in the population of genomes. Occupancy of only a fraction of the possible sites may explain the lack of a typical MNase partial digestion band ladder pattern for HSV DNA during lytic infection. On average, DNA encoding Immediate Early (IE), Early (E) and Late (L) genes appear to have a similar density of nucleosomes.

Topics: Carbocyanines; Cell Line, Tumor; Cell Nucleus; Cluster Analysis; Comparative Genomic Hybridization; DNA Probes; DNA, Viral; Genes, Immediate-Early; Genome, Viral; Herpesvirus 1, Human; Humans; Micrococcal Nuclease; Nucleosomes; Virus Replication

We experimentally demonstrate a label-free biosensor for the ERBB2 cancer gene DNA target based on the distance-dependent detection of surface-enhanced fluorescence (SEF) on nanoporous gold disk (NPGD) plasmonic nanoparticles. We achieve detection of 2.4 zeptomole of DNA target on the NPGD substrate with an upper concentration detection limit of 1 nM. Without the use of molecular spacers, the NPGD substrate as an SEF platform was shown to provide higher net fluorescence for visible and NIR fluorophores compared to glass and non-porous gold substrates. The enhanced fluorescence signals in patterned nanoporous gold nanoparticles make NPGD a viable material for further reducing detection limits for biomolecular targets used in clinical assays. With patterned nanoporous gold disk (NPGD) plasmonic nanoparticles, a label-free biosensor that makes use of distance-dependent detection of surface-enhanced fluorescence (SEF) is constructed and tested for zeptomole detection of ERBB2 cancer gene DNA targets.

Topics: Base Sequence; Biomarkers, Tumor; Carbocyanines; DNA, Single-Stranded; Gold; Limit of Detection; Metal Nanoparticles; Nucleic Acid Hybridization; Spectrometry, Fluorescence; Sulfur; Surface Properties

We herein describe a novel in-stem molecular beacon (ISMB) containing multiple Cy3-quencher pairs on d-threoninol scaffolds in the stem region. The designed Cy3 derivative was not significantly quenched by the adjacent nucleobases, self-quenching of the fluorophore was minimal, and the fluorophore did not severely destabilize the duplex. Using newly designed Cy3, we synthesized ISMBs containing two Cy3 moieties. The signal to background ratio of the ISMB containing two Cy3 moieties was above 100, whereas that with one Cy3 was 30. A Cy3-derivative containing ISMB used in a fluorescence in situ hybridization (FISH) detected endogenous β-actin mRNA in fixed cells without need for washing procedures.

Topics: Actins; Amino Alcohols; Base Sequence; Butylene Glycols; Carbocyanines; Fluorescent Dyes; HeLa Cells; Humans; In Situ Hybridization, Fluorescence; Oligonucleotide Probes; RNA, Messenger

When modeling single-molecule fluorescence lifetime experimental data, the analysis often involves fitting a biexponential distribution to binned data. When dealing with small sample sizes, there is the potential for convergence failure in numerical optimization, for convergence to local optima resulting in physically unreasonable parameter estimates, and also for overfitting the data.. To avoid the problems that arise in small sample sizes, we have developed a gamma conversion method to estimate the lifetime components. The key idea is to use a gamma distribution for initial numerical optimization and then convert the gamma parameters to biexponential ones via moment matching. A simulation study is undertaken with 30 unique configurations of parameter values. We also performed the same analysis on data obtained from a fluorescence lifetime experiment using the fluorophore Cy3. In both the simulation study and the real data analysis, fitting the biexponential directly led to a large number of data sets whose estimates were physically unreasonable, while using the gamma conversion yielded estimates consistently close to the true values.. Our analysis shows that using numerical optimization methods to fit the biexponential distribution directly can lead to failure to converge, convergence to physically unreasonable parameter estimates, and overfitting the data. The proposed gamma conversion method avoids these numerical difficulties, yielding better results.

Topics: Carbocyanines; Computer Simulation; DNA; Fluorescence; Fluorescent Dyes; Humans; Models, Statistical; Photons; Sample Size; Spectrometry, Fluorescence

RNA interference holds tremendous potential as one of the most powerful therapeutic strategies. However, the properties of short interfering RNA (siRNA), such as hydrophilicity, negative charge, and instability in serum have limited its applications; therefore, significant efforts have been undertaken to improve its cellular uptake. Cell penetrating peptides have been utilized to deliver various biologically active molecules, such as proteins, liposomes, nanoparticles, peptide nucleic acids, and recently small interfering RNAs. Here, we introduce a new cell penetrating peptide GL1(Ac-GLWRAWLWKAFLASNWRRLLRLLR-NH2) to improve the intracellular uptake of siRNA. This peptide consists of four tryptophan residues that facilitated its binding with the cell membrane, five arginine residues and one lysine residue which are positively charged at physiological pH, which induced the formation of peptide-siRNA complexes and enhanced the affinity of the peptide and cell membrane. Moreover, GL1 adopted helical secondary structure due to the altered distribution of polar and nonpolar residues in the sequence. In this study, we investigated the effect of peptide/siRNA molar ratio on the particle size, surface charge, secondary structure, and uptake efficiency. The results showed that GL1 formed stable complexes with siRNA mainly through electrostatic interaction and hydrophobic interaction, and the complexes displayed a spherical shape with the size of ~100 nm and positive surface charge. Utilizing the techniques of fluorescence microscopy and flow cytometry, the intracellular localization of Cy3-labeled GAPDH siRNA was visualized and the cellular uptake was quantified. It is worth noting that in the serum free environment, compared to Lipofectamine 2000, GL1 achieved higher cellular uptake of siRNA (~95%); in the presence of serum, GL1 retained the same level of siRNA cellular uptake (~84%) as Lipofectamine 2000. In addition, the viability of cells treated by GL1 in all studied molar ratios was >85%, which was significantly higher than that treated by Lipofectamine 2000 (~70%). Taken together, the peptide GL1 demonstrated promise as a siRNA delivery system.

Topics: Amino Acid Sequence; Animals; Carbocyanines; Cell Survival; Cell-Penetrating Peptides; CHO Cells; Cricetulus; Gene Silencing; Genetic Vectors; Hydrophobic and Hydrophilic Interactions; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Molecular Sequence Data; Particle Size; Protein Structure, Secondary; RNA, Small Interfering

Spliceosomes are multimegadalton RNA-protein complexes responsible for the faithful removal of noncoding segments (introns) from pre-messenger RNAs (pre-mRNAs), a process critical for the maturation of eukaryotic mRNAs for subsequent translation by the ribosome. Both the spliceosome and ribosome, as well as many other RNA and DNA processing machineries, contain central RNA components that endow biomolecular complexes with precise, sequence-specific nucleic acid recognition, and versatile structural dynamics. Single-molecule fluorescence (or Förster) resonance energy transfer (smFRET) microscopy is a powerful tool for the study of local and global conformational changes of both simple and complex biomolecular systems involving RNA. The integration of biochemical tools such as immunoprecipitation with advanced methods in smFRET microscopy and data analysis has opened up entirely new avenues toward studying the mechanisms of biomolecular machines isolated directly from complex biological specimens, such as cell extracts. Here, we detail the general steps for using prism-based total internal reflection fluorescence microscopy in exemplary single-molecule pull-down FRET studies of the yeast spliceosome and discuss the broad application potential of this technique.

Topics: Carbocyanines; Exons; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Immunoprecipitation; Introns; Microscopy, Fluorescence; Nucleic Acid Conformation; Ribosomes; RNA Precursors; RNA Splicing; RNA, Fungal; Saccharomyces cerevisiae; Spliceosomes; Staining and Labeling

Serological proteome analysis (SERPA) combines classical proteomic technology with effective separation of cellular protein extracts on two-dimensional gel electrophoresis, western blotting, and identification of the antigenic spot of interest by mass spectrometry. A critical point is related to the antigenic target characterization by mass spectrometry, which depends on the accuracy of the matching of antigenic reactivities on the protein spots during the 2D immunoproteomic procedures. The superimposition, based essentially on visual criteria of antigenic and protein spots, remains the major limitation of SERPA. The introduction of fluorescent dyes in proteomic strategies, commonly known as 2D-DIGE (differential in-gel electrophoresis), has boosted the qualitative capabilities of 2D electrophoresis. Based on this 2D-DIGE strategy, we have improved the conventional SERPA by developing a new and entirely fluorescence-based bi-dimensional immunoproteomic (FBIP) analysis, performed with three fluorescent dyes. To optimize the alignment of the different antigenic maps, we introduced a landmark map composed of a combination of specific antibodies. This methodological development allows simultaneous revelation of the antigenic, landmark and proteomic maps on each immunoblot. A computer-assisted process using commercially available software automatically leads to the superimposition of the different maps, ensuring accurate localization of antigenic spots of interest.

Topics: Animals; Antibodies, Monoclonal; Autoantibodies; Autoantigens; Blotting, Western; Carbocyanines; Fluorescent Dyes; Hep G2 Cells; Humans; Image Processing, Computer-Assisted; Immunoglobulin G; Isoelectric Focusing; Luminescent Measurements; Lupus Erythematosus, Systemic; Mice; Molecular Weight; Proteomics; Two-Dimensional Difference Gel Electrophoresis

The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis have been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with ECL Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27 kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract.

Topics: Animals; Antibodies, Monoclonal; Blotting, Western; Carbocyanines; Electrophoresis, Gel, Two-Dimensional; Fluorescent Dyes; HSP27 Heat-Shock Proteins; Humans; Immunoblotting; Immunoconjugates; Isoelectric Focusing; Proteins; Proteomics; Workflow

Fluorescence-based single-vesicle fusion assays provide a powerful method for studying mechanisms underlying complex biological processes of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated vesicle fusion and neurotransmitter release. A crucial element of these assays is the ability of the fluorescent probe(s) to reliably detect key intermediate events of fusion pore opening and content release/mixing. Here, we report a new, reliable, and efficient single-vesicle content-mixing assay using a high affinity, fluorophore tagged host-guest pair, cucurbit[7]uril-Cy3 and adamantane-Cy5 as a fluorescence resonance energy transfer (FRET) pair. The power of these probes is demonstrated by the first successful observation of flickering dynamics of the fusion pore by in vitro assay using neuronal SNARE-reconstituted vesicles.

Topics: Adamantane; Animals; Bridged-Ring Compounds; Carbocyanines; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Imidazoles; Membrane Fusion; Neurons; SNARE Proteins

γ-Tilmanocept ((99m)Tc-labeled-tilmanocept or [(99m)Tc]-tilmanocept) is the first mannose-containing, receptor-directed, radiolabeled tracer for the highly sensitive imaging of sentinel lymph nodes in solid tumor staging. To elucidate the mannose-binding receptor that retains tilmanocept in this microenvironment, human macrophages were used that have high expression of the C-type lectin mannose receptor (MR; CD206). Cy3-labeled tilmanocept exhibited high specificity binding to macrophages that was nearly abolished in competitive inhibition experiments. Furthermore, Cy3-tilmanocept binding was markedly reduced on macrophages deficient in the MR by small interfering RNA treatment and was increased on MR-transfected HEK 293 cells. Finally, confocal microscopy revealed colocalization of Cy3-tilmanocept with the macrophage membrane MR and binding of labeled tilmanocept to MR(+) cells (macrophages and/or dendritic cells) in human sentinel lymph node tissues. Together these data provide strong evidence that CD206 is a major binding receptor for γ-tilmanocept. Identification of CD206 as the γ-tilmanocept-binding receptor enables opportunities for designing receptor-targeted advanced imaging agents and therapeutics for cancer and other diseases.

Topics: Carbocyanines; Cells, Cultured; Dextrans; Flow Cytometry; HEK293 Cells; Humans; Immunohistochemistry; Lectins, C-Type; Lymph Nodes; Macrophages; Mannans; Mannose Receptor; Mannose-Binding Lectins; Microscopy, Confocal; Molecular Structure; Neoplasms; Protein Binding; Radiopharmaceuticals; Receptors, Cell Surface; RNA Interference; Sentinel Lymph Node Biopsy; Technetium Tc 99m Pentetate

Paper is a promising platform for the development of decentralized diagnostic assays owing to the low cost and ease of use of paper-based analytical devices (PADs). It can be challenging to detect on PADs very low concentrations of nucleic acid biomarkers of lengths as used in clinical assays. Herein we report the use of thermophilic helicase-dependent amplification (tHDA) in combination with a paper-based platform for fluorescence detection of probe-target hybridization. Paper substrates were patterned using wax printing. The cellulosic fibers were chemically derivatized with imidazole groups for the assembly of the transduction interface that consisted of immobilized quantum dot (QD)-probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as the acceptor dye in a fluorescence resonance energy transfer (FRET)-based transduction method. After probe-target hybridization, a further hybridization event with a reporter sequence brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs, triggering a FRET sensitized emission that served as an analytical signal. Ratiometric detection was evaluated using both an epifluorescence microscope and a low-cost iPad camera as detectors. Addition of the tHDA method for target amplification to produce sequences of ∼100 base length allowed for the detection of zmol quantities of nucleic acid targets using the two detection platforms. The ratiometric QD-FRET transduction method not only offered improved assay precision, but also lowered the limit of detection of the assay when compared with the non-ratiometric QD-FRET transduction method. The selectivity of the hybridization assays was demonstrated by the detection of single nucleotide polymorphism.

Topics: Biomarkers; Carbocyanines; Fluorescence Resonance Energy Transfer; Humans; Nucleic Acid Hybridization; Oligonucleotides; Paper; Polymorphism, Single Nucleotide; Quantum Dots

Protein-induced fluorescence enhancement (PIFE) is a term used to describe the increase in fluorescence intensity observed when a protein binds to a nucleic acid in the proximity of a fluorescent probe. PIFE using the single-molecule dye Cy3 is gaining popularity as an approach to investigate the dynamics of proteins that interact with nucleic acids. In this work, we used complexes of DNA and Klenow fragment and a combination of time-resolved fluorescence and transient spectroscopy techniques to elucidate the photophysical mechanism that leads to protein-enhanced fluorescence emission of Cy3 when in close proximity to a protein (PIFE). By monitoring the formation of the cis isomer directly, we proved that the enhancement of Cy3 fluorescence correlates with a decrease in the efficiency of photoisomerization, and occurs in conditions where the dye is sterically constrained by the protein.

Topics: Carbocyanines; DNA; DNA Polymerase I; Fluorescent Dyes; Isomerism; Light; Proteins; Spectrometry, Fluorescence

Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.

Topics: Animals; Calmodulin; Carbocyanines; Cattle; Fluorescence Resonance Energy Transfer; Nitric Oxide Synthase Type I; Protein Structure, Secondary; Protein Structure, Tertiary; Spectrophotometry, Ultraviolet; Time Factors

To develop a biodegradable polymeric drug delivery system for the treatment of ovarian cancer with the capacity for non-invasive fate monitoring, we designed and synthesized N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-epirubicin (EPI) conjugates. The polymer backbone was labeled with acceptor fluorophore Cy5, while donor fluorophores (Cy3 or EPI) were attached to HPMA copolymer side chains via an enzyme-cleavable GFLG linker. This design allows elucidating separately the fate of the drug and of the polymer backbone using fluorescence resonance energy transfer (FRET). The degradable diblock conjugate (2P-EPI) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization using a bifunctional chain transfer agent (Peptide2CTA). The pharmacokinetics (PK) and therapeutic effect of 2P-EPI (Mw ~100 kDa) were determined in mice bearing human ovarian carcinoma A2780 xenografts. Compared to 1st generation conjugate (P-EPI, Mw <50 kDa), 2P-EPI demonstrated remarkably improved PK such as fourfold terminal half-life (33.22 ± 3.18 h for 2P-EPI vs. 7.55 ± 3.18 h for P-EPI), which is primarily attributed to the increased molecular weight of the polymer carrier. Notably, complete tumor remission and long-term inhibition of tumorigenesis (100 days) were achieved in mice (n=5) treated with 2P-EPI. Moreover, in vitro cell uptake and intracellular drug release were determined via FRET intensity changes. The results establish a solid foundation for future in vivo tracking of drug delivery and chain scission of polymeric conjugates by FRET imaging.

Topics: Animals; Antibiotics, Antineoplastic; Carbocyanines; Cell Line, Tumor; Cell Survival; Epirubicin; Female; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Methacrylates; Mice; Mice, Nude; Ovarian Neoplasms; Tumor Burden

Topics: Biochemistry; Carbocyanines; Catalase; Deoxyribonucleases; Free Radical Scavengers; Oxygen; Photobleaching; Protocatechuate-3,4-Dioxygenase

Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ(70)-dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator-DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)--a Cu(+)-responsive MerR-family metalloregulator--modulates RNAP interactions with CueR's cognate suboptimal promoter PcopA, and how RNAP affects CueR-PcopA interactions. We find that RNAP can form two noninterconverting complexes at PcopA in the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a "biased sampling" instead of "dynamic equilibrium shifting" mechanism in regulating transcription initiation; it modulates RNAP's binding-unbinding kinetics, without allowing interconversions between the dead-end and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopA into its specific binding mode, likely helping amplify the differences between apo- and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription.

Topics: Adenosine Triphosphatases; Algorithms; Base Sequence; Carbocyanines; Cation Transport Proteins; Copper-Transporting ATPases; DNA-Directed RNA Polymerases; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Fluorescence Resonance Energy Transfer; Kinetics; Models, Genetic; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; Sigma Factor; Trans-Activators; Transcription, Genetic

Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β2-adrenergic receptor (β2AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs.

Topics: Binding Sites; Carbocyanines; Humans; Molecular Dynamics Simulation; Propanolamines; Receptors, Adrenergic, beta-2

Far-field optical nanoscopy has been widely used to image small objects with sub-diffraction-limit spatial resolution. Particularly, reversible saturable optical fluorescence transition (RESOLFT) nanoscopy with photoswitchable fluorescent proteins is a powerful method for super-resolution imaging of living cells with low light intensity. Here we demonstrate for the first time the implementation of RESOLFT nanoscopy for a biological system using organic fluorophores, which are smaller in size and easier to be chemically modified. With a covalently-linked dye pair of Cy3 and Alexa647 to label subcellular structures in fixed cells and by optimizing the imaging buffer and optical parameters, our RESOLFT nanoscopy achieved a spatial resolution of ~74 nm in the focal plane. This method provides a powerful alternative for low light intensity RESOLFT nanoscopy, which enables biological imaging with small organic probes at nanoscale resolution.

Topics: Animals; Buffers; Carbocyanines; Dimerization; Fluorescence; Fluorescent Dyes; Humans; Imaging, Three-Dimensional; Light; Male; Mice; Molecular Probes; Nanotechnology; NIH 3T3 Cells; Organic Chemicals; Subcellular Fractions

Self-assembled, biodegradable materials that embed fragile, soluble, or insoluble compounds of therapeutic interest have potential use as drug delivery systems. The bead-forming peptide Ac-X3-gT can embed hydrophobic and hydrophilic payloads. Loaded peptide beads were internalized by human acute monocytic leukemia cell line (THP-1) macrophages, THP-1 monocytes, and hepatocellular carcinoma cells (Huh7). Furthermore, paclitaxel and doxorubicin coencapsulated in the peptide beads were delivered to THP-1 monocytes, causing a decrease in cell viability due to the activity of the anticancer drugs. In addition to the bead-forming peptide Ac-X3-gT, the use of a positively charged peptide analogue increased the RNA/DNA embedding efficiency to 99% by charge compensation and micellar complexation. Internalization of the resulting gene delivery systems by Huh7 cells led to specific gene silencing either by embedded small interfering RNA or by plasmid-encoding small hairpin RNA delivered in cells. The new class of purely peptidic material caused no measurable toxicity during in vitro experiments, thereby indicating potential use as a drug delivery system for multidrug delivery and gene therapy.

Topics: Biocompatible Materials; Carbocyanines; Cell Line, Tumor; Cell Survival; Drug Delivery Systems; Endocytosis; Gene Expression Regulation, Neoplastic; Gene Silencing; Gene Transfer Techniques; Hepatocytes; Humans; Micelles; Microspheres; Nanoparticles; Peptides; RNA, Small Interfering

We reported an optical DNA/protein microfluidic sensor which consists of single stranded (ss) DNA-Cy3 probes on gold surface and simple line-shape microfluidic channel. These ssDNA-Cy3 probes with random sequence in bulk solution or on gold surface exhibits fluorescence enhancement after binding with complementary ssDNA (cssDNA) targets. Particularly it did not require complicated design or hairpin-like stem-loop conformation, which made it easier to be made and applied in analytes detection by fluorescence switching techniques. Using ssDNA-cy3 probes attached on gold surface in a microfluidic channel, strong fluorescence enhancement was measured by ssDNA with cssDNA binding or ssDNA with cssDNA-biotin binding. The following introduction of streptavidin resulted in fluorescence quenching (fluorescence decrease) because of the binding of hybridized DNA-biotin with streptavidin. This sensor showed strong affinity and high sensitivity toward the streptavidin, the minimum detectable concentration for streptavidin was 1 pM, equating to an absolute detection limit of 60 amol in this microfluidic channel. Microfluidic channel height and flow rate is optimized to increase surface reaction efficiency and fluorescence switching efficiency. In contrast to previously reported optical molecular beacon approach, this sensor can be used not only for the detection of cssDNA target, but also for the detection of streptavidin. This microfluidic sensor offers the promise of analyzing kinds of molecular targets or immunoreactions.

Topics: Biosensing Techniques; Biotin; Carbocyanines; DNA, Complementary; Fluorescence; Limit of Detection; Microfluidic Analytical Techniques; Nucleic Acid Hybridization; Streptavidin

The use of small-interfering RNA (siRNA) has great potential for the development of drugs designed to knock down the expression of damage- or disease-causing genes. However, because of the high molecular weight and negative charge of siRNA, it is restricted from crossing the blood-cochlear barrier, which limits the concentration and size of molecules that are able to gain access to cells of the inner ear. Intratympanic approaches, which deliver siRNA to the middle ear, rely on permeation through the round window for access to the structures of the inner ear. We developed an innovative siRNA delivery recombination protein, TAT double-stranded RNA-binding domains (TAT-DRBDs), which can transfect Cy3-labeled siRNA into cells of the inner ear, including the inner and outer hair cells, crista ampullaris, macula utriculi and macula sacculi, through intact round-window permeation in the chinchilla in vivo, and there were no apparent morphological damages for the time of observation. We also found that Cy3-labeled siRNA could directly enter spiral ganglion neurons and the epithelium of the stria vascularis independently; however, the mechanism is unknown. Therefore, as a non-viral vector, TAT-DRBD is a good candidate for the delivery of double-stranded siRNAs for treating various inner ear ailments and preservation of hearing function.

Topics: Animals; Carbocyanines; Chinchilla; Genetic Vectors; Recombinant Proteins; RNA, Double-Stranded; RNA, Small Interfering; Round Window, Ear; Spiral Ganglion; Stria Vascularis; Transfection

Photophysical measurements are reported for Cy3-DNA constructs in which both Cy3 nitrogen atoms are attached to the DNA backbone by short linkers. While this linking was thought to rigidify the orientation of the dye and hinder cis-isomerization, the relatively low fluorescence quantum yield and the presence of a short component in the time-resolved fluorescence decay of the dye indicated that cis-isomerization remained possible. Fluorescence correlation spectroscopy and transient absorption experiments showed that photoisomerization occurred with high efficiency. Molecular dynamics simulations of the trans dye system indicated the presence of stacked and unstacked states, and free energy simulations showed that the barriers for stacking/unstacking were low. In addition, simulations showed that the ground cis state was feasible without DNA distortions. Based on these observations, a model is put forward in which the doubly linked dye can photoisomerize in the unstacked state.

Topics: Carbocyanines; DNA; Models, Molecular; Molecular Dynamics Simulation; Molecular Structure; Photochemical Processes; Spectrometry, Fluorescence

Non human antibodies administered to human patients often generate anti-antibody responses, leading in extreme cases to anaphylactic shock. Completely human antibodies are therefore favored over their murine, chimeric and humanized counterparts. However, the accurate evaluation of human antibodies on human tissue samples cannot be achieved using indirect immunohistochemical methods because of endogenous immunoglobulins that are co-detected by the secondary antibodies. Direct detection is often used instead, but this lacks the signal amplification conferred by the secondary antibody and is therefore less sensitive. We developed a simple fluorescence-based indirect immunohistochemical method that allows human primary antibodies bound specifically to their target antigens in human tissue samples to be detected clearly and without interfering background staining. This approach involves a biotinylated human primary antibody (H10(Biotin)) and Cy3-conjugated streptavidin (Strep(Cy3)). We tested the protocol using a human carcinoembryonic antigen (CEA) specific IgG1 (H10). We identified an exposure time threshold that allowed the elimination of low Strep(Cy3) background staining, yet achieved sufficient signal amplification to make our approach four times more sensitive than comparable direct immunohistochemical procedures. The principle of this indirect immunohistochemical assay should be transferable to other species allowing the specific and sensitive detection of any primary antibody on homologous tissues.

Topics: Animals; Antibodies, Monoclonal; Biotin; Biotinylation; Carbocyanines; Carcinoembryonic Antigen; Carcinoma; CHO Cells; Colonic Neoplasms; Cricetulus; Fluorescent Antibody Technique, Indirect; HEK293 Cells; Humans; Immunoglobulin G; Mice; Sensitivity and Specificity; Staining and Labeling; Streptavidin

Visualization of cancer cells requires distinguishing malignant from normal cells by objective criteria with high specificity. For several years, tumor markers expressed on the surface of cancer cells have been characterized as cancer signatures, and their labeling with specific imaging probes has revolutionized cancer diagnosis. This specific labeling is also an important tool in surgery tumor ablation. The present study considers the tumor labeling potential of an aptamer that specifically recognizes the epithelial cancer biomarker mucin1 (MUC1). This anti-MUC1 aptamer was investigated in vitro in a three-dimensional (3D) environment and compared to an anti-MUC1 antibody for its capacity to visualize cancer cells. Multicellular spheroids of breast cancer MCF-7 cells were used as tumor models and anti-MUC1 fluorescent aptamer and antibody were visualized by fluorescence imaging. Results showed that the antibodies interacted only with cells located on the surface of the spheroid, whereas the anti-MUC1 aptamers were able to penetrate inside these 3D tumor models and thereafter internalized into the cancer cells. Due to their lack of immunogenicity and their facility to be chemically modified, aptamers may replace advantageously the use of antibodies in diagnosis based on imaging setup thanks to their specific detection of cancer cells without invasive surgical procedures or during clinical intraoperative intervention.

Topics: Antibodies, Monoclonal; Aptamers, Nucleotide; Biological Transport; Biomarkers, Tumor; Carbocyanines; Female; Fluorescent Dyes; Gene Expression; Humans; MCF-7 Cells; Microscopy, Fluorescence, Multiphoton; Molecular Imaging; Mucin-1; Spheroids, Cellular

DNA constructs labeled with cyanine fluorescent dyes are important substrates for single-molecule (sm) studies of the functional activity of protein-DNA complexes. We previously studied the local DNA backbone fluctuations of replication fork and primer-template DNA constructs labeled with Cy3/Cy5 donor-acceptor Förster resonance energy transfer (FRET) chromophore pairs and showed that, contrary to dyes linked 'externally' to the bases with flexible tethers, direct 'internal' (and rigid) insertion of the chromophores into the sugar-phosphate backbones resulted in DNA constructs that could be used to study intrinsic and protein-induced DNA backbone fluctuations by both smFRET and sm Fluorescent Linear Dichroism (smFLD). Here we show that these rigidly inserted Cy3/Cy5 chromophores also exhibit two additional useful properties, showing both high photo-stability and minimal effects on the local thermodynamic stability of the DNA constructs. The increased photo-stability of the internal labels significantly reduces the proportion of false positive smFRET conversion 'background' signals, thereby simplifying interpretations of both smFRET and smFLD experiments, while the decreased effects of the internal probes on local thermodynamic stability also make fluctuations sensed by these probes more representative of the unperturbed DNA structure. We suggest that internal probe labeling may be useful in studies of many DNA-protein interaction systems.

Topics: Carbocyanines; Circular Dichroism; DNA Probes; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Staining and Labeling; Thermodynamics

A simple strategy for the immobilization of Cy3-labeled single strand DNA (Cy3-ssDNA) on a Si(001) surface and its release under control of both light and pH stimuli is presented. In order to prepare a dual pH/light-triggered surface, positively chargeable azobenzene molecules are self-assembled on the Si(001) surface. The surface wettability of this substrate can be changed under influence of both light and pH conditions. The substrates can be positively charged under mildly acidic conditions. The pH-sensitive behavior of the film allows binding of Cy3-ssDNA on the functionalized Si(001) surface through effective electrostatic interactions with the negatively charged polynucleotide backbone. Moreover, irradiation of the film with UVA light induces trans-cis isomerization of the azobenzene units on the surface. As a result, the binding affinity for DNA decreases due to the changing surface hydrophilicity. In order to understand and control the reversible photoswitchable mechanism of this surface, water contact angles are measured after UVA and visible light irradiation. The release of DNA from a dual pH/light-sensitive sample is performed using fluorescence microscopy. The results show that irradiation of the film with UVA light induces trans-cis isomerization of the photoresponsive azobenzene units; this leads to significant changes in the surface hydrophilicity and reduces the binding affinity for DNA.

Topics: Adsorption; Carbocyanines; DNA, Single-Stranded; Hydrogen-Ion Concentration; Immobilized Nucleic Acids; Light; Optical Phenomena; Surface Properties

There is a critical need for techniques that directly monitor protein synthesis within cells isolated from normal and diseased tissue. Fibrotic disease, for which there is no drug treatment, is characterized by the overexpression of collagens. Here, we use a bioinformatics approach to identify a pair of glycine and proline isoacceptor tRNAs as being specific for the decoding of collagen mRNAs, leading to development of a FRET-based approach, dicodon monitoring of protein synthesis (DiCoMPS), that directly monitors the synthesis of collagen. DiCoMPS aimed at detecting collagen synthesis will be helpful in identifying novel anti-fibrotic compounds in cells derived from patients with fibrosis of any etiology, and, suitably adapted, should be widely applicable in monitoring the synthesis of other proteins in cells.

Topics: Animals; Carbocyanines; Cells, Cultured; Collagen; Fibroblasts; Fibrosis; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Kinetics; Mice; Mice, Knockout; Microscopy, Confocal; PTEN Phosphohydrolase; RNA, Transfer, Gly; RNA, Transfer, Pro; Transfection

Curcumin (CUR), a naturally derived anti-cancer cocktail is arguably the most widely studied neutraceutical. Despite a lot of promises, it is yet to reach the market as an active anti-cancer formulation. In the present study, we have prepared highly soluble (3 mg/ml) CUR-γ-hydroxypropyl cyclodextrin (CUR-CD) hollow spheres. CUR-CD hollow spheres were prepared by a novel and scalable spray drying method. CUR-CD was then encapsulated into positively charged biodegradable chitosan (CUR-CD-CS) nanoparticles. The CUR-CD-CS nanoparticles were characterised by TEM, SEM, DLS, drug loading and in vitro release. We tested the efficacy of these CUR-CD-CS nanoparticles in SCC25 cell lines using MTT assay and investigated its cellular uptake mechanism. We also studied Oligo DNA loading in CUR-CD-CS nanoparticles and its delivery via confocal imaging and FACS analysis. Our results demonstrated that CUR-CD-CS nanoparticles showed superior in vitro release performance and higher cytotoxicity in SCC25 cell line amongst all tested formulations. The cytotoxicity results were corroborated by cell cycle analysis and apoptosis test, showing nearly 100% apoptotic cell death in the case of CUR-CD-CS nanoparticles. Compared to CS nanoparticles, CS-CD nanoformulation showed higher cellular delivery of Cy3-Oligo DNA which was tested quantitatively using flowcytometry analysis, indicating that CD not only enhanced CUR solubility but also boosted the cellular uptake. Our study shows that rationally designed bio-degradable natural biomaterials have great potential as next generation nano-carriers for hydrophobic drug delivery such as CUR with potential of dual drug-gene delivery.

Topics: Calorimetry, Differential Scanning; Carbocyanines; Cell Cycle; Cell Death; Cell Line, Tumor; Cell Survival; Chitosan; Curcumin; gamma-Cyclodextrins; Humans; Light; Nanoconjugates; Scattering, Radiation; Solubility

Förster resonance energy transfer (FRET) technology relies on the close proximity of two compatible fluorophores for energy transfer. Tagged (Cy3 and Cy5) complementary DNA strands forming a stable duplex and a doubly-tagged single strand were shown to demonstrate FRET outside of a cellular environment. FRET was also observed after transfecting these DNA strands into fixed and live cells using methods such as microinjection and electroporation, but not when using lipid based transfection reagents, unless in the presence of the endosomal acidification inhibitor bafilomycin. Avoiding the endocytosis pathway is essential for efficient delivery of intact DNA probes into cells.

Topics: Animals; Carbocyanines; Cell Survival; CHO Cells; Cricetinae; Cricetulus; DNA Probes; DNA, Single-Stranded; Electroporation; Fluorescence Resonance Energy Transfer; Microinjections; Microscopy, Confocal; Transfection

Supramolecular fibers are prominent structures in biology and chemistry. A quantitative understanding of molecular exchange pathways in these one-dimensional aggregates was obtained by a combination of super-resolution stochastic optical reconstruction microscopy and stochastic simulation. The potential of this methodology is demonstrated with a set of well-defined synthetic building blocks that self-assemble into supramolecular fibrils. Previous ensemble measurements hid all molecular phenomena underpinning monomer exchange, but the molecular pathway determined from single-aggregate studies revealed unexpected homogeneous exchange along the polymer backbone. These results pave the way for experimental investigation of the structure and exchange pathways of synthetic and natural supramolecular fibers.

Topics: Actin Cytoskeleton; Benzamides; Biopolymers; Carbocyanines; Fluorescent Dyes; Microscopy; Molecular Imaging; Polyethylene Glycols; Polymers

The effects of "molecular crowding" on elementary biochemical processes due to high solute concentrations are poorly understood and yet clearly essential to the folding of nucleic acids and proteins into correct, native structures. The present work presents, to our knowledge, first results on the single-molecule kinetics of solute molecular crowding, specifically focusing on GAAA tetraloop-receptor folding to isolate a single RNA tertiary interaction using time-correlated single-photon counting and confocal single-molecule FRET microscopy. The impact of crowding by high-molecular-weight polyethylene glycol on the RNA folding thermodynamics is dramatic, with up to ΔΔG° ∼ -2.5 kcal/mol changes in free energy and thus >60-fold increase in the folding equilibrium constant (Keq) for excluded volume fractions of 15%. Most importantly, time-correlated single-molecule methods permit crowding effects on the kinetics of RNA folding/unfolding to be explored for the first time (to our knowledge), which reveal that this large jump in Keq is dominated by a 35-fold increase in tetraloop-receptor folding rate, with only a modest decrease in the corresponding unfolding rate. This is further explored with temperature-dependent single-molecule RNA folding measurements, which identify that crowding effects are dominated by entropic rather than enthalpic contributions to the overall free energy change. Finally, a simple "hard-sphere" treatment of the solute excluded volume is invoked to model the observed kinetic trends, and which predict ΔΔG° ∼ -5 kcal/mol free-energy stabilization at excluded volume fractions of 30%.

Topics: Base Sequence; Carbocyanines; Entropy; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Kinetics; Microscopy, Confocal; Nucleic Acid Conformation; Protein Folding; Proteins; RNA; RNA Folding; Solutions; Thermodynamics

We present label-free, in situ monitoring of individual DNA hybridization in microfluidics. By immobilizing molecular sentinel probes on nanoporous gold disks, we demonstrate sensitivity approaching the single-molecule limit via surface-enhanced Raman scattering which provides robust signals without photobleaching for more than an hour. We further demonstrate that a target concentration as low as 20 pM can be detected within 10 min under diffusion-limited transport.

Topics: Breast Neoplasms; Carbocyanines; Diffusion; DNA; DNA, Single-Stranded; Female; Gold; Humans; Light; Metal Nanoparticles; Microfluidic Analytical Techniques; Microfluidics; Nanostructures; Nanotechnology; Nucleic Acid Hybridization; Porosity; Receptor, ErbB-2; Spectrum Analysis, Raman; Surface Plasmon Resonance

Degradable aliphatic polyesters are the cornerstones of nanoparticle (NP)-based therapeutics. In this paradigm, covalent modification of the NP with cell-targeting motifs and dyes can aid in guiding the NP to its destination and gaining visual confirmation. Therefore, strategies to impart chemistries along the polymer backbone that are amenable to easy modification, such as 1,3-dipolar cycloaddition of an azide to an alkyne (the "click reaction"), could be significant. Here we present a simple and efficient way to introduce alkyne groups at high density in aliphatic polyesters without compromising their crystallinity via the copolymerization of cyclic lactones with propargyl 3-methylpentenoate oxide (PMPO). Copolymers of lactic acid and ε-caprolactone with PMPO were synthesized with up to 9 mol % alkyne content, and accessibility of the alkyne groups to the click reaction was demonstrated using several dyes commonly employed in fluorescence microscopy and imaging (Cy3, ATTO-740, and coumarin 343). In order to establish the suitability of these copolymers as nanocarriers, copolymers were formulated into NPs, and cytocompatibility, cellular uptake, and visualization studies undertaken in HeLa cells. Dye-modified NPs exhibited no quenching, remained stable in solution for at least 10 days, showed no cytotoxicity, and were readily taken up by HeLa cells. Furthermore, in addition to enabling the incorporation of multiple fluorophores within the same NP through blending of individual dye-modified copolymers, dye-modified polyesters offer advantages over physical entrapment of dye, including improved signal to noise ratio and localization of the fluorescence signal within cells, and possess the necessary prerequisites for drug delivery and imaging.

Topics: Alkynes; Biocompatible Materials; Carbocyanines; Click Chemistry; Coumarins; Epoxy Compounds; Fluorescent Dyes; HeLa Cells; Humans; Lactones; Nanoparticles; Polyesters; Polymerization

In this unit, we describe a multiplex microsphere quantitative PCR. The system is based on the use of two additional oligonucleotides within a single tube PCR reaction. The first oligonucleotide is modified with a single base pair mismatch and is otherwise equivalent to a universal sequence added to the forward PCR primer. Further, this first extra oligonucleotide is coupled to Luminex microspheres. The second additional oligonucleotide is designed to be complementary to the universal sequence, and is modified with the fluorescent dye Cy3. As the PCR reaction proceeds, the second oligonucleotide is able to bind to the microspheres. Thus, quantitative monitoring of PCR progress takes place. The microsphere-mediated Cy3-detection is measured using flow cytometry directly after the PCR reaction. This allows a flow cytometer analysis from up to 150 different spheres and, therefore, multiple genes in one reaction. The multiplex microsphere qPCR is demonstrated using three target genes from Influenza A and Neisseria meningitidis. The multiplex microsphere system will enable a higher degree of multiplexing than is possible with currently available qPCR systems.

Topics: Animals; Carbocyanines; DNA Primers; Influenza A virus; Microspheres; Multiplex Polymerase Chain Reaction; Neisseria meningitidis

An aptamer-based competitive binding assay for one-step (i.e. no requirement of pre-treatment) quantitation of target molecules of interest has been developed. This method has been successfully employed for the fast and sensitive detection of the surface antigen of the hepatitis B virus (HBsAg). The key features of our method include its low intrinsic background noise, low costs, high resolution, and high sensitivity, enabling the detection of as low as 1.25 mIU mL(-1), approximately 40-fold better than that of the most widely used Abbott Architect assay for HBsAg detection, without the tedious extraction and/or washing procedures. Moreover, this assay has better recovery and accuracy than that of conventional competitive binding assay or others for HBsAg quantitation.

Topics: Aptamers, Nucleotide; Binding, Competitive; Carbocyanines; Fluorescence Resonance Energy Transfer; Hepatitis B; Hepatitis B Surface Antigens; Hepatitis B virus; Humans; Limit of Detection

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm x cm(2) on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10(-3) cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Topics: Animals; Astrocytes; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport, Active; Blood-Brain Barrier; Carbocyanines; Cell Culture Techniques; Cell Membrane Permeability; Coculture Techniques; Endothelial Cells; Female; Male; Models, Animal; Rats; Rats, Wistar; Reproducibility of Results; Rhodamine 123

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

Topics: Carbocyanines; Comet Assay; DNA; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Nucleic Acid Conformation; Polymerase Chain Reaction

We have adapted a method to map cell surface proteins and to monitor the effect of specific regulatory RNAs on the surface composition of the bacteria. This method involves direct labeling of surface proteins of living bacteria using fluorescent dyes and a subsequent separation of the crude extract by 2D gel electrophoresis. The strategy yields a substantial enrichment in surface proteins over cytoplasmic proteins. We validated this method by monitoring the effect of the regulatory RNA MicA in Escherichia coli, which regulates the synthesis of several outer membrane proteins, and highlighted the role of Staphylococcus aureus RNAIII for the maintenance of cell wall integrity.

Topics: Bacterial Outer Membrane Proteins; Bacterial Proteins; Base Sequence; Carbocyanines; Cell Membrane; Cell Wall; Electrophoresis, Gel, Two-Dimensional; Escherichia coli; Microscopy, Confocal; Molecular Sequence Data; Reproducibility of Results; RNA, Bacterial; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staining and Labeling; Staphylococcus aureus

Replication protein A (RPA) is a eukaryotic single-stranded DNA (ssDNA) binding protein that plays critical roles in most aspects of genome maintenance, including replication, recombination and repair. RPA binds ssDNA with high affinity, destabilizes DNA secondary structure and facilitates binding of other proteins to ssDNA. However, RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of RPA-DNA interactions, we combined ensemble and single-molecule fluorescence approaches to examine human RPA (hRPA) diffusion along ssDNA and find that an hRPA heterotrimer can diffuse rapidly along ssDNA. Diffusion of hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a one-dimensional diffusion coefficient of hRPA on ssDNA of D1~5000nt(2) s(-1) at 37°C. Diffusion of hRPA while bound to ssDNA enables it to be readily repositioned to allow other proteins access to ssDNA.

Topics: Carbocyanines; DNA Repair; DNA Replication; DNA, Single-Stranded; Fluorescent Dyes; Gene Rearrangement; Humans; Nucleic Acid Denaturation; Protein Binding; Recombination, Genetic; Replication Protein A

A complete fingerprint of a tissue sample requires a detailed description of its cellular and extracellular components while minimizing artifacts. We introduce the application of a novel scanning electron microscope (airSEM™) in conjunction with light microscopy for functional analysis of tissue preparations at nanometric resolution (<10 nm) and under ambient conditions. Our metal-staining protocols enable easy and detailed visualization of tissues and their extracellular scaffolds. A multimodality imaging setup, featuring airSEM™ and a light microscope on the same platform, provides a convenient and easy-to-use system for obtaining structural and functional correlative data. The airSEM™ imaging station complements other existing imaging solutions and shows great potential for studies of complex biological systems.

Topics: Animals; Carbocyanines; Collagen Type I; Coloring Agents; Electron Microscope Tomography; Extracellular Matrix; Imaging, Three-Dimensional; Lung; Mice; Microscopy, Electron, Scanning; Organometallic Compounds; Rats; Ruthenium Red; Staining and Labeling; Tail

We performed comprehensive glycome analysis of a large set of human pluripotent stem cells (hPSCs) using a high-density lectin microarray. We found that a recombinant lectin, rBC2LCN, binds exclusively to all of the undifferentiated hPSCs tested, but not to differentiated somatic cells. rBC2LCN can be used for both the staining and sorting of fixed and live hPSCs. rBC2LCN could serve as a novel detection reagent for hPSCs, particularly given that rBC2LCN is cost effective and, unlike conventional antibodies which require mammalian cells for their production, is easy to produce in a large amount (0.1 g/L) in an Escherichia coli expression system. Here we describe protocols for the fluorescence staining of fixed and live hPSCs and their detection by flow cytometry.

Topics: Carbocyanines; Cell Differentiation; Cell Survival; Flow Cytometry; Fluorescein-5-isothiocyanate; Glycomics; Humans; Lectins; Microarray Analysis; Molecular Imaging; Molecular Probes; Phycoerythrin; Pluripotent Stem Cells; Staining and Labeling

The aquaporin family comprises a large family of integral membrane proteins that enable the movement of water and other small, neutral solutes across plasma membranes. Although function and mechanism of aquaporins in central nervous system injury have been reported, the pathophysiologic role of aquaporin 1 (AQP1) in peripheral nerve has not been extensively documented. In the present study, we aimed to study the temporal and spatial distribution of AQP1 in spinal cord and dorsal root ganglia after sciatic nerve injury.. Forty-eight adult female mice were randomly divided into four groups (intact controls, sham operated, cut injury, and crush injury). Animals receiving cut or crush injuries were sacrificed at the 2nd, 24th, and 48th postoperative hours. Spinal cord samples at the level of lumbosacral intumescences and corresponding dorsal root ganglia on the experimental and contralateral side were dissected free and proceeded to AQP1 immunohistochemistry.. Our quantitative estimations revealed that a sharp increase in AQP1 immunoreactivity at the 24th postoperative hour was observed. This sharp increase was no more evident at 48 h after sciatic nerve injury. Identical peak was observed after both cut and crush injuries.. We demonstrated that there was a temporal relationship with an increased expression of AQP1 following injury sustained to the sciatic nerve that was significantly observed in dorsal root ganglia and spinal cord. Those expressions were also subsided over time.

Topics: Animals; Aquaporin 1; Carbocyanines; Disease Models, Animal; Female; Ganglia, Spinal; Gene Expression Regulation; Image Processing, Computer-Assisted; Mice; Mice, Inbred BALB C; Sciatic Neuropathy; Spinal Cord; Time Factors

We prepared the bioactive liquid crystal (LC) droplets by decorating an oligopeptide at the LC/aqueous interface to stabilize the LC droplets. These LC droplets can be used to detect proteases through the specific cleavage of protease on an oligopeptide leading to a bipolar-to-radial transition of LC configuration inside the droplets.

Topics: Amino Acid Sequence; Carbocyanines; Chymotrypsin; Fluorescent Dyes; Liquid Crystals; Molecular Sequence Data; Oligopeptides; Peptide Hydrolases; Spectrometry, Fluorescence

To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red).

Topics: Acetylation; Amino Acid Sequence; Animals; Carbocyanines; Chromosomes; Fluorescent Dyes; HeLa Cells; Histones; Humans; Hybridomas; Immunoconjugates; Immunoglobulin Fab Fragments; Maleimides; Mice; Mitosis; Molecular Imaging; Molecular Sequence Data; Phosphorylation; Protein Processing, Post-Translational; Staining and Labeling

The mechanical flexibility of ssDNA is crucial for understanding the biological machinery but its characterization has been difficult due to the lack of an experimental tool that measures the structure of ssDNA in the nanometer scale. Here, we demonstrate that single-molecule FRET can be used to probe the structures of a flexible ssDNA. We designed a dsDNA with various lengths of single-stranded overhang and determined the flexibility of the single-stranded segment by measuring the FRET value. We found that three of our ssDNAs with lengths shorter than the persistence length are indeed long enough to undergo folding. Since metal ions present in solution can affect the flexibility of DNA, we employed Na(+) and Mg(2+) at different concentrations. We found that there is no significant effect of charge screening by metal ion when the ssDNA is less than 9 bases in length but it becomes appreciable for longer ssDNAs.

Topics: Base Sequence; Carbocyanines; DNA, Single-Stranded; Fluorescence Resonance Energy Transfer; Ions; Magnesium; Sodium

Metallic nanoparticles (MNPs) are known to alter the emission of vicinal fluorophores through the near-field interaction, leading to either fluorescence quenching or enhancement. Much ambiguity remains in the experimental outcome of such a near-field interaction, particularly for bulk colloidal solution. It is hypothesized that the strong far-field interference from the inner filter effect of the MNPs could mask the true near-field MNP-fluorophore interaction significantly. Thus, in this work, a reliable internal control capable of decoupling the near-field interaction from far-field interference is established by the use of the DNA toehold concept to mediate the in situ assembly and disassembly of the MNP-fluorophore conjugate. A model gold nanoparticle (AuNP)-Cy3 system is used to investigate our proposed toehold-mediated internal control system. The maximum fluorescence enhancement is obtained for large-sized AuNP (58 nm) separated from Cy3 at an intermediate distance of 6.8 nm, while fluorescence quenching is observed for smaller-sized AuNP (11 nm and 23 nm), which is in agreement with the theoretical values reported in the literature. This work shows that the toehold-mediated internal control design can serve as a central system for evaluating the near-field interaction of other MNP-fluorophore combinations and facilitate the rational design of specific MNP-fluorophore systems for various applications.

Topics: Carbocyanines; Colloids; DNA; Fluorescent Dyes; Gold; Kinetics; Metal Nanoparticles; Nanotechnology; Oligonucleotides; Particle Size; Spectrometry, Fluorescence; Sulfhydryl Compounds

The ability to recognize small organic molecules and chemical modifications of host molecules is an essential capability of the adaptive immune system, which until now was thought to be mediated mainly by B cell antigen receptors. Here we report that small molecules, such as cyanine 3 (Cy3), a synthetic fluorescent molecule, and 4-hydroxy-3-nitrophenylacetyl (NP), one of the most noted haptens, are γδ T cell antigens, recognized directly by specific γδ TCRs. Immunization with Cy3 conjugates induces a rapid Cy3-specific γδ T cell IL-17 response. These results expand the role of small molecules and chemical modifications in immunity and underscore the role of γδ T cells as unique adaptive immune cells that couple B cell-like antigen recognition capability with T cell effector function.

Topics: Animals; Carbocyanines; Cell Membrane; Haptens; Immunity; Immunization; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Receptors, Antigen, T-Cell, gamma-delta

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (>200 µm), localization precision of 2-3 nm can be achieved.

Topics: Animals; Carbocyanines; Cornea; DNA; Immobilized Nucleic Acids; Microscopy, Fluorescence; Phase Transition; Quantum Dots; Rabbits

Accurate tracing of cell viability is critical for optimizing delivery methods and evaluating the efficacy and safety of cell therapeutics. A nanoparticle-based cell tracker is developed to image cell fate from live to dead. The particle is fabricated from two types of optically quenched polyelectrolytes, a life indicator and a death indicator, through electrostatic interactions. On incubation with cells, the fabricated bifunctional nanoprobes are taken up efficiently and the first colour is produced by normal intracellular proteolysis, reflecting the healthy status of the cells. Depending on the number of coated layers, the signal can persist for several replication cycles. However, as the cells begin dying, the second colour appears quickly to reflect the new cell status. Using this chameleon-like cell tracker, live cells can be distinguished from apoptotic and necrotic cells instantly and definitively.

Topics: Annexin A5; Apoptosis; Biotechnology; Carbocyanines; Cell Line, Tumor; Cell Lineage; Cell Survival; Electrolytes; HeLa Cells; Humans; Microscopy, Fluorescence; Molecular Probes; Molecular Weight; Nanoparticles; Necrosis; Reactive Oxygen Species; Signal Transduction; Static Electricity; Time Factors

A new DNA building block bearing a push-pull-substituted diaryltetrazole linked to the 5-position of 2'-deoxyuridine through an aminopropynyl group was synthesized. The accordingly modified oligonucleotide allows postsynthetic labeling with a maleimide-modified sulfo-Cy3 dye, N-methylmaleimide, and methylmethacrylate as dipolarophiles by irradiation at 365 nm (LED). The determined rate constant of (23±7) M(-1)  s(-1) is remarkably high with respect to other copper-free bioorthogonal reactions and comparable with the copper-catalyzed cycloaddition between azides and acetylenes.

Topics: Azoles; Base Sequence; Carbocyanines; Cycloaddition Reaction; Deoxyuridine; DNA; Light; Maleimides

A novel reduction and pH dual-sensitive nonviral vector for long-circulating and tumor-targeted siRNA delivery is described. The nanomedicine is negatively charged at neutral pH of bloodstream whereas it is positively charged at lower pH of tumor tissue (ca. 6.8). Interlayer crosslinking with disulfide bonds stabilizes the nanomedicine during blood circulation and allows quick intracellular siRNA release after endocytosis.

Topics: Animals; Carbocyanines; Cell Line, Tumor; Endocytosis; Genetic Vectors; Humans; Hydrogen-Ion Concentration; Mice; Mice, Nude; Microscopy, Confocal; Neoplasms; Optical Imaging; Oxidation-Reduction; Polyethylene Glycols; Polymers; RNA, Small Interfering; Spectroscopy, Fourier Transform Infrared; Transfection; Transplantation, Heterologous

Cy3 and Cy5 cyanine dyes terminally attached to the 5'C end (C1) of the DNA oligonucleotide were studied by metadynamics (MTD), molecular dynamics (MD), and density-functional methods with dispersion corrections (DFT-D). MTD simulations explored the free energy surface (FES) of the dye-DNA interactions, which included stacking and major groove binding motifs and unstacked structures. Dynamics of the stacked structures was studied by the MD simulations. All possible combinations of stacking interactions between the two indole rings of the dyes and the neighbor guanine and cytosine rings were observed. The most probable interaction included the stacking between the dye's distal indole ring and the guanine base. In ∼10% of the structures the delocalized π-electrons of the dyes' polymethine linkers played a key role in the dye-DNA dispersion interactions. The stacked conformers of the Cy3 dye were confirmed as true minima by DFT-D full optimizations. The stacked dye decreased flexibility up to two neighbor base pairs.

Topics: Carbocyanines; DNA; Electrons; Entropy; Models, Chemical; Molecular Dynamics Simulation; Molecular Structure; Pliability

The growing maturity of DNA-based architectures has raised considerable interest in applying them to create photoactive light harvesting and sensing devices. Toward optimizing efficiency in such structures, resonant energy transfer was systematically examined in a series of dye-labeled DNA duplexes where donor-acceptor separation was incrementally changed from 0 to 16 base pairs. Cyanine dyes were localized on the DNA using double phosphoramidite attachment chemistry. Steady state spectroscopy, single-pair fluorescence, time-resolved fluorescence, and ultrafast two-color pump-probe methods were utilized to examine the energy transfer processes. Energy transfer rates were found to be more sensitive to the distance between the Cy3 donor and Cy5 acceptor dye molecules than efficiency measurements. Picosecond energy transfer and near-unity efficiencies were observed for the closest separations. Comparison between our measurements and the predictions of Förster theory based on structural modeling of the dye-labeled DNA duplex suggest that the double phosphoramidite linkage leads to a distribution of intercalated and nonintercalated dye orientations. Deviations from the predictions of Förster theory point to a failure of the point dipole approximation for separations of less than 10 base pairs. Interactions between the dyes that alter their optical properties and violate the weak-coupling assumption of Förster theory were observed for separations of less than four base pairs, suggesting the removal of nucleobases causes DNA deformation and leads to enhanced dye-dye interaction.

Topics: Carbocyanines; DNA; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Models, Molecular; Nucleic Acid Conformation; Nucleic Acid Heteroduplexes; Spectrometry, Fluorescence; Spectrum Analysis

The in vivo application of aptamers as therapeutics could be improved by enhancing target-specific accumulation while minimizing off-target uptake. We designed a light-triggered system that permits spatiotemporal regulation of aptamer activity in vitro and in vivo. Cell binding by the aptamer was prevented by hybridizing the aptamer to a photo-labile complementary oligonucleotide. Upon irradiation at the tumor site, the aptamer was liberated, leading to prolonged intratumoral retention. The relative distribution of the aptamer to the liver and kidney was also significantly decreased, compared to that of the free aptamer.

Topics: Animals; Aptamers, Nucleotide; Base Sequence; Carbocyanines; Cell Line, Tumor; Fluorescence Resonance Energy Transfer; Mammary Neoplasms, Experimental; Mice, Nude; Microscopy, Fluorescence; Nucleic Acid Hybridization; Oligonucleotides; Skin; Tissue Distribution; Ultraviolet Rays

Rapid and specific on-site detection of disease-causing or toxin-producing organisms is essential to public health and safety. Many molecular recognition methods target ribosomal RNA sequences due to their specificity and abundance in the cell. In this work RNA targets were identified and quantified using a colorimetric bioassay. Peptide nucleic acid (PNA) probes were used to capture RNA targets, and a micrococcal nuclease digestion was performed to remove all non-target nucleic acids, including single base mismatches flanked by adenines or uracils. Perfectly-matched PNA-RNA hybrids remained intact and were detected using the symmetrical cyanine dye 3,3'-diethylthiadicarbocyanine iodide (DiSC2(5)). Assay applicability to complex samples was demonstrated using mixtures containing RNA sequences from two related, harmful algal bloom-causing Alexandrium species. Target RNA was detected even in mixtures with mismatched sequences in excess of the perfect match. The fieldability of the assay was tested with a portable two-wavelength colorimeter developed to quantify the dye-indicated hybridization signal. The colorimeter sensing performance was shown to be comparable to a laboratory spectrophotometer. This quick, inexpensive and robust system has the potential to replace laborious identification schemes in field environments.

Topics: Biological Assay; Biosensing Techniques; Carbocyanines; Colorimetry; Harmful Algal Bloom; Nucleic Acid Hybridization; Peptide Nucleic Acids; RNA, Ribosomal

Glutaraldehyde causes especially high autofluorescence. It reacted with proteins and peptides to generate visible to near-IR emitters. A model indicated that ethylenediamine and a secondary amine in the molecule were key components for the formation of emissive species. The mechanism enables us to control the generation and elimination of autofluorescence.

Topics: Amines; Amino Acid Sequence; Carbocyanines; Cell Line, Tumor; Ethylenediamines; Glutaral; Humans; Hydrogen-Ion Concentration; Molecular Sequence Data; Peptides; Proteins; Spectrophotometry

Branched Poly(ethylenimine) (bPEI) is a commercially available cationic polymer, which is well-studied due to its superior gene transfection efficacies. However, its toxicity is a major concern for its use in clinical applications. Therefore, bPEI is modified with various non-ionic, non-toxic moieties in an effort to reduce its toxicity. Ternary complexes of PEI with anionic polymers have also been used to decrease its toxicity. In this report, we have prepared pH-sensitive glycopolymers of linear and hyperbranched architecture via reversible addition-fragmentation chain transfer polymerization (RAFT). These anionic glycopolymers were complexed with bPEI at varying weight by weight (w/w) ratios to reduce the toxicity of PEI in vitro. In addition, these PEI-anionic glycopolymer complexes showed improved buffering capacity, as compared to PEI alone. The interactions of anionic glycopolymers with Hep G2 and HEK 293T cells were then studied as a function of time. The cellular uptake and gene expression of PEI polyplexes in the presence of anionic glycopolymers was directly related to the interactions of anionic glycopolymers with Hep G2 and HEK 293T cells.

Topics: Anions; Carbocyanines; Carbohydrates; Cell Line; Cell Survival; Chromatography, Gel; DNA; Endocytosis; HEK293 Cells; Hep G2 Cells; Humans; Light; Magnetic Resonance Spectroscopy; Molecular Weight; Polyethyleneimine; Polymerization; Scattering, Radiation; Static Electricity; Transfection

DNA methylation, catalyzed by methylases, plays a critical role in many biological processes, and many methylases have been regarded as promising targets for antimicrobial drugs. In this work, we report a stimulus responsive, self-regulating anticancer drug release platform, comprising a multifunctional DNA that upon methylation by methyltransferase (MTase) releases 5-fluorouracil (5-Fu) and in turn inhibits subsequent expression of MTase. The multifunctional DNA with anticancer drug are first methylated by DNA adenine methylation (DAM) methyltransferase (MTase) and then cut by the methylation-sensitive restriction endonuclease Dpn I. Removal of duplex from the functional DNA by the methylation/cleavage process will release the anticancer drug, resulting in inhibition of the activity of DAM in turn. Consequently, the enzyme activity of DAM MTase can be self-regulated. Furthermore, we found that the inhibition efficiency of 5-Fu significantly increase as it is functionalized with DNA.

Topics: Adenine; Antineoplastic Agents, Alkylating; Carbocyanines; DNA; DNA Methylation; DNA Restriction Enzymes; DNA, Single-Stranded; Fluorouracil; HeLa Cells; Humans; Microscopy, Confocal; Site-Specific DNA-Methyltransferase (Adenine-Specific); Spectrometry, Fluorescence

We have developed a fluorescence-based fiber-optical biosensor, which can selectively detect different antibodies in serial at preselected positions inside a single piece of fiber. The fiber is a microstructured polymer optical fiber fabricated from TOPAS cyclic olefin copolymer, which allows for UV activation of localized sensor layers inside the holes of the fiber. Serial fluorescence-based selective sensing of Cy3-labelled α-streptavidin and Cy5-labelled α-CRP antibodies is demonstrated.

Topics: Antibodies; Biosensing Techniques; Carbocyanines; Fluorescence; Humans; Optical Fibers; Polymers; Streptavidin

Oligonucleotide assisted tri(8-hydroxyquinoline) aluminium (Alq3) microrods were prepared for the first time. When hybridized with oligonucleotide labeled by Cy3 fluorescent dye, a significant photoluminescence variation of the Alq3 microrods was observed due to Förster resonance energy transfer, unlike when Cy5-oligonucleotide was used. Versatile nucleotide manipulation would open up wider applications of Alq3-based materials, based on this fundamental observation.

Topics: Carbocyanines; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Oligonucleotides; Organometallic Compounds; X-Ray Diffraction

Progressive dynamic, relative quantitative changes were compared in glycans associated with retinal proteins of wild type (wt) and retinal degeneration 1 (rd1) mice during neonatal development and degeneration of retinae.. Proteins extracted from retinae of postnatal days 2 (PN2), PN7, and PN14 wt and rd1 mice were labeled with Cy3-fluorescent dye. Glycome of these proteins was quantified relatively by lectin microarray technique. Net fluorescence emitted by individual complexes formed between 45 lectins and Cy3-labeled proteins was measured by evanescent-field fluorescence-assisted microarray reader.. GlcNAcβ1-oligomer and high-mannose/Manα1-6Man were major glycans associated with the proteins of PN2, PN7, and PN14 wt and rd1 mice retinae. Gal/GalNAc/Man3-core-bi-/tri-antennary-complex, Sia2-3Galβ1-4GlcNAc, and high-mannose glycans were conjugated mainly to proteins from PN7 rd1 and PN14 wt retinae, respectively. With increasing neonatal age, mannosylated, GlcNAcβ, and sialylated (minor component) glycans were increased, and fucosylated GlcNAc/Galβ glycans were decreased significantly in wt retinal proteins. This trend was less evident in PN14 rd1 retinal proteins. Mouse retina was almost devoid of Siaα2-6 (except WGA bound Sia), Fucα1-2, and Gal/GalNAc-containing glycans. STL reacting GlcNAc oligomers were high in PN2 rd1 retinae.. Quantitative dynamic, relative variation in high-mannose and GlcNAc glycans, Siaα2-3Galβ1-4GlcNAc associated with proteins from PN2, PN7, and PN14 wt and rd1 mice retinae suggested that these glycans participate in retinal development and degeneration, and may be used as markers for retinal electrophysiologic integrity during transplantation/therapy studies; Siaα2-3Galβ1-4GlcNAc-specific Agrocybe cylindracea lectin and other lectins may be used to enrich/purify retinal ribbon synapse glycoproteins and other glycoproteins including rhodopsin. Further investigations are required.

Topics: Animals; Animals, Newborn; Carbocyanines; Disease Models, Animal; Eye Proteins; Fluorescent Dyes; Glycomics; Glycoproteins; Lectins; Mice; Polysaccharides; Protein Array Analysis; Retina; Retinitis Pigmentosa

The cell is a spatially organized system whose function emerges from the complex interaction of molecular components. Such local interaction of nanometer-sized molecules generates patterns that span throughout the cell. Those patterns, in turn, regulate the molecular interactions. Understanding such simultaneous bidirectional causation requires quantifying the spatio-temporal progression of biochemical reactions in the context of a living cell. Due to its ability to resolve micrometer-sized structures, biological microscopy has been instrumental to the discovery and understanding of living systems. Functional fluorescence microscopy allows a cellular dynamic topographic map of proteins to be overlaid with topological information on the causality that determines protein state. Here we describe how Förster/fluorescence resonance energy transfer (FRET) can be used to measure the phosphorylation state of proteins in the context of the cell.

Topics: Animals; Carbocyanines; Cell Line; Cell Physiological Phenomena; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; Humans; Microscopy, Fluorescence; Transfection

Recent advances in fluorescence microscopy have enabled high-resolution imaging and tracking of single proteins and biomolecules in cells. To achieve high spatial resolutions in the nanometer range, bright and photostable fluorescent probes are critically required. From this view, there is a strong need for development of advanced fluorescent probes with molecular-scale dimensions for fluorescence imaging. Polymer-based dendrimer nanoconjugates hold strong potential to serve as versatile fluorescent probes due to an intrinsic capacity for tailored spectral properties such as brightness and emission wavelength. In this work, we report a new, to our knowledge, class of molecular probes based on dye-conjugated dendrimers for fluorescence imaging and single-molecule fluorescence microscopy. We engineered fluorescent dendritic nanoprobes (FDNs) to contain multiple organic dyes and reactive groups for target-specific biomolecule labeling. The photophysical properties of dye-conjugated FDNs (Cy5-FDNs and Cy3-FDNs) were characterized using single-molecule fluorescence microscopy, which revealed greatly enhanced photostability, increased probe brightness, and improved localization precision in high-resolution fluorescence imaging compared to single organic dyes. As proof-of-principle demonstration, Cy5-FDNs were used to assay single-molecule nucleic acid hybridization and for immunofluorescence imaging of microtubules in cytoskeletal networks. In addition, Cy5-FDNs were used as reporter probes in a single-molecule protein pull-down assay to characterize antibody binding and target protein capture. In all cases, the photophysical properties of FDNs resulted in enhanced fluorescence imaging via improved brightness and/or photostability.

Topics: Biological Transport; Carbocyanines; Dendrimers; Fluorescent Dyes; HEK293 Cells; Humans; Kinetics; Light; Microscopy, Fluorescence; Nanostructures; Nucleic Acid Hybridization

Lactosylated gramicidin-containing lipid nanoparticles (Lac-GLN) were developed for delivery of anti-microRNA-155 (anti-miR-155) to hepatocellular carcinoma (HCC) cells. MiR-155 is an oncomiR frequently elevated in HCC. The Lac-GLN formulation contained N-lactobionyl-dioleoyl phosphatidylethanolamine (Lac-DOPE), a ligand for the asialoglycoprotein receptor (ASGR), and an antibiotic peptide gramicidin A. The nanoparticles exhibited a mean particle diameter of 73 nm, zeta potential of +3.5mV, anti-miR encapsulation efficiency of 88%, and excellent colloidal stability at 4°C. Lac-GLN effectively delivered anti-miR-155 to HCC cells with a 16.1- and 4.1-fold up-regulation of miR-155 targets C/EBPβ and FOXP3 genes, respectively, and exhibited significant greater efficiency over Lipofectamine 2000. In mice, intravenous injection of Lac-GLN containing Cy3-anti-miR-155 led to preferential accumulation of the anti-miR-155 in hepatocytes. Intravenous administration of 1.5 mg/kg anti-miR-155 loaded Lac-GLN resulted in up-regulation of C/EBPβ and FOXP3 by 6.9- and 2.2-fold, respectively. These results suggest potential application of Lac-GLN as a liver-specific delivery vehicle for anti-miR therapy.

Topics: Animals; Anti-Bacterial Agents; Antibodies; Carbocyanines; CCAAT-Enhancer-Binding Proteins; Cell Line, Tumor; Cell Survival; Drug Carriers; Fluorescent Dyes; Forkhead Transcription Factors; Gramicidin; Hep G2 Cells; Humans; Lactose; Lipids; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Nanoparticles; Tissue Distribution

Lengthening smFRET lifetimes: We investigated various photoprotection system combinations to find the combination that optimally extended the photobleach lifetime of a Cy3/Cy5 smFRET pair attached to a DNA hairpin in a single-molecule environment. We found that the glucose/glucose oxygen-scavenging solution in combination with redox-based photostabilization solutions yielded the longest average photobleaching lifetimes.

Topics: Carbocyanines; DNA; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Glucose; Oxidation-Reduction; Photobleaching

Cyanine dyes are widely used to study the folding and structural transformations of nucleic acids using fluorescence resonance energy transfer (FRET). The extent to which FRET can be used to extract inter- and intramolecular distances has been the subject of considerable debate in the literature; the contribution of dye and linker dynamics to the observed FRET signal is particularly troublesome. We used molecular dynamics (MD) simulations to study the dynamics of the indocarbocyanine dyes Cy3 and Cy5 attached variously to the 3' or 5' terminal bases of a 16-base-pair RNA duplex. We then used Monte Carlo modeling of dye photophysics to predict the results of single-molecule-sensitive FRET measurements of these same molecules. Our results show that the average value of FRET depends on both the terminal base and the linker position. In particular, 3' attached dyes typically explore a wide region of configuration space, and the relative orientation factor, κ(2), has a distribution that approaches that of free-rotators. This is in contrast to 5' attached dyes, which spend a significant fraction of their time in one or more configurations that are effectively stacked on the ends of the RNA duplex. The presence of distinct dye configurations for 5' attached dyes is consistent with observations, made by others, of multiple fluorescence lifetimes of Cy3 on nucleic acids. Although FRET is frequently used as a molecular "ruler" to measure intramolecular distances, the unambiguous measurement of distances typically relies on the assumption that the rotational degrees of freedom of the dyes can be averaged out and that the donor lifetime in the absence of the acceptor is a constant. We demonstrate that even for the relatively free 3' attached dyes, the correlation time of κ(2) is still too long to justify the use of a free-rotation approximation. We further explore the consequences of multiple donor lifetimes on the predicted value of FRET.

Topics: Carbocyanines; Fluorescence Resonance Energy Transfer; Molecular Dynamics Simulation; RNA

Inhalation of airborne particulate matter (PM), such as silicon dioxide (SiO2) and titanium dioxide (TiO2), induces acute lung inflammation. siRNA therapy has been proposed as a method to repair acute lung inflammation. To determine whether DEXA-PEI/MIF siRNA contributes to SiO2-induced acute lung inflammation repair, we administered Dexa-PEI/MIF siRNA in SiO2-treated Beas-2b cells and instilled DEXA-PEI-MIF siRNA intratracheally in mice with SiO2-induced acute lung inflammation. Using genetic (MIF mRNA RT-PCR), histological (H&E and PAS) and immunohistochemical (MIF and Muc5ac) analyses, we estimated the acute lung inflammation in Beas-2b cells and BALB/c mice. Cells and mice treated with SiO2 particles demonstrated pulmonary inflammation. DEXA-PEI/MIF siRNA restricted the extent of the pulmonary inflammation reaction to SiO2 in cells and mice. In case of SiO2-treated Beas-2b cells, only DEXA-PEI treatment failed to effectively regulate MIF mRNA release. At the same time, only DEXA-PEI treatment adjusted the amount of MIF mRNA to some extent in SiO2-treated BALB/c mice. siRNA treatment did not markedly control MIF mRNA release in mice. We also observed that the amount of MIF mRNA was decreased in cells and mice treated with DEXA-PEI/MIF siRNA. The increase of MIF mRNA markedly increased Muc5ac; in contrast, the decrease of MIF mRNA using DEXA-PEI/MIF siRNA effectively lowered Muc5ac in SiO2-treated cells and mice. These results suggest that DEXA-PEI plays a role in delivering siRNA to the nucleus as a carrier and limits the extent of acute lung inflammation. MIF siRNA also contributed to the reparative lung response in SiO2-induced pulmonary inflammation.

Topics: Animals; Bronchoalveolar Lavage Fluid; Carbocyanines; Dexamethasone; Female; Gene Expression Regulation; Gene Knockdown Techniques; Humans; Inflammation; Lung; Macrophage Migration-Inhibitory Factors; Mice; Mice, Inbred BALB C; Mucin 5AC; Particulate Matter; Polyethyleneimine; RNA, Messenger; RNA, Small Interfering; Static Electricity; Transfection

We measure the stiffness of tiled DNA nanotubes (HX-tubes) as a function of their (defined) circumference by analyzing their micrometer-scale thermal deformations using fluorescence microscopy. We derive a model that relates nanoscale features of HX-tube architecture to the measured persistence lengths. Given the known stiffness of double-stranded DNA, we use this model to constrain the average spacing between and effective stiffness of individual DNA duplexes in the tube. A key structural feature of tiled nanotubes that can affect stiffness is their potential to form with discrete amounts of twist of the DNA duplexes about the tube axis (supertwist). We visualize the supertwist of HX-tubes using electron microscopy of gold nanoparticles, attached to specific sites along the nanotube. This method reveals that HX-tubes tend not to form with supertwist unless forced by sequence design, and, even when forced, supertwist is reduced by elastic deformations of the underlying DNA lattice. We compare the hybridization energy gained upon closing a duplex sheet into a tube with the elastic energy paid for deforming the sheet to allow closure. In estimating the elastic energy we account for bending and twisting of the individual duplexes as well as shearing between them. We find the minimum supertwist state has minimum free energy, and global untwisting of forced supertwist is energetically favorable, consistent with our experimental data. Finally, we show that attachment of Cy3 dyes or changing counterions can cause nanotubes to adopt a permanent writhe with micrometer-scale pitch and amplitude. We propose that the coupling of local twist and global counter-twist may be useful in characterizing perturbations of DNA structure.

Topics: Algorithms; Anisotropy; Carbocyanines; Colloids; DNA; Drug Delivery Systems; Elasticity; Gold; Ligands; Metal Nanoparticles; Microscopy, Electron; Microscopy, Fluorescence; Nanotechnology; Nanotubes; Nucleic Acid Conformation; Temperature; Time Factors

Design and development of a new formulation as a unique assembly of distinct fluorescent reporters with nonoverlapping fluorescence spectra and a F19 magnetic resonance imaging agent into colloidally and optically stable triphasic nanoemulsion are reported. Specifically, a cyanine dye-perfluorocarbon (PFC) conjugate was introduced into the PFC phase of the nanoemulsion and a near-infrared dye was introduced into the hydrocarbon (HC) layer. To the best of our knowledge, this is the first report of a triphasic nanoemulsion system where each oil phase, HC, and PFC are fluorescently labeled and formulated into an optically and colloidally stable nanosystem. Having, each oil phase separately labeled by a fluorescent dye allows for improved correlation between in vivo imaging and histological data. Further, dual fluorescent labeling can improve intracellular tracking of the nanodroplets and help assess the fate of the nanoemulsion in biologically relevant media. The nanoemulsions were produced by high shear processing (microfluidization) and stabilized with biocompatible nonionic surfactants resulting in mono-modal size distribution with average droplet size less than 200 nm. Nanoemulsions demonstrate excellent colloidal stability and only moderate changes in the fluorescence signal for both dyes. Confocal fluorescence microscopy of macrophages exposed to nanoemulsions shows the presence of both fluorescence agents in the cytoplasm.

Topics: Animals; Carbocyanines; Cell Line; Cell Survival; Drug Stability; Emulsions; Ethers; Fluorescent Dyes; Fluorocarbons; Magnetic Resonance Imaging; Mice; Nanoparticles; Optical Imaging; Spectroscopy, Near-Infrared

The choroid plexus (CP) is present on the ventricular walls of the brain, produces cerebrospinal fluid (CSF), contains many blood vessels, and is a major functional component of the blood-CSF barrier. The CP is an important site in the pathophysiology of various neurological diseases, including Alzheimer's disease and meningeal amyloidosis. We performed gene silencing in the CP in vivo by using an antisense oligonucleotide (ASO). A short ASO of length 12 nucleotides was intravenously injected into rats. The ASO was not delivered to neurons or glia in the central nervous system, but was successfully delivered into the CP, and resulted in a significant reduction of endogenous target gene expression in epithelial cells within the CP. Although the mechanism of uptake of the ASO by the CP was not elucidated, the ASO bound to albumin in vivo, and the distribution of ASO delivery was similar to that of albumin delivery. These findings suggest that we inhibited target gene expression in the epithelial cells of the CP via albumin-ASO conjugates. This strategy should be useful for investigations of the function of CP, and for the development of new gene-silencing therapies for diseases with pathophysiology related to the CP.

Topics: Albumins; Animals; Carbocyanines; Cell Culture Techniques; Cell Line; Choroid Plexus; Drug Delivery Systems; Epithelial Cells; Feasibility Studies; Female; Fluorescent Dyes; Gene Silencing; Gene Targeting; Injections, Intravenous; Mice; Mice, Inbred Strains; Microscopy, Confocal; Oligonucleotides; Oligonucleotides, Antisense; Phosphorothioate Oligonucleotides; Protein Binding; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Spectrometry, Fluorescence; Superoxide Dismutase; Superoxide Dismutase-1

The CyDye family of fluorescent dyes is currently the overwhelming choice for applications in proteomic analysis, using two-dimensional difference gel electrophoresis (2D-DIGE). Protein labeling with CyDyes is hampered by protein precipitation and gel smearing when used above minimal labeling. The solubility of labeled protein may be improved by introducing water solubilizing groups on the dye such as cysteic acids. However, addition of a negatively charged functionality will have the undesired effect of shifting the pI in relation to the unlabeled protein. These limitations have been addressed through the synthesis of highly water-soluble and pI balancing zwitterionic CyDye fluorophores (Z-CyDyes). The new dyes feature a cysteic acid motif, a titratable amine functionality and a NHS activated ester group. In side by side 2D-DIGE comparisons of Z-CyDyes and CyDyes, the new dyes significantly enhanced protein spot volume and the number of spots that were detected. Z-CyDyes have the potential to enhance the depth of proteome coverage and provide a general strategy for improving the performance of protein tagging reagents.

Topics: Archaeal Proteins; Carbocyanines; Cysteine; Electrophoresis, Gel, Two-Dimensional; Fluorescent Dyes; Proteomics; Solubility; Staining and Labeling; Sulfolobus solfataricus; Water

Novel silica nanoparticles mimicking virus surface topography are prepared. It is demonstrated that increases in nanoscale surface roughness promote both binding of biomolecules and cellular uptake; thus, the cellular delivery efficiency is significantly increased (scale bars 20 μm).

Topics: Biocompatible Materials; Carbocyanines; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Drug Carriers; HeLa Cells; Humans; MCF-7 Cells; Microscopy, Confocal; Nanoparticles; Oligonucleotides; Polo-Like Kinase 1; Polyethyleneimine; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; rab GTP-Binding Proteins; RNA Interference; RNA, Small Interfering; Silicon Dioxide; Surface Properties

A multi-functional mesoporous silica nanoparticle (MSN)-based boron neutron capture therapy (BNCT) agent, designated as T-Gal-B-Cy3@MSN, was synthesized with hydrophobic mesopores for incorporating a large amount of o-carborane (almost 60% (w/w) boron atoms per MSN), and the amines on the external surface were conjugated with trivalent galactosyl ligands and fluorescent dyes for cell targeting and imaging, respectively. The polar and hydrophilic galactosyl ligands enhance the water dispersibility of the BNCT agent and inhibit the possible leakage of o-carborane loaded in the MSN. Confocal microscopic images showed that T-Gal-B-Cy3@MSNs were endocytosed by cells and were then released from lysosomes into the cytoplasm of cells. Moreover, in comparison with the commonly used clinical BNCT agent, sodium borocaptate (BSH), T-Gal-B-Cy3@MSN provides a higher delivery efficiency (over 40-50 fold) of boron atoms and a better effect of BNCT in neutron irradiation experiments. MTT assays show a very low cytotoxicity for T-Gal-B-Cy3@MSN over a 2 h incubation time. The results are promising for the design of multifunctional MSNs as potential BNCT agents for clinical use.

Topics: Boranes; Boron Neutron Capture Therapy; Carbocyanines; Cell Survival; Fluorescent Dyes; Galactose; Hep G2 Cells; Humans; Microscopy, Confocal; Nanoparticles; Porosity; Silicon Dioxide

We demonstrate here a DNA-based conformationally switchable nanodumbell structure that modulates the interparticle distance between two gold-silver core-shell nanoparticles to induce significant changes in the SERS signal.

Topics: Carbocyanines; DNA; Gold; Metal Nanoparticles; Nucleic Acid Conformation; Particle Size; Silver; Spectrum Analysis, Raman

Phagocytosis--the engulfment of cells and foreign bodies--is an important cellular process in innate immunity, development, and disease. Quantification of various stages of phagocytosis, especially in a rapid screening fashion, is an invaluable tool for elucidating protein function during this process. However, current methods for assessing phagocytosis are largely limited to flow cytometry and manual image-based assays, providing limited information. Here, we present an image-based, semi-automated phagocytosis assay to rapidly quantitate three distinct stages during the early engulfment of opsonized beads. Captured images are analyzed using the image-processing software ImageJ and quantified using a macro. Modifications to this method allowed quantification of phagocytosis only in fluorescently labeled transfected cells. Additionally, the time course of bead internalization could be measured using this approach. The assay could discriminate perturbations to stages of phagocytosis induced by known pharmacological inhibitors of filamentous actin and phosphoinositol-3-kinase. Our methodology offers the ability to automatically categorize large amounts of image data into the three early stages of phagocytosis within minutes, clearly demonstrating its potential value in investigating aberrant phagocytosis when manipulating proteins of interest in drug screens and disease.

Topics: Animals; Carbocyanines; Cell Line; Chromones; Cytochalasin D; Cytological Techniques; Erythrocytes; Fluorescent Dyes; High-Throughput Screening Assays; Image Processing, Computer-Assisted; Immunoglobulin G; Mice; Microscopy, Fluorescence; Microspheres; Morpholines; Phagocytosis; Reproducibility of Results; Sheep

We analyzed the enzymatic profiles of on-chip DNA ligation as we controlled the lateral spacing of surface-immobilized DNA substrates using dendron molecules with different sizes at the nanoscale. Enzymatic on-chip DNA ligation was performed on the dendron-coated surface within 20 min with no need for post-ligation gel electrophoresis. The enzymatic DNA repair was assessed by the fluorescence intensity at the repaired DNA duplex after thermally dissociating the unligated Cy3-labeled DNA from the DNA duplex, in which the Cy3-labeled DNA was hybridized prior to the on-chip DNA ligation. The rate of the nick-sealing reaction on the 27-acid dendron surface was 3-fold higher than that on the 9-acid dendron surface, suggesting that the wider lateral spacing determined by the larger dendron molecule could facilitate the access of DNA ligase to the nick site. The performance of on-chip DNA ligation was dropped to 10% and 3% when the nick was replaced by one- and two-nucleotide-long gaps, respectively. The 5' terminal phosphorylation of DNA strands by polynucleotide kinase and the on-chip DNA cleavage by endonucleases were also quantitatively monitored throughout the on-chip DNA ligation on the dendron-coated surface. A better understanding of the enzymatic kinetics of on-chip DNA ligation will contribute to a more reliable performance of various on-chip DNA ligation-based assays.

Topics: Carbocyanines; Dendrimers; DNA; DNA Ligases; Genetic Techniques; Kinetics; Oligonucleotide Array Sequence Analysis

Irradiation of solutions of the cyanine dyes Cy3, Cy3B, and Cy5 in the presence of Mn(2+) causes an increase in the yield of formation of the triplet state of the dye. This results in increased photobleaching and triplet blinking. Experiments with other divalent ions and paramagnetic molecules suggest that the enhancement in the intersystem-crossing rate is related to the paramagnetic nature of the Mn(2+) cation. The results are consistent with a model in which the formation of a weak collisional complex between the dye and the ion results in mixing of the singlet and triplet states of the dye. These findings are particularly significant in single-molecule spectroscopy and super-resolution imaging methods, in which photobleaching and blinking play an important role.

Topics: Benzenesulfonates; Carbocyanines; Fluorescent Dyes; Ions; Manganese; Photobleaching; Quantum Theory

Although a link between excess lipid storage and aberrant glucose metabolism has been recognized for many years, little is known what role lipid storage droplets and associated proteins such as Plin2 play in managing cellular glucose levels. To address this issue, the influence of Plin2 on glucose uptake was examined using 2-NBD-Glucose and [(3)H]-2-deoxyglucose to show that insulin-mediated glucose uptake was decreased 1.7- and 1.8-fold, respectively in L cell fibroblasts overexpressing Plin2. Conversely, suppression of Plin2 levels by RNAi-mediated knockdown increased 2-NBD-Glucose uptake several fold in transfected L cells and differentiated 3T3-L1 cells. The effect of Plin2 expression on proteins involved in glucose uptake and transport was also examined. Expression of the SNARE protein SNAP23 was increased 1.6-fold while levels of syntaxin-5 were decreased 1.7-fold in Plin2 overexpression cells with no significant changes observed in lipid droplet associated proteins Plin1 or FSP27 or with the insulin receptor, GLUT1, or VAMP4. FRET experiments revealed a close proximity of Plin2 to SNAP23 on lipid droplets to within an intramolecular distance of 51 Å. The extent of targeting of SNAP23 to lipid droplets was determined by co-localization and co-immunoprecipitation experiments to show increased partitioning of SNAP23 to lipid droplets when Plin2 was overexpressed. Taken together, these results suggest that Plin2 inhibits glucose uptake by interacting with, and regulating cellular targeting of SNAP23 to lipid droplets. In summary, the current study for the first time provides direct evidence for the role of Plin2 in mediating cellular glucose uptake.

Topics: 3T3-L1 Cells; Animals; Biological Transport; Carbocyanines; Cytochalasin B; Cytoplasmic Granules; Deoxyglucose; Fibroblasts; Fluorescence Resonance Energy Transfer; Glucose; Glucose Transporter Type 1; Immunoblotting; Insulin; L Cells; Lipids; Membrane Proteins; Mice; Microscopy, Confocal; Models, Biological; Perilipin-2; Protein Binding; Qb-SNARE Proteins; Qc-SNARE Proteins; RNA Interference

DNA "breathing" is a thermally driven process in which base-paired DNA sequences transiently adopt local conformations that depart from their most stable structures. Polymerases and other proteins of genome expression require access to single-stranded DNA coding templates located in the double-stranded DNA "interior," and it is likely that fluctuations of the sugar-phosphate backbones of dsDNA that result in mechanistically useful local base pair opening reactions can be exploited by such DNA regulatory proteins. Such motions are difficult to observe in bulk measurements, both because they are infrequent and because they often occur on microsecond time scales that are not easy to access experimentally. We report single-molecule fluorescence experiments with polarized light, in which tens-of-microseconds rotational motions of internally labeled iCy3/iCy5 donor-acceptor Förster resonance energy transfer fluorophore pairs that have been rigidly inserted into the backbones of replication fork constructs are simultaneously detected using single-molecule Förster resonance energy transfer and single-molecule fluorescence-detected linear dichroism signals. Our results reveal significant local motions in the ∼100-μs range, a reasonable time scale for DNA breathing fluctuations of potential relevance for DNA-protein interactions. Moreover, we show that both the magnitudes and the relaxation times of these backbone breathing fluctuations are significantly perturbed by interactions of the fork construct with a nonprocessive, weakly binding bacteriophage T4-coded helicase hexamer initiation complex, suggesting that these motions may play a fundamental role in the initial binding, assembly, and function of the processive helicase-primase (primosome) component of the bacteriophage T4-coded DNA replication complex.

Topics: Algorithms; Bacteriophage T4; Carbocyanines; DNA; DNA Replication; DNA, Single-Stranded; DNA, Viral; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Kinetics; Models, Genetic; Models, Molecular; Nucleic Acid Conformation; Protein Multimerization; Viral Proteins

In this work, we have successfully prepared a series of new sterol-based cationic lipids (1-4) via an efficient 'Click' chemistry approach. The pDNA binding affinity of these lipids was examined by EB displacement and agarose-gel retardant assay. The average particle sizes and surface charges of the sterol-based cationic lipids/pDNA lipoplexes were analyzed by dynamic laser light scattering instrument (DLS), and the morphologies of the lipoplexes were observed by atomic force microscopy (AFM). The cytotoxicity of the lipids were examined by MTT and LDH assay, and the gene transfection efficiencies of these lipid carriers were investigated by luciferase gene transfection assay in various cell lines. In addition, the intracellular uptake and trafficking/localization behavior of the Cy3-DNA loaded lipoplexes were preliminarily studied by fluorescence microscopy. The results demonstrated that the pDNA loading capacity, lipoplex particle size, zeta potential and morphology of the sterol lipids/pDNA lipoplexes depended largely on the molecular structure factors including sterol-skeletons and headgroups. Furthermore, the sterol-based lipids showed quite different cytotoxicity and gene transfection efficacy in A549 and HeLa cells. Interestingly, it was found that the cholesterol-bearing lipids 1 and 2 showed 7-10(4) times higher transfection capability than their lithocholate-bearing counterparts 3 and 4 in A549 and HeLa cell lines, suggested that the gene transfection capacity strongly relied on the structure of sterol skeletons. Moreover, the study on the structure-activity relationships of these sterol-based cationic lipid gene carriers provided a possible approach for developing low cytotoxic and high efficient lipid gene carriers by selecting suitable sterol hydrophobes and cationic headgroups.

Topics: Carbocyanines; Cations; Cell Line, Tumor; Cell Survival; Click Chemistry; DNA; HeLa Cells; Humans; Lipids; Microscopy, Atomic Force; Sterols; Structure-Activity Relationship; Transfection

Topics: Biotin; Carbocyanines; Click Chemistry; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; MicroRNAs; Organophosphorus Compounds

The era of molecular medicine has ushered in the development of microscopic methods that can report molecular processes in thick tissues with high spatial resolution. A commonality in deep-tissue microscopy is the use of near-infrared (NIR) lasers with single- or multiphoton excitations. However, the relationship between different NIR excitation microscopic techniques and the imaging depths in tissue has not been established. We compared such depth limits for three NIR excitation techniques: NIR single-photon confocal microscopy (NIR SPCM), NIR multiphoton excitation with visible detection (NIR/VIS MPM), and all-NIR multiphoton excitation with NIR detection (NIR/NIR MPM). Homologous cyanine dyes provided the fluorescence. Intact kidneys were harvested after administration of kidney-clearing cyanine dyes in mice. NIR SPCM and NIR/VIS MPM achieved similar maximum imaging depth of ∼100 μm. The NIR/NIR MPM enabled greater than fivefold imaging depth (>500 μm) using the harvested kidneys. Although the NIR/NIR MPM used 1550-nm excitation where water absorption is relatively high, cell viability and histology studies demonstrate that the laser did not induce photothermal damage at the low laser powers used for the kidney imaging. This study provides guidance on the imaging depth capabilities of NIR excitation-based microscopic techniques and reveals the potential to multiplex information using these platforms.

Topics: Animals; Carbocyanines; Cell Line, Tumor; Cell Survival; Contrast Media; Emulsions; Fluorescent Dyes; Histocytochemistry; Image Processing, Computer-Assisted; Kidney; Lasers; Male; Mice; Microscopy, Fluorescence, Multiphoton; Phantoms, Imaging; Phospholipids; Soybean Oil; Spectroscopy, Near-Infrared

Adenosine triphosphate (ATP) analogues labelled with two dyes suitable for undergoing Förster Resonance Energy Transfer (FRET) have the potential to be valuable tools to continuously study the enzymatic activity of ATP consuming enzymes. Here, we present a synthesis strategy that allows obtaining these ATP analogues in a straight-forward manner. Earlier studies indicate that modifying ATP at the O2'- and the γ-position is a very promising starting point for the design of these probes. We synthesized probes modified with five different combinations of dyes attached to these positions and investigated their fluorescence characteristics in the non-cleaved state as well as after enzymatic hydrolysis. All presented probes largely change their fluorescence characteristics upon cleavage. They include ratiometric FRET probes as well as dark quenched analogues. For typical in vitro applications a combination of the sulfonated polymethine dyes Sulfo-Cy3 and Sulfo-Cy5 seems to be most promising due to their excellent solubility in aqueous buffer and a large change of fluorescence characteristics upon cleavage. For this combination of dyes we also synthesized analogues modified at the γ- and the C2- or the O3'-position, respectively, as these attachment sites are also well accepted by certain ATP consuming enzymes. These analogues show comparably large changes in fluorescence characteristics. Overall, we present new ATP-based FRET probes that have the potential to enable monitoring the enzymatic activity of ATP consuming enzymes.

Topics: Adenosine Triphosphate; Carbocyanines; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Hydrolysis

Reliable and efficient identification of DNA is a major goal in on-site diagnostics. One dimensional nanostructures like nanowires (NW) represent potential sensor structures due to their extreme surface-to-bulk ratio, enabling enhanced biomolecule binding which results in optimal signals. While silicon NW are already well studied, NW made from other materials with promising properties like ZnO are not yet established as NW sensor material for bioanalytics. Here we demonstrate the DNA functionalization of ZnO NW even at the single NW level and their successful application in a DNA hybridization assay.

Topics: Carbocyanines; DNA; Fluorescent Dyes; Immobilized Nucleic Acids; Nanotechnology; Nanowires; Nucleic Acid Hybridization; Optical Phenomena; Silicon; Transducers; Zinc Oxide

Based on cyanine dye probe oxazole yellow (YO) and Cy(3), a series of novel styryl cyanine dyes were designed and synthesized. Carbazole was inserted into the structures of YO and Cy(3) to act as a bridge to link the benzoxazole and indole group. This modification resulted in a novel kind of benzoxazole indole styryl cyanine dye with a carbazole-bridged chain. The dyes were characterized by (1)HNMR and MS. The spectra of the novel dyes were also performed and the results showed that the maximum emission wavelength of the carbazole styryl cyanine dye was shifted red, the Stokes shift increased and the fluorescence intensity enhanced compared with those of YO and Cy(3). These results indicated that the novel dye could be used as an excellent fluorescent probe in biological labeling.

Topics: Animals; Benzoxazoles; Carbazoles; Carbocyanines; Cattle; Chemistry Techniques, Synthetic; Fluorescent Dyes; Quinolines; Serum Albumin, Bovine; Spectrometry, Fluorescence

The fluorescence resonant energy transfer (FRET) from a donor to an acceptor via transition dipole-dipole interactions decreases the donor's fluorescent lifetime. The donor's fluorescent lifetime decreases as the FRET efficiency increases, following the equation: E(FRET) = 1 - τ(DA)/τ(D), where τ(D) and τ(DA) are the donor fluorescence lifetime without FRET and with FRET. Accordingly, the FRET time trajectories associated with single-molecule conformational dynamics can be recorded by measuring the donor's lifetime fluctuations. In this article, we report our work on the use of a Cy3/Cy5-labeled enzyme, HPPK to demonstrate probing single-molecule conformational dynamics in an enzymatic reaction by measuring single-molecule FRET donor lifetime time trajectories. Compared with single-molecule fluorescence intensity-based FRET measurements, single-molecule lifetime-based FRET measurements are independent of fluorescence intensity. The latter has an advantage in terms of eliminating the analysis background noise from the acceptor fluorescence detection leak through noise, excitation light intensity noise, or light scattering noise due to local environmental factors, for example, in a AFM-tip correlated single-molecule FRET measurements. Furthermore, lifetime-based FRET also supports simultaneous single-molecule fluorescence anisotropy.

Topics: Carbocyanines; Diphosphotransferases; Fluorescence Resonance Energy Transfer; Molecular Dynamics Simulation; Photons; Protein Structure, Tertiary

Actin filament severing is critical for the dynamic turnover of cellular actin networks. Cofilin severs filaments, but additional factors may be required to increase severing efficiency in vivo. Srv2/cyclase-associated protein (CAP) is a widely expressed protein with a role in binding and recycling actin monomers ascribed to domains in its C-terminus (C-Srv2). In this paper, we report a new biochemical and cellular function for Srv2/CAP in directly catalyzing cofilin-mediated severing of filaments. This function is mediated by its N-terminal half (N-Srv2), and is physically and genetically separable from C-Srv2 activities. Using dual-color total internal reflection fluorescence microscopy, we determined that N-Srv2 stimulates filament disassembly by increasing the frequency of cofilin-mediated severing without affecting cofilin binding to filaments. Structural analysis shows that N-Srv2 forms novel hexameric star-shaped structures, and disrupting oligomerization impairs N-Srv2 activities and in vivo function. Further, genetic analysis shows that the combined activities of N-Srv2 and Aip1 are essential in vivo. These observations define a novel mechanism by which the combined activities of cofilin and Srv2/CAP lead to enhanced filament severing and support an emerging view that actin disassembly is controlled not by cofilin alone, but by a more complex set of factors working in concert.

Topics: Actin Cytoskeleton; Adaptor Proteins, Signal Transducing; Carbocyanines; Catalysis; Cofilin 1; Cytoskeletal Proteins; Microfilament Proteins; Microscopy, Electron; Microscopy, Fluorescence; Models, Molecular; Mutation; Protein Binding; Protein Multimerization; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

Cell research often requires combinational detection of RNA and DNA by fluorescence in situ hybridization (RNA-DNA FISH). However, it is difficult to preserve the fragile RNA signals through the harsh conditions used to denature the DNA template in DNA FISH. The current protocols of RNA-DNA FISH still cannot work robustly in all experiments. RNA-DNA FISH remains as a technically challenging and tedious experiment. By incorporating protein components into the signal detection steps of RNA FISH, which is then followed by a post-fixation step, we established an improved protocol of RNA-DNA FISH. The established method worked satisfyingly and robustly in our studies on Xist (inactivated X chromosome specific transcript) RNA and Terra (telomeric repeat-containing RNA). Our results provided the direct evidence to show that, not all the telomeres are associated with Terra, and a significant fraction of Terra foci do not overlap with telomere DNA in interphase cell nuclei. The improved method of simultaneous RNA-DNA FISH is reliable and time-efficient. It can be used in a variety of biological studies.

Topics: Animals; Biotin; Carbocyanines; Cells, Cultured; DNA; Female; Fluorescent Antibody Technique; In Situ Hybridization, Fluorescence; Male; Mice; Models, Biological; Preservation, Biological; RNA; RNA, Long Noncoding; Signal Processing, Computer-Assisted; Specimen Handling

We report the first demonstration of surface-initiated enzymatic polymerization (SIEP) for the direct detection of RNA in a fluorescence microarray format. This new method incorporates multiple fluorophores into an RNA strand using the two-step sequential and complementary reactions catalyzed by yeast poly(A) polymerase (PaP) to incorporate deoxyadenosine triphosphate (dATP) at the 3'-OH of an RNA molecule, followed by terminal deoxynucleotidyl transferase (TdT) to catalyze the sequential addition of a mixture of natural and fluorescent deoxynucleotides (dNTPs) at the 3'-OH of an RNA-DNA hybrid. We found that the 3'-end of RNA can be efficiently converted into DNA (∼50% conversion) by polymerization of dATP using yeast PaP, and the short DNA strand appended to the end of the RNA by PaP acts as the initiator for the TdT-catalyzed polymerization of longer DNA strands from a mixture of natural and fluorescent dNTPs that contain up to ∼45 Cy3 fluorophores per 1 kb DNA. We obtained an ∼2 pM limit of detection (LOD) and a 3 log-linear dynamic range for hybridization of a short 21 base-long RNA target to an immobilized peptide nucleic acid probe, while fragmented mRNA targets from three different full length mRNA transcripts yielded a ∼10 pM LOD with a similar dynamic range in a microarray format.

Topics: Carbocyanines; Exoribonucleases; Fluorescent Dyes; Nucleic Acid Amplification Techniques; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Polymerization; RNA; Saccharomyces cerevisiae; Surface Properties

We report the rapid and highly sensitive trace analysis of mercury(ii) ions in water using a surface-enhanced Raman scattering (SERS)-based microdroplet sensor. Aptamer-modified Au/Ag core-shell nanoparticles have been fabricated and utilized as highly functional sensing probes. All detection processes for the reaction between mercury(II) ions and aptamer-modified nanoparticles were performed in a specially designed microdroplet channel. Small water droplets that included sample reagents were separated from each other by an oil phase that continuously flowed along the channel. This two-phase liquid-liquid segmented flow system prevented the adsorption of aggregated colloids to the channel walls due to localized reagents within encapsulated droplets. The result was reduced residence time distributions. The limit of detection (LOD) of mercury(II) ions in water was determined by the SERS-based microdroplet sensor to be below 10 pM, which is three orders below the EPA-defined maximum contaminant level. This combination of a SERS-based microfluidic sensor with aptamer-based functional nanoprobes can be used for in-the-field sensing platforms, due to its size and simplicity.

Topics: Aptamers, Nucleotide; Carbocyanines; Gold; Ions; Mercury; Metal Nanoparticles; Microfluidic Analytical Techniques; Oils; Silver; Spectrum Analysis, Raman; Water

A paper-based solid-phase assay is presented for transduction of nucleic acid hybridization using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) were FRET-paired with Cy3 acceptor. Hybridization of Cy3-labeled oligonucleotide targets provided the proximity required for FRET-sensitized emission from Cy3, which served as an analytical signal. The assay exhibited rapid transduction of nucleic acid hybridization within minutes. Without any amplification steps, the limit of detection of the assay was found to be 300 fmol with the upper limit of the dynamic range at 5 pmol. The implementation of glutathione-coated QDs for the development of nucleic acid hybridization assay integrated on a paper-based platform exhibited excellent resistance to nonspecific adsorption of oligonucleotides and showed no reduction in the performance of the assay in the presence of large quantities of noncomplementary DNA. The selectivity of nucleic acid hybridization was demonstrated by single-nucleotide polymorphism (SNP) detection at a contrast ratio of 19 to 1. The reuse of paper over multiple cycles of hybridization and dehybridization was possible, with less than 20% reduction in the performance of the assay in five cycles. This work provides an important framework for the development of paper-based solid-phase QD-FRET nucleic acid hybridization assays that make use of a ratiometric approach for detection and analysis.

Topics: Carbocyanines; Chromatography, Paper; Fluorescence Resonance Energy Transfer; Nucleic Acid Hybridization; Polymorphism, Single Nucleotide; Quantum Dots

Methods have been developed for the solid-phase detection of nucleic acids using mixed films of quantum dots (QDs) and oligonucleotide probes in microtiter plates. Polystyrene microwells were functionalized with multidentate imidazole ligands to immobilize QDs. Oligonucleotide hybridization was transduced using QDs as donors in fluorescence resonance energy transfer (FRET). One detection channel paired green-emitting QD donors with Cy3 acceptors and served as an internal standard. A second detection channel paired red-emitting QDs with Alexa Fluor 647 acceptors and served as the primary detection channel. A selective assay for multiple targets was demonstrated using a 96-well plate format, which combined the advantages of two-plex QD-FRET with the high-throughput capability and convenience of microtiter plates. The assay had excellent resistance to the nonspecific adsorption of DNA and discriminated between fully complementary and single base-pair mismatched sequences with a contrast ratio >2. Under optimal conditions for a single color (green QD) assay format, the limit of detection (LOD) was 4 nM, and the dynamic range was from 20 to 300 nM. In a two-color assay, the detection channel (red QD) exhibited linear response between 4 and 100 nM and a LOD of 4 nM.

Topics: Carbocyanines; Fluorescence Resonance Energy Transfer; Imidazoles; Ligands; Microarray Analysis; Nucleic Acid Hybridization; Nucleic Acids; Oligonucleotide Probes; Polystyrenes; Quantum Dots

Labeling of viruses can be used to reveal viral infection pathways and screen potential anti-viral drugs. Complex procedures, including virus cultivation, purification and labeling are involved in traditional virus labeling methods. And the manipulation of living virus brings risk to researcher health. In this work, we report a general method for site-specific labeling of the envelope virus in an integrated microfluidic device with simple procedures and high security. Site-specific labeling of virus was achieved by fusing the biotin acceptor peptide (AP-tag) and the biotin ligase enzyme (BirA enzyme) with the envelope protein GP64 of baculovirus. The AP-tag could be modified by BirA enzyme to introduce the biotin moiety onto the viral envelope. Western blots and fluorescence colocalization analysis proved that the baculoviruses were biotinylated and labeled with high efficiency. The integrated device incorporated several operation steps including cell seeding, cell culture, cell transfection, virus culture and virus labeling. Since virus biotinylation was achieved during the process of virus cultivation, the complex procedures of virus labeling were simplified in our device. Furthermore the whole process could be completed in the integrated microfluidic device, and direct contact between viruses and researchers could be eliminated in our method, which could greatly reduce the risk to researcher health during living virus labeling.

Topics: Animals; Baculoviridae; Biotin; Blotting, Western; Carbocyanines; Carbon-Nitrogen Ligases; Escherichia coli Proteins; Glycoproteins; Green Fluorescent Proteins; Microfluidic Analytical Techniques; Peptides; Repressor Proteins; Sf9 Cells; Streptavidin; Viral Envelope Proteins

During viral replication, HIV-1 reverse transcriptase (RT) plays a pivotal role in converting genomic RNA into proviral DNA. While the biologically relevant form of RT is the p66-p51 heterodimer, two recombinant homodimer forms of RT, p66-p66 and p51-p51, are also catalytically active. Here we investigate the binding of the three RT isoforms to a fluorescently labeled 19/50-nucleotide primer/template DNA duplex by exploiting single-molecule protein-induced fluorescence enhancement (SM-PIFE). PIFE, which does not require labeling of the protein, allows us to directly visualize the binding/unbinding of RT to a double-stranded DNA substrate. We provide values for the association and dissociation rate constants of the RT homodimers p66-p66 and p51-p51 with a double-stranded DNA substrate and compare those to the values recorded for the RT heterodimer p66-p51. We also report values for the equilibrium dissociation constant for the three isoforms. Our data reveal great similarities in the intrinsic binding affinities of p66-p51 and p66-p66, with characteristic Kd values in the nanomolar range, much smaller (50-100-fold) than that of p51-p51. Our data also show discrepancies in the association/dissociation dynamics among the three dimeric RT isoforms. Our results further show that the apparent binding affinity of p51-p51 for its DNA substrate is to a great extent time-dependent when compared to that of p66-p66 and p66-p51, and is more likely determined by the dimer dissociation into its constituent monomers rather than the intrinsic binding affinity of dimeric RT.

Topics: Carbocyanines; DNA; DNA Primers; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; HIV Reverse Transcriptase; Humans; Kinetics; Nucleic Acid Hybridization; Protein Binding; Protein Isoforms; Recombinant Proteins; RNA; Substrate Specificity

Large-scale RNAi-based screens are a major technology, but require adequate prioritization and validation of candidate genes from the primary screen. In this work, we performed a large-scale pooled shRNA screen in mouse embryonic stem cells (ESCs) to discover genes associated with oxidative stress resistance and found several candidates. We then developed a bioinformatics pipeline to prioritize these candidates incorporating effect sizes, functional enrichment analysis, interaction networks and gene expression information. To validate candidates, we mixed normal cells with cells expressing the shRNA coupled to a fluorescent protein, which allows control cells to be used as an internal standard, and thus we could detect shRNAs with subtle effects. Although we did not identify genes associated with oxidative stress resistance, as a proof-of-concept of our pipeline we demonstrate a detrimental role of Edd1 silencing in ESC growth. Our methods may be useful for candidate gene prioritization of large-scale RNAi-based screens.

Topics: Animals; Carbocyanines; Embryonic Stem Cells; Mice; Oligonucleotide Array Sequence Analysis; Oxidative Stress; RNA Interference; RNA, Small Interfering

To optimize cellular delivery and specific labeling of tagged cytosolic proteins by biarsenical fluorescent probes built around a cyanine dye (Cy3) scaffold, we have systematically varied the polarity of the N-alkyl chain (i.e., 4-5 methylene groups appended by a sulfonate or methoxy ester moiety) and arsenic capping reagent (ethanedithiol versus benzenedithiol). Optimal live-cell labeling and visualization of tagged cytosolic proteins is reported using an ethanedithiol capping reagent with the uncharged methoxy ester functionalized N-alkyl chains. These measurements demonstrate the general utility of this new class of photostable and highly fluorescent biarsenical probes based on the cyanine dye scaffold for in vivo labeling of tagged cellular proteins for live cell imaging measurements of protein dynamics.

Topics: Amino Acid Sequence; Carbocyanines; DNA-Directed RNA Polymerases; Escherichia coli; Escherichia coli Proteins; Fluorescent Dyes; Microscopy, Fluorescence; Permeability; Spectrometry, Fluorescence; Staining and Labeling

2'-Deoxyinosine (1) and thymidine (7) were N-alkylated with geranyl and farnesyl moieties. These hydrophobic derivatives, 3a and 3b, and 9a and 9b, respectively, represent the first synthetic biomimetic nucleoterpenes and were subsequently 5'-protected and converted into the corresponding 3'-O-phosphoramidites, 5a and 5b and 11a and 11b, respectively. The latter were used to prepare a series of lipophilized oligonucleotide dodecamers, a part of which were additionally labelled with indocarbocyanine fluorescent dyes (Cy3 or Cy5), 18-23. The insertion of the lipooligonucleotides into, as well as duplex formation at artificial lipid bilayers was studied by single-molecule fluorescence spectroscopy and fluorescence microscopy.

Topics: Biomimetic Materials; Carbocyanines; Inosine; Lipid Bilayers; Microscopy, Fluorescence; Oligonucleotides; Spectrometry, Fluorescence; Thymidine

M2 muscarinic acetylcholine receptors modulate cardiac rhythm via regulation of the inward potassium current. To increase our understanding of M2 receptor physiology we used Total Internal Reflection Fluorescence Microscopy to visualize individual receptors at the plasma membrane of transformed CHO(M2) cells, a cardiac cell line (HL-1), primary cardiomyocytes and tissue slices from pre- and post-natal mice. Receptor expression levels between individual cells in dissociated cardiomyocytes and heart slices were highly variable and only 10% of murine cardiomyocytes expressed muscarinic receptors. M2 receptors were evenly distributed across individual cells and their density in freshly isolated embryonic cardiomyocytes was ~1μm(-2), increasing at birth (to ~3μm(-2)) and decreasing back to ~1μm(-2) after birth. M2 receptors were primarily monomeric but formed reversible dimers. They diffused freely at the plasma membrane, moving approximately 4-times faster in heart slices than in cultured cardiomyocytes. Knowledge of receptor density and mobility has allowed receptor collision rate to be modeled by Monte Carlo simulations. Our estimated encounter rate of 5-10 collisions per second, may explain the latency between acetylcholine application and GIRK channel opening.

Topics: Animals; Carbocyanines; CHO Cells; Cricetinae; Fluorescent Dyes; Mice; Microscopy, Fluorescence; Myocardium; Myocytes, Cardiac; Organ Specificity; Primary Cell Culture; Protein Structure, Quaternary; Protein Transport; Receptor, Muscarinic M2; Staining and Labeling

Reversible disulfide oxidation between proximal cysteines in proteins represents a common regulatory control mechanism to modulate flux through metabolic pathways in response to changing environmental conditions. To enable in vivo measurements of cellular redox changes linked to disulfide bond formation, we have synthesized a cell-permeable thiol-reactive affinity probe (TRAP) consisting of a monosubstituted cyanine dye derivatized with arsenic (i.e., TRAP_Cy3) to trap and visualize dithiols in cytosolic proteins. Alkylation of reactive thiols prior to displacement of the bound TRAP_Cy3 by ethanedithiol permits facile protein capture and mass spectrometric identification of proximal reduced dithiols to the exclusion of individual cysteines. Applying TRAP_Cy3 to evaluate cellular responses to increases in oxygen and light levels in the photosynthetic microbe Synechococcus sp. PCC7002, we observe large decreases in the abundance of reduced dithiols in cellular proteins, which suggest redox-dependent mechanisms involving the oxidation of proximal disulfides. Under these same growth conditions that result in the oxidation of proximal thiols, there is a reduction in the abundance of post-translational oxidative protein modifications involving methionine sulfoxide and nitrotyrosine. These results suggest that the redox status of proximal cysteines responds to environmental conditions, acting to regulate metabolic flux and minimize the formation of reactive oxygen species to decrease oxidative protein damage.

Topics: Arsenic; Carbocyanines; Fluorescent Dyes; Molecular Structure; Oxidation-Reduction; Sulfhydryl Compounds; Synechococcus

Recently, we have demonstrated that DNA hybridization using acoustic streaming induced by two piezoelectric transducers provides higher DNA hybridization efficiency than the conventional method. In this work, we refine acoustic streaming system for DNA hybridization by inserting an additional piezoelectric transducer and redesigning the locations of the transducers. The Comsol® Multiphysics was used to design and simulate the velocity field generated by the piezoelectric agitation. The simulated velocity vector followed a spiral vortex flow field with an average direction outward from the center of the transducers. These vortices caused the lower signal intensity in the middle of the microarray for the two-piezoelectric disk design. On the contrary, the problem almost disappeared in the three-piezoelectric-disk system. The optimum condition for controlling the piezoelectric was obtained from the dye experiments with different activation settings for the transducers. The best setting was to activate the side disks and middle disk alternatively with 1 second activating time and 3 second non-activating time for both sets of transducers. DNA hybridization using microarrays for the malaria parasite Plasmodium falciparum from the optimized process yielded a three-fold enhancement of the signal compared to the conventional method. Moreover, a greater number of spots passed quality control in the optimized device, which could greatly improve biological interpretation of DNA hybridization data.

Topics: Acoustics; Carbocyanines; DNA Probes; DNA, Protozoan; Finite Element Analysis; Models, Molecular; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Plasmodium falciparum; Signal Processing, Computer-Assisted; Transducers

The development of rapid detection assays for malaria diagnostics is an area of intensive research, as the traditional microscopic analysis of blood smears is cumbersome and requires skilled personnel. Here, we describe a simple and sensitive immunoassay that successfully detects malaria antigens in infected blood cultures. This homogeneous assay is based on the fluorescence quenching of cyanine 3B (Cy3B)-labeled recombinant Plasmodium falciparum heat shock protein 70 (PfHsp70) upon binding to gold nanoparticles (AuNPs) functionalized with an anti-Hsp70 monoclonal antibody. Upon competition with the free antigen, the Cy3B-labeled recombinant PfHsp70 is released to solution resulting in an increase of fluorescence intensity. Two types of AuNP-antibody conjugates were used as probes, one obtained by electrostatic adsorption of the antibody on AuNPs surface and the other by covalent bonding using protein cross-linking agents. In comparison with cross-linked antibodies, electrostatic adsorption of the antibodies to the AuNPs surfaces generated conjugates with increased activity and linearity of response, within a range of antigen concentration from 8.2 to 23.8 μg.mL(-1). The estimated LOD for the assay is 2.4 μg.mL(-1) and the LOQ is 7.3 μg.mL(-1). The fluorescence immunoassay was successfully applied to the detection of antigen in malaria-infected human blood cultures at a 3% parasitemia level, and is assumed to detect parasite densities as low as 1,000 parasites.μL(-1).

Topics: Antibodies, Monoclonal; Antigens, Protozoan; Carbocyanines; Fluorescent Antibody Technique; Fluorescent Dyes; Gold; HSP70 Heat-Shock Proteins; Humans; Malaria; Nanoparticles; Plasmodium falciparum; Protozoan Proteins; Recombinant Proteins; Sensitivity and Specificity

Integration of viral DNA into the host genome is an essential step in retroviral replication that is mediated by a stable nucleoprotein complex comprising a tetramer of integrase bridging the two ends of the viral DNA in a stable synaptic complex (SSC) or intasome. Assembly of HIV-1 intasomes requires several hundred base pairs of nonspecific internal DNA in addition to the terminal viral DNA sequence that is protected in footprinting experiments. We find that only one of the viral DNA ends in the intasome requires long-nonspecific internal DNA for intasome assembly. Although intasomes are unstable in solution when the nonspecific internal DNA is cut off after assembly, they are stable in agarose gels. These complexes are indistinguishable from SSCs with nonspecific internal DNA in Förster resonance energy transfer (FRET) experiments suggesting the interactions with the viral DNA and integrase tetramer are the same regardless of the presence of nonspecific internal DNA. We discuss models of how the internal DNA contributes to intasome assembly and stability. FRET is exquisitely sensitive to the distance between the fluorophores and given certain assumptions can be translated to distance measurements. We anticipated that a set of such distance constraints would provide a map of the DNA path within the intasome. In reality, the constraints we could impose from the FRET data were quite weak allowing a wide envelope for the possible path. We discuss the difficulties of converting the FRET signal to absolute distance within nucleoprotein complexes.

Topics: Base Sequence; Carbocyanines; DNA; DNA Footprinting; DNA, Viral; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; HIV-1; Humans; Integrases; Protein Multimerization; Protein Stability; Virus Integration

Intensity fluctuations between an ON-state and an OFF-state, also called blinking, are common to all luminescent objects when studied at the level of individuals. We studied blinking of three dyes from a homologous series (Cy3, Cy5, Cy7). The underlying radical anion states were induced by removing oxidants (i.e. oxygen) and by adding the reductant ascorbic acid. We find that for different conditions with distinct levels of oxidants in solution the OFF-state lifetime always increases in the order Cy3

Topics: Carbocyanines; Oxidation-Reduction; Oxygen; Spectrometry, Fluorescence; Temperature

Single molecule experiments were used to investigate the photobleaching mechanism of single Cy3 molecules encapsulated in large unilamellar vesicles. Analysis of thousands of individual fluorescence traces evidenced that the survival probability distribution is monoexponential with an average survival time depending on the laser intensity, the concentration of molecular oxygen, the temperature and the rigidity of the medium. Taken together, our data suggest that the photobleaching of Cy3 molecules is governed by two parallel pathways, namely photooxidation and a thermally activated structural rearrangement of the Cy3 molecules in their excited state.

Topics: Carbocyanines; Photobleaching; Spectrometry, Fluorescence; Temperature; Thermodynamics; Unilamellar Liposomes

The goal of the study was to assess the efficacy of combined extracellular matrix metalloprotease inducer (EMMPRIN)- and death receptor 5 (DR5)-targeted therapy for pancreatic adenocarcinoma in orthotopic mouse models with multimodal imaging. Cytotoxicity of anti-EMMPRIN antibody and anti-DR5 antibody (TRA-8) in MIA PaCa-2 and PANC-1 cell lines was measured by ATPlite assay in vitro. The distributions of Cy5.5-labeled TRA-8 and Cy3-labeled anti-EMMPRIN antibody in the 2 cell lines were analyzed by fluorescence imaging in vitro. Groups 1 to 12 of severe combined immunodeficient mice bearing orthotopic MIA PaCa-2 (groups 1-8) or PANC-1 (groups 9-12) tumors were used for in vivo studies. Dynamic contrast-enhanced-MRI was applied in group 1 (untreated) or group 2 (anti-EMMPRIN antibody). The tumor uptake of Tc-99m-labeled TRA-8 was measured in group 3 (untreated) and group 4 (anti-EMMPRIN antibody). Positron emission tomography/computed tomography imaging with (18)F-FDG was applied in groups 5 to 12. Groups 5 to 8 (or groups 9 to 12) were untreated or treated with anti-EMMPRIN antibody, TRA-8, and combination, respectively. TRA-8 showed high killing efficacy for both MIA PaCa-2 and PANC-1 cells in vitro, but additional anti-EMMPRIN treatment did not improve the cytotoxicity. Cy5.5-TRA-8 formed cellular caps in both the cell lines, whereas the maximum signal intensity was correlated with TRA-8 cytotoxicity. Anti-EMMPRIN therapy significantly enhanced the tumor delivery of the MR contrast agent, but not Tc-99m-TRA-8. Tumor growth was significantly suppressed by the combination therapy, and the additive effect of the combination was shown in both MIA PaCa-2 and PANC-1 tumor models.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Basigin; Carbocyanines; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Drug Therapy, Combination; Female; Fluorodeoxyglucose F18; Humans; Magnetic Resonance Imaging; Mice; Mice, Inbred BALB C; Mice, SCID; Microscopy, Fluorescence; Multimodal Imaging; Pancreatic Neoplasms; Positron-Emission Tomography; Receptors, TNF-Related Apoptosis-Inducing Ligand; Tomography, X-Ray Computed; Xenograft Model Antitumor Assays

In the present study, we report a novel aptasensor based on silver nanoparticle enhanced fluorescence for the detection of adenosine. First, the distance dependence nature of silver nanoparticle enhanced fluorescence was investigated through fluorescent dyes modified oligonucleotides to control the spacing distance between dyes and AgNP. The results showed that the fluorescence intensity reached the maximum value with the spacing distance of dyes about 8 nm from AgNP surface. The fluorescence intensity decreases when the spacing distance is either above or below this value. Based on this result, a fluorescence switch is constructed. In the "OFF" state, without the target molecules, there is a greater spacing distance between the Cy3 dyes and the AgNP giving comparatively lower fluorescence intensity. While in the "ON" state, in the presence of target molecules, the fluorescence signals increased for the conformation structure change of the aptamer which shorten the spacing distance between the Cy3 dyes and the AgNP to 8 nm. Using adenosine as target, the aptasensor produced a linear range from 200 nM to 200 μM with a correlation coefficient of 0.9949 and the detection limit was 48 nM estimated using 3σ. The aptasensor was also found to be specific in targeting adenosine. The presented method shows a new strategy of combining aptamer recognition and silver nanoparticle for fluorescence signal enhancement and increasing sensitivity.

Topics: Adenosine; Aptamers, Nucleotide; Biosensing Techniques; Carbocyanines; Equipment Design; Fluorescence; Fluorescent Dyes; Limit of Detection; Metal Nanoparticles; Silver

MicroRNAs (miRNAs) are short noncoding ribonucleic acids known to affect gene expression at the translational level and there is mounting evidence that miRNAs play a role in the function of tumor-associated macrophages (TAMs). To aid the functional analyses of miRNAs in an in-vitro model of TAMs known as M2 macrophages, a transfection method to introduce artificial miRNA constructs or miRNA molecules into primary human monocytes is needed. Unlike differentiated macrophages or dendritic cells, undifferentiated primary human monocytes have been known to show resistance to lentiviral transduction. To circumvent this challenge, other techniques such as electroporation and chemical transfection have been used in other applications to deliver small gene constructs into human monocytes. To date, no studies have compared these two methods objectively to evaluate their suitability in the miRNA functional analysis of M2 macrophages. Of the methods tested, the electroporation of miRNA-construct containing plasmids and the chemical transfection of miRNA precursor molecules are the most efficient approaches. The use of a silencer siRNA labeling kit (Ambion) to conjugate Cy 3 fluorescence dyes to the precursor molecules allowed the isolation of successfully transfected cells with fluorescence-activated cell sorting. The chemical transfection of these dye-conjugated miRNA precursors yield an efficiency of 37.5 ± 0.6% and a cell viability of 74 ± 1%. RNA purified from the isolated cells demonstrated good quality, and was fit for subsequent mRNA expression qPCR analysis. While electroporation of plasmids containing miRNA constructs yield transfection efficiencies comparable to chemical transfection of miRNA precursors, these electroporated primary monocytes seemed to have lost their potential for differentiation. Among the most common methods of transfection, the chemical transfection of dye-conjugated miRNA precursors was determined to be the best-suited approach for the functional analysis of M2 macrophages.

Topics: Carbocyanines; Cell Differentiation; Cell Line, Tumor; Cell Survival; Cells, Cultured; Electroporation; Fluorescent Dyes; Humans; Macrophages; MicroRNAs; Monocytes; RNA Precursors; Transfection; U937 Cells

Experiments in rodents revealed neuropeptide S (NPS) to constitute a potential novel treatment option for anxiety diseases such as panic and post-traumatic stress disorder. However, both its cerebral target sites and the molecular underpinnings of NPS-mediated effects still remain elusive. By administration of fluorophore-conjugated NPS, we pinpointed NPS target neurons in distinct regions throughout the entire brain. We demonstrated their functional relevance in the hippocampus. In the CA1 region, NPS modulates synaptic transmission and plasticity. NPS is taken up into NPS receptor-expressing neurons by internalization of the receptor-ligand complex as we confirmed by subsequent cell culture studies. Furthermore, we tracked internalization of intranasally applied NPS at the single-neuron level and additionally demonstrate that it is delivered into the mouse brain without losing its anxiolytic properties. Finally, we show that NPS differentially modulates the expression of proteins of the glutamatergic system involved inter alia in synaptic plasticity. These results not only enlighten the path of NPS in the brain, but also establish a non-invasive method for NPS administration in mice, thus strongly encouraging translation into a novel therapeutic approach for pathological anxiety in humans.

Topics: Analysis of Variance; Animals; Anti-Anxiety Agents; Anxiety; CA1 Region, Hippocampal; Carbocyanines; Cell Line, Transformed; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Electrophysiology; Evoked Potentials; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Humans; In Vitro Techniques; Intravitreal Injections; Male; Maze Learning; Mice; Mice, Inbred C57BL; Neurofilament Proteins; Neurons; Neuropeptides; Oxazolidinones; Protein Transport; Pyrazines; Receptors, AMPA; Receptors, Neuropeptide; Rhodamines; RNA, Messenger; Synapsins; Transfection

Tuning the luminescence intensity of fluorophores using nanoparticles has shown great potential for the detection of inorganic metal ions, viruses, and proteins. The enhancement or quenching of a dye's fluorescence intensity is strongly dependent on the spatial separation of the dye from the nanoparticle surface. To extend luminescence probing from the solution platform to the solid-state platform, we explored and performed dye quenching assessment using an array format in this study. We report the distance-dependent fluorescence behavior of Au-DNA conjugates prepared by equilibrating phosphine-stabilized gold nanoparticles (AuNPs) of 10-nm size with the designed spacer ds-DNA consisting of thiol-modified target and Cy3-labeled complementary probe of different lengths (5-20 nm). The Cy3-labeled products were immobilized onto MPTMS (3-mercaptopropyltrimethoxysilane)-modified glass substrates and then excited with a 532-nm laser source. Quenching efficiency of AuNPs with increasing Au-to-dye distance was assessed using ligand exchange of the thiolated oligonucleotide by 2-mercaptoethanol (ME) to obtain free Cy3-DNA probe, thus eliminating nanoparticle effect on the dye's luminescence intensity. Effective exchange, revealed by UV-vis absorption and fluorescence profiles, was achieved in a few minutes. It was observed that fluorescence quenching of Au-DNA-Cy3 assessed using the array format was consistent with the result in solution phase for the conjugates with up to 10-nm Au-to-Cy3 separation distance.

Topics: Carbocyanines; Coated Materials, Biocompatible; DNA; Fluorescent Dyes; Gold; Metal Nanoparticles; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Solutions

Topics: Animals; Carbocyanines; Dendrimers; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; HeLa Cells; Humans; Microscopy, Fluorescence; Nanoparticles; Ultraviolet Rays; Zebrafish

Degradation of mRNA by RNA interference is one of the most powerful and specific mechanisms for gene silencing. However, insufficient cellular uptake and poor stability have limited its usefulness. Here, we report efficient delivery of siRNA via the use of biodegradable nanoparticles (NPs) made from monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly-l-lysine (mPEG-PLGA-PLL) triblock copolymers. Various physicochemical properties of mPEG-PLGA-PLL NPs, including morphology, size, surface charge, siRNA encapsulation efficiency, and in vitro release profile of siRNA from NPs, were characterized by scanning electron microscope, particle size and zeta potential analyzer, and high performance liquid chromatography. The levels of siRNA uptake and targeted gene inhibition were detected in human lung cancer SPC-A1-GFP cells stably expressing green fluorescent protein. Examination of the cultured SPC-A1-GFP cells with fluorescent microscope and flow cytometry showed NPs loading Cy3-labeled siRNA had much higher intracellular siRNA delivery efficiencies than siRNA alone and Lipofectamine-siRNA complexes. The gene silencing efficiency of mPEG-PLGA-PLL NPs was higher than that of commercially available transfecting agent Lipofectamine while showing no cytotoxicity. Thus, the current study demonstrates that biodegradable NPs of mPEG-PLGA-PLL triblock copolymers can be potentially applied as novel non-viral vectors for improving siRNA delivery and gene silencing.

Topics: Biocompatible Materials; Carbocyanines; Cell Line, Tumor; Cell Survival; Drug Carriers; Humans; Lipids; Microscopy, Fluorescence; Nanoparticles; Particle Size; Polyesters; Polyethylene Glycols; Polyglactin 910; RNA Interference; RNA, Small Interfering; Transfection

The glassy winged sharpshooter, Homalodisca vitripennis, is an unusually robust and efficient leafhopper vector of Xylella fastidiosa. X. fastidiosa is the causative agent of Pierce's disease, almond scorch, citrus variegated chlorosis and other serious plant diseases. The present study was conducted to establish whether RNA interference (RNAi) was induced in nymphal H. vitripennis that were injected with actin dsRNAs and other dsRNAs.. A dramatic reduction in target H. vitripennis actin mRNAs and the formation of small interfering RNAs (siRNAs), hallmarks of RNAi, were found following the injection of actin dsRNAs. Quantitative reverse transcription PCR indicated an 80% reduction in actin mRNA levels by 5 days post-injection. Western blot analysis showed a dramatic drop in actin protein levels by 3 days post-injection. Biological effects such as incomplete nymphal-adult ecdysis and > 95% mortality were also found following the injection of fifth-instar nymphs with actin dsRNA. Dramatic reductions in target mRNA levels were also found following the injection of other dsRNAs into fifth-instar H. vitripennis.. The findings indicate that RNAi is induced in post-embryonic leafhoppers by dsRNA. The present system can be used to screen potential gene-silencing targets that can be used for reducing the vector competence of H. vitripennis and other leafhoppers.

Topics: Actins; Animals; Carbocyanines; Genetic Engineering; Hemiptera; Injections; Nymph; RNA Interference; RNA, Double-Stranded; RNA, Messenger

A near-infrared fluorochrome, GPU-311, was designed, synthesized and evaluated for its application in non-invasive imaging of tumor hypoxia. Efficient synthesis was achieved by nucleophilic substitution and click chemistry ring using the bifunctional tetraethylene glycol linker 2 containing thiol and azide groups for the conjugation of the propargylated nitroimidazole 1 and the heptamethine cyanine dye 3 bearing a 2-chloro-1-cyclohexenyl ring. GPU-311 exhibited long excitation and emission wavelength (Ex/Em=785/802 nm) and a decent quantum yield (0.05). The water solubility and hydrophilicity of GPU-311 increased. After in vitro treatment of SUIT-2/HRE-Luc pancreatic cancer cells with GPU-311, a higher level of fluorescence was observed selectively in hypoxia than in normoxia. However, in vivo fluorescence imaging of a mouse xenograft model after GPU-311 administration revealed inadequate accumulation of GPU-311 in tumors due to its rapid elimination through the liver.

Topics: Animals; Carbocyanines; Cell Hypoxia; Cell Line, Tumor; Fluorescence; Fluorescent Dyes; Humans; Hydrophobic and Hydrophilic Interactions; Mice; Neoplasms; Nitroimidazoles; Solubility; Spectroscopy, Near-Infrared

Biomolecules inside a microfluidic system can be used to solve computational problems, such as theorem proving, which is an important class of logical reasoning problems. In this article, the Boolean variables (literals) were represented using single-stranded DNA molecules, and theorem proving was performed by the hybridization and ligation of these variables into a double-stranded "solution" DNA. Then, a novel sequential reaction mixing method in a microfluidic chip was designed to solve a theorem proving problem, where a reaction loop and three additional chambers were integrated and controlled by pneumatic valves. DNA hybridization, ligation, toehold-mediated DNA strand displacement, exonuclease I digestion, and fluorescence detection of the double-stranded DNA were sequentially performed using this platform. Depending on the computational result, detection of the correct answer was demonstrated based on the presence of a fluorescence signal. This result is the first demonstration that microfluidics can be used to facilitate DNA-based logical inference.

Topics: Carbocyanines; Computers, Molecular; DNA, Single-Stranded; Equipment Design; Fluorescent Dyes; Logic; Microfluidic Analytical Techniques; Models, Chemical; Spectrometry, Fluorescence

Cy3B is an extremely bright and stable fluorescent dye, which is only available for coupling to nucleic acids post-synthetically. This severely limits its use in the fields of genomics, biology and nanotechnology. We have optimized the synthesis of Cy3B, and for the first time produced a diverse range of Cy3B monomers for use in solid-phase oligonucleotide synthesis. This molecular toolkit includes phosphoramidite monomers with Cy3B linked to deoxyribose, to the 5-position of thymine, and to a hexynyl linker, in addition to an oligonucleotide synthesis resin in which Cy3B is linked to deoxyribose. These monomers have been used to incorporate single and multiple Cy3B units into oligonucleotides internally and at both termini. Cy3B Taqman probes, Scorpions and HyBeacons have been synthesized and used successfully in mutation detection, and a dual Cy3B Molecular Beacon was synthesized and found to be superior to the corresponding Cy3B/DABCYL Beacon. Attachment of Cy3, Cy3B and Cy5 to the 5-position of thymidine by an ethynyl linker enabled the synthesis of an oligonucleotide FRET system. The rigid linker between the dye and nucleobase minimizes dye-dye and dye-DNA interactions and reduces fluorescence quenching. These reagents open up new future applications of Cy3B, including more sensitive single-molecule and cell-imaging studies.

Topics: Benzenesulfonates; Carbocyanines; DNA Probes; Fluorescent Dyes; Oligonucleotide Probes; Organophosphorus Compounds; Polymerase Chain Reaction

Free flow electrophoresis (FFE) has been applied in numerous studies as a protein separation technique due to its multiple advantages such as fast and efficient sample recovery, high resolving power, high reproducibility, and wide applicability to protein classes. As a stand-alone platform however, its utility in comparative proteomic analysis is limited as protein samples must be run sequentially rather than simultaneously which introduces inherent variability when attempting to perform quantitative analysis. Here we describe an approach combining fluorescent CyDye technology (DIGE) with FFE to simultaneously separate and identify differentially expressed proteins in a model cell system.

Topics: Buffers; Carbocyanines; Cell Extracts; Denaturing Gradient Gel Electrophoresis; Densitometry; Fluorescent Dyes; HT29 Cells; Humans; Hydrogen-Ion Concentration; Image Processing, Computer-Assisted; Isoelectric Point; Proteins; Staining and Labeling

Two-dimensional difference gel electrophoresis (2D DIGE) is a modified form of 2D electrophoresis (2DE) that allows one to compare two or three protein samples simultaneously on the same gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis. Proteins that are common to the samples appear as "spots" with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios. With the appropriate imaging system, difference gel electrophoresis (DIGE) is capable of reliably detecting as little as 0.2 fmol of protein, and protein differences down to ±15%, over a ∼20,000-fold protein concentration range. DIGE combined with digital image analysis therefore greatly improves the statistical assessment of proteome variation. Here we describe a protocol for conducting DIGE experiments, which takes 2-3 days to complete.

Topics: Animals; Buffers; Carbocyanines; Cell Extracts; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Proteins; Staining and Labeling; Two-Dimensional Difference Gel Electrophoresis

Bio-switchable optofluidic lasers based on DNA Holliday junctions were demonstrated. Nearly 100% wavelength switching was achieved through reversible conformational change of the Holliday junction controlled by magnesium ionic strength.

Topics: Carbocyanines; DNA, Cruciform; Lasers; Magnesium; Microfluidic Analytical Techniques; Nucleic Acid Conformation; Optics and Photonics; Osmolar Concentration

The copper(I) catalyzed azide-alkyne cycloaddition 'click' reaction yields a specific product under mild conditions and in some of the most chemically complex environments. This reaction has been used extensively to tag DNA, proteins, glycans and only recently RNA. Click reactions in aqueous buffer typically include a ligand for Cu(I), however we find that acetonitrile as a minor co-solvent can serve this role. Here we investigate the click labeling of RNA and DNA in aqueous buffer to determine the relationship between the stoichoimetry of Cu(I) and the acetonitrile co-solvent that affects nucleic acid stability. We find that very low concentrations of acetonitrile perform equally well and obviate the need for any additional Cu(I) stabilizing ligand. These pseudo-ligandless reaction conditions are optimal for nucleic acids click conjugations.

Topics: Acetonitriles; Alkynes; Azides; Carbocyanines; Catalysis; Click Chemistry; Copper; Cyclization; DNA; Ligands; RNA; Solvents

The micro-viscosity and molecular crowding experienced by specific proteins can regulate their dynamics and function within live cells. Taking advantage of the emerging TMP-tag technology, we present the design, synthesis and application of a hybrid genetic-chemical molecular rotor probe whose fluorescence lifetime can report protein-specific micro-environments in live cells.

Topics: Carbocyanines; Cell Survival; Escherichia coli; Fluorescent Dyes; HEK293 Cells; Histones; Humans; Microscopy, Fluorescence; Recombinant Fusion Proteins; Tetrahydrofolate Dehydrogenase; Transfection; Trimethoprim; Viscosity

Quantitation of poly(A)-RNA, time-dependent visualization of intracellular poly(A)(+)-RNA localization in living mammalian cells, and time-resolved intracellular binding dynamics of molecular beacons at the single-molecule level using a fluorescence resonance energy transfer (FRET)-based molecular beacon are described. FRET-based molecular beacons were designed as poly(A)-targeting probes to be oligonucleotides that contained Cy5 and Cy3 fluorescent dyes at the strand ends and a poly(A)-targeting sequence inside the strand. Our ratiometric analysis using poly(A)-targeting probes allowed for highly specific and wide-ranging detection (from 1.25nM to 0.5μM) of poly(A)-RNA, as well as for determination of K(d) values, and revealed a distribution of the probe itself and localization of the target RNA sequence in cells. Furthermore, time-dependent FRET-mediated fluorescence changes at the single-molecule level caused by the folding-induced gradual conformation changes in live cells were observed.

Topics: Carbocyanines; Fluorescence Resonance Energy Transfer; HeLa Cells; Humans; Microscopy, Fluorescence; Nucleic Acid Hybridization; Oligonucleotide Probes; Poly A; RNA, Messenger; Time Factors

Asymmetric dye clusters with a single fluorophore (Cy3) and multiple quenchers (4'-methylthioazobenzene-4-carboxylate, methyl red, and 4'-dimethylamino-2-nitroazobenzene-4-carboxylate) were prepared. The dye and one-to-five quenchers were tethered through D-threoninol to opposite strands of a DNA duplex. NMR analysis revealed that the clusters with a single fluorophore and two quenchers formed a sandwich-like structure (antiparallel H-aggregates). The melting temperatures of all the heteroclusters were almost the same, although structural distortion should become larger, as the number of quenchers increased. An asymmetric heterocluster of a single fluorophore and two quenchers showed larger excitonic interaction (i.e., hypochromicity of Cy3), than did a single Cy3 and a single quencher. Due to the larger exciton coupling between the dyes, the 1:2 heterocluster suppressed the background emission more efficiently than the 1:1 cluster. However, more quenchers did not enhance quenching efficiency due to the saturation of exciton coupling with two quenchers. Finally, this asymmetric 1:2 heterocluster was introduced into the stem region of a molecular beacon (MB; also known as an in-stem MB) targeting the fusion site in the L6 BCR-ABL fusion gene. With this MB design, the signal/background ratio was as high as 68 due to efficient suppression of background emission resulting from the maximized excitonic interaction.

Topics: Amino Alcohols; Azo Compounds; Base Sequence; Butylene Glycols; Carbocyanines; Carboxylic Acids; DNA; Fluorescent Dyes; Genes, abl; Humans; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Conformation; Molecular Probes; Molecular Sequence Data; Nucleic Acid Denaturation; Organic Chemicals; Spectrometry, Fluorescence; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Temperature

We introduce a simple and effective method to tailor the wetting and adhesion properties of thiolene-based microfluidic devices. This one-step lithographic scheme combines most of the advantages offered by the current methods employed to pattern microchannels: (i) the channel walls can be modified in situ or ex situ, (ii) their wettability can be varied in a continuous manner, (iii) heterogeneous patterning can be easily accomplished, with contact-angle contrasts extending from 0 to 90° for pure water, (iv) the surface modification has proven to be highly stable upon aging and heating. We first characterize the wetting properties of the modified surfaces. We then provide the details of two complementary methods to achieve surface patterning. Finally, we demonstrate the two methods with three examples of applications: the capillary guiding of fluids, the production of double emulsions, and the culture of cells on adhesive micropatterns.

Topics: Animals; Carbocyanines; Cell Adhesion; Cells, Cultured; Fibronectins; Mice; Microfluidic Analytical Techniques; Microscopy, Fluorescence; Porosity; Surface Properties; Wettability

Sequencing-by-ligation (SBL) is one of the next-generation sequencing methods for massive parallel sequencing. The ligated probes used in SBL should be accurately cleaved for a better ligation in the next cycle. Here, a novel kind of oligonucleotide probe that could be accurately cleaved at the given position was proposed. Deoxynucleoside phosphorothioates were introduced into the deoxyoxanosine-containing oligonucleotide probes in order to increase the cleavage accuracy of endonuclease V on double-stranded DNA templates. The results illustrated that incorporating deoxynucleoside phosphorothioates could greatly reduce the effect of the nonsynchronous sequencing primer, and the queried bases of the DNA templates were unambiguously identified with 5 cycles of sequencing ligations. Additionally, the read length can reach up to 25 bp with high accuracy. The SBL-based method is inexpensive, has high-throughput, and is easy to operate allowing massive scale-up, miniaturization and automation.

Topics: Carbocyanines; Deoxyribonuclease (Pyrimidine Dimer); DNA Cleavage; Inosine; Oligonucleotide Probes; Phosphorothioate Oligonucleotides; Sequence Analysis, DNA

FRET has been used as a powerful tool in biological fields as biosensors, bioimaging, protein folding/unfolding monitoring, biomolecular interactions, and so on. It is also important to applying FRET to high hydrostatic pressure studies on biosystems or biorelated systems. Herein, we construct a FRET system by labeling Cy3 on C-phycocyanin (C-PC) to investigate the effect of hydrostatic pressure on the fluorescence and FRET behavior between them. The fluorescence spectra of individual Cy3, C-PC, and integrated Cy3/C-PC system are measured separately under compression. An enhanced FRET efficiency under compression is concluded based on fluorescence behavior differences between them. To further reveal the origination of the enhanced FRET efficiency with pressure, the overlap integral between Cy3 emission and C-PC absorption is also calculated, and several possible explanations are proposed.

Topics: Carbocyanines; Fluorescence; Fluorescence Resonance Energy Transfer; Hydrostatic Pressure; Models, Molecular; Phycocyanin

Single-molecule fluorescence methods offer the promise of ultrasensitive detection of biomolecules, but the passive immobilization methods commonly employed require analyte concentrations in the picomolar range. Here, we demonstrate that superparamagnetic Fe(3)O(4) nanoparticles (NPs) can be used with an external magnetic field as a simple strategy to enhance the immobilization efficiency and thereby decrease the detection limit. Inorganic NPs functionalized with streptavidin were bound to biotinylated single-stranded DNA oligonucleotides, which were in turn annealed to complementary oligonucleotides labeled with a Cy3 fluorescent dye. Using an external magnetic field, the superparamagnetic nanoparticles were localized to a specific region within the flow chamber surface. From the single-molecule fluorescence time traces, single-step photobleaching indicated that the surface-immobilized NPs were primarily bound with a single Cy3-labeled oligonucleotide. This strategy gave a concentration detection limit for the Cy3-labeled oligonucleotide of 100aM, 3000-fold lower than that from an analogous strategy with passive immobilization. With a sample volume of 25μl, this method achieved a mole detection limit of approximately 2.5zmol (∼1500 molecules). Together, the results support that idea that single-molecule fluorescence methods could be used for biological applications such as detection and measurements of nucleic acids from biological or clinical samples without polymerase chain reaction amplification.

Topics: Carbocyanines; DNA; Ferrosoferric Oxide; Fluorescent Dyes; Immobilized Nucleic Acids; Magnetic Fields; Magnetite Nanoparticles; Microscopy, Fluorescence; Nucleic Acid Hybridization; Oligonucleotides; Photobleaching; Streptavidin; Surface Properties

The antihypertensive peptides, Val-Pro-Pro and Ile-Pro-Pro, were successfully detected in the aorta of spontaneously hypertensive rats after orally administering these peptides by a guanidine-thiocyanate treatment to prevent proteolysis. Cy3-labeled versions of both peptides were localized in the endothelial cells of arterial vessels in the rats. The accumulation of both peptides in the endothelial cells suggested in vivo inhibitory activity of the angiotensin I-converting enzyme.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Arteries; Blood Pressure; Carbocyanines; Endothelial Cells; Guanidines; Hypertension; Isotope Labeling; Male; Microscopy, Fluorescence; Oligopeptides; Peptidyl-Dipeptidase A; Rats; Rats, Inbred SHR; Thiocyanates

Optical trapping and single-molecule fluorescence are two major single-molecule approaches. Their combination has begun to show greater capability to study more complex systems than either method alone, but met many fundamental and technical challenges. We built an instrument that combines base-pair resolution dual-trap optical tweezers with single-molecule fluorescence microscopy. The instrument has complementary design and functionalities compared with similar microscopes previously described. The optical tweezers can be operated in constant force mode for easy data interpretation or in variable force mode for maximum spatiotemporal resolution. The single-molecule fluorescence detection can be implemented in either wide-field or confocal imaging configuration. To demonstrate the capabilities of the new instrument, we imaged a single stretched λ DNA molecule and investigated the dynamics of a DNA hairpin molecule in the presence of fluorophore-labeled complementary oligonucleotide. We simultaneously observed changes in the fluorescence signal and pauses in fast extension hopping of the hairpin due to association and dissociation of individual oligonucleotides. The combined versatile microscopy allows for greater flexibility to study molecular machines or assemblies at a single-molecule level.

Topics: Bacteriophage lambda; Base Sequence; Carbocyanines; DNA, Viral; Inverted Repeat Sequences; Microscopy, Fluorescence; Optical Tweezers

Dual functional hierarchically assembled nanostructures, with two unique functions of carrying therapeutic cargo electrostatically and maintaining radiolabeled imaging agents covalently within separate component building blocks, have been developed via the supramolecular assembly of several spherical cationic shell cross-linked nanoparticles clustered around a central anionic shell cross-linked cylinder. The shells of the cationic nanoparticles and the hydrophobic core domain of the anionic central cylindrical nanostructure of the assemblies were utilized to complex negatively charged nucleic acids (siRNA) and to undergo radiolabeling, respectively, for potential theranostic applications. The assemblies exhibited exceptional cell transfection and radiolabeling efficiencies, providing an overall advantage over the individual components, which could each facilitate only one or the other of the functions.

Topics: Carbocyanines; Drug Delivery Systems; Molecular Structure; Nanostructures; Particle Size; Radiopharmaceuticals; RNA, Small Interfering; Static Electricity

This paper presents highly sensitive fluorescence detections of avidin and streptavidin using an optical interference mirror (OIM) slide consisting of a plane mirror covered with an optical interference layer. Compared with a common glass slide, the OIM slide can enhance the fluorescence from a dye by more than 100-fold. We fabricated an OIM slide by depositing an optical interference layer of Al(2)O(3) on an Ag mirror. To enhance the fluorescence maximally, the optimal thickness of the Al(2)O(3) layer was estimated from optical interference theory. For detections of protein, avidin/streptavidin labeled with fluorescein, Cy3, and Cy5 were detected with biotin immobilized on an OIM slide with the optimal Al(2)O(3) thickness. We achieved a sensitivity improvement of more than 50-fold, comparing with a glass slide. Such a high degree of improvement would be a significant contribution to further progress in biomedical research and medical diagnostics.

Topics: Adsorption; Aluminum Oxide; Avidin; Biosensing Techniques; Biotin; Carbocyanines; Glass; Immobilized Proteins; Optical Devices; Silver; Solutions; Spectrometry, Fluorescence; Streptavidin

We report a label-free fluorescent strategy to study the distribution and stability of DNA origami nanostructures in live, cellular systems, using carbazole-based biscyanine as a probe molecule which has the characteristic property of restriction of intramolecular rotation (RIR) induced emission.

Topics: Carbocyanines; DNA; Fluorescent Dyes; Humans; MCF-7 Cells; Microscopy, Fluorescence; Nanostructures

RNA interference has emerged as a powerful tool in biological and pharmaceutical research; however, the enzymatic degradation and polyanionic nature of short interfering RNAs (siRNAs) lead to their poor cellular uptake and eventual biological effects. Among nonviral delivery systems, cell-penetrating peptides have been recently employed to improve the siRNA delivery efficiency. Here we introduce an 18-mer amphipathic, amino-acid-pairing peptide, C6, as an siRNA delivery carrier. Peptide C6 adopted a helical structure upon coassembling with siRNA. The C6-siRNA coassembly showed a size distribution between 50 and 250 nm, confirmed by dynamic light scattering and atomic force microscopy. The C6-siRNA interaction enthalpy and stoichiometry were 8.8 kJ·mol(-1) and 6.5, respectively, obtained by isothermal titration calorimetry. A minimum C6/siRNA molar ratio of 10:1 was required to form stable coassemblies/complexes, indicated by agarose gel shift assay and fluorescence spectroscopy. Peptide C6 showed lower toxicity and higher efficiency in cellular uptake of siRNA compared with Lipofectamine 2000. Fluorescence microscopy images also confirmed the localization of C6-siRNA complexes in the cytoplasm using Cy3-labeled siRNAs. These results indicate high capabilities of C6 in forming safe and stable complexes with siRNA and enhancing its cellular uptake.

Topics: Animals; Carbocyanines; Cell Survival; Cell-Penetrating Peptides; CHO Cells; Cricetinae; Cricetulus; Drug Carriers; Lipids; Particle Size; RNA Interference; RNA, Small Interfering; Thermodynamics; Transfection

Sub-diffraction-limit imaging can be achieved by sequential localization of photoactivatable fluorophores, for which the image resolution depends on the number of photons detected per localization. We report a strategy for fluorophore caging that creates photoactivatable probes with high photon yields. Upon photoactivation, these probes can provide 10(4)-10(6) photons per localization and allow imaging of fixed samples with resolutions of several nanometers. This strategy can be applied to many fluorophores across the visible spectrum.

Topics: Bacteriophage M13; Carbocyanines; Cell Line; Fluorescent Dyes; Humans; Microscopy, Fluorescence; Microtubules; Oxidation-Reduction; Photochemical Processes; Photons

Photoswitchable fluorescent probes capable of the targeted labeling of tagged proteins are of significant interest due to their ability to enable in situ imaging of protein complexes within native biomolecular assemblies. Here we describe the synthesis of a fluorescent probe (AsCy3Cy5) and demonstrate the targeted labeling and super-resolution imaging of a tagged protein within a supramolecular protein complex.

Topics: Arsenicals; Carbocyanines; Fluorescence; Fluorescent Dyes; Molecular Structure; Spectrometry, Fluorescence

We present that modulation of fluorescence emission by linearly polarized excitation light can allow us to resolve spatially two fluorescent molecules within a diffraction limit and to determine simultaneously their precise dipole directions. Using polarization-dependent photoswitching, we imaged the 2D geometry of the DNA Holliday junction in a 10-nm length scale by measuring both the distance and the in-plane dipole angle between Cy3 emitters stacked onto the ends of two adjacent branches of the Holliday junction. The proposed polarization-modulated imaging technique provides a simple and nonstochastic imaging process to visualize the nanostructure, including directional information, of biomolecules beyond the diffraction limit.

Topics: Carbocyanines; DNA; Fluorescent Dyes; Holliday Junction Resolvases; Models, Chemical; Photochemical Processes

A new, fast, simple and cost-effective sensing device for monitoring H(2)S has been developed. Proof-of-principle results showing that a commercial and cheap Myoglobin (Mb) can be successfully used as a biological probe for a fluorescence biosensor for H(2)S detection are reported. The two different commercial labels Cy3 and Atto620 were selected for this study. A high selectivity for detecting H(2)S against other thiols was found. The applicability of the proposed sensing system was successfully explored not only in solution but also when applied in the form of a solid state device.

Topics: Animals; Biosensing Techniques; Carbocyanines; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Hydrogen Sulfide; Immobilized Proteins; Myoglobin; Temperature

Polymer microarrays as platforms for cell-based assays are presented, offering a unique approach to high-throughput cellular analysis. These high-throughput (HT) platforms are used for the screening of new materials with the purpose of first finding substrates upon which a specific cell line would adhere and second to gain a rapid understanding of the interactions between cells and biomaterials. Arrays presented here are fabricated using pre-synthesised polymers by contact printing via a robotic microarrayer. These large arrays of polymers are then incubated with cell cultures and the results obtained are used to significantly help the design of synthetic biomaterials, implant surfaces and tissue-engineering scaffolds by finding correlations between their chemical structure and their biological performance. The flexibility of polymer microarrays analysis not only greatly refines our knowledge of multitude of cell-biomaterial interactions but could also be used in biocompatibility assessments as novel biomarkers.

Topics: Animals; Benzimidazoles; Biocompatible Materials; Carbocyanines; Cell Adhesion; Cell Culture Techniques; Cell Line; Cells, Cultured; Fluorescein-5-isothiocyanate; Fluorescent Dyes; High-Throughput Screening Assays; Humans; Indoles; K562 Cells; L Cells; Mice; Microarray Analysis; Phalloidine; Polymers; Rhodamines; Substrate Specificity

Metal-enhanced fluorescence (MEF) increased total photon emission of Cy3- and Cy5-labeled ribosomal initiation complexes near 50 nm silver particles 4- and 5.5-fold, respectively. Fluorescence intensity fluctuations above shot noise, at 0.1-5 Hz, were greater on silver particles. Overall signal-to-noise ratio was similar or slightly improved near the particles. Proximity to silver particles did not compromise ribosome function, as measured by codon-dependent binding of fluorescent tRNA, dynamics of fluorescence resonance energy transfer between adjacent tRNAs in the ribosome, and tRNA translocation induced by elongation factor G.

Topics: Base Sequence; Carbocyanines; Colloids; Fluorescence Resonance Energy Transfer; Metal Nanoparticles; Photobleaching; Photons; Protein Biosynthesis; Ribosomes; RNA, Messenger; RNA, Transfer; Silver

This paper reports that water molecules around F-actin, a polymerized form of actin, are more mobile than those around G-actin or in bulk water. A measurement using pulse-field gradient spin-echo (1)H NMR showed that the self-diffusion coefficient of water in aqueous F-actin solution increased with actin concentration by ∼5%, whereas that in G-actin solution was close to that of pure water. This indicates that an F-actin/water interaction is responsible for the high self-diffusion of water. The local viscosity around actin was also investigated by fluorescence measurements of Cy3, a fluorescent dye, conjugated to Cys 374 of actin. The steady-state fluorescence anisotropy of Cy3 attached to F-actin was 0.270, which was lower than that for G-actin, 0.334. Taking into account the fluorescence lifetimes of the Cy3 bound to actin, their rotational correlation times were estimated to be 3.8 and 9.1ns for F- and G-actin, respectively. This indicates that Cy3 bound to F-actin rotates more freely than that bound to G-actin, and therefore the local water viscosity is lower around F-actin than around G-actin.

Topics: Actin Cytoskeleton; Animals; Buffers; Carbocyanines; Cysteine; Fluorescent Dyes; Nuclear Magnetic Resonance, Biomolecular; Spectrometry, Fluorescence; Water

Integration of human immunodeficiency virus cDNA ends by integrase (IN) into host chromosomes involves a concerted integration mechanism. IN juxtaposes two DNA blunt ends to form the synaptic complex, which is the intermediate in the concerted integration pathway. The synaptic complex is inactivated by strand transfer inhibitors (STI) with IC(50) values of ∼20 nM for inhibition of concerted integration. We detected a new nucleoprotein complex on a native agarose gel that was produced in the presence of >200 nM STI, termed the IN-single DNA (ISD) complex. Two IN dimers appear to bind in a parallel fashion at the DNA terminus, producing an ∼32-bp DNase I protective footprint. In the presence of raltegravir (RAL), MK-2048, and L-841,411, IN incorporated ∼20-25% of the input blunt-ended DNA substrate into the stabilized ISD complex. Seven other STI also produced the ISD complex (≤5% of input DNA). The formation of the ISD complex was not dependent on 3'OH processing, and the DNA was predominantly blunt ended in the complex. The RAL-resistant IN mutant N155H weakly forms the ISD complex in the presence of RAL at ∼25% level of wild-type IN. In contrast, MK-2048 and L-841,411 produced ∼3-fold to 5-fold more ISD than RAL with N155H IN, which is susceptible to these two inhibitors. The results suggest that STI are slow-binding inhibitors and that the potency to form and stabilize the ISD complex is not always related to inhibition of concerted integration. Rather, the apparent binding and dissociation properties of each STI influenced the production of the ISD complex.

Topics: Base Pairing; Biocatalysis; Carbocyanines; Deoxyribonuclease I; DNA; DNA Footprinting; Drug Resistance, Viral; Electrophoresis, Agar Gel; Fluorescent Dyes; HIV Integrase; HIV Long Terminal Repeat; HIV-1; Humans; Inhibitory Concentration 50; Integrase Inhibitors; Keto Acids; Mutant Proteins; Protein Binding; Protein Multimerization; Pyrrolidinones; Raltegravir Potassium; Substrate Specificity

We used optogenetics to determine the global respiratory effects produced by selectively stimulating raphe obscurus (RO) serotonergic neurons in anesthetized mice and to test whether these neurons detect changes in the partial pressure of CO(2), and hence function as central respiratory chemoreceptors. Channelrhodopsin-2 (ChR2) was selectively (∼97%) incorporated into ∼50% of RO serotonergic neurons by injecting AAV2 DIO ChR2-mCherry (adeno-associated viral vector double-floxed inverse open reading frame of ChR2-mCherry) into the RO of ePet-Cre mice. The transfected neurons heavily innervated lower brainstem and spinal cord regions involved in autonomic and somatic motor control plus breathing but eschewed sensory related regions. Pulsed laser photostimulation of ChR2-transfected serotonergic neurons increased respiratory frequency (fR) and diaphragmatic EMG (dEMG) amplitude in relation to the duration and frequency of the light pulses (half saturation, 1 ms; 5-10 Hz). dEMG amplitude and fR increased slowly (half saturation after 10-15 s) and relaxed monoexponentially (tau, 13-15 s). The breathing stimulation was reduced ∼55% by methysergide (broad spectrum serotonin antagonist) and potentiated (∼16%) at elevated levels of inspired CO(2) (8%). RO serotonergic neurons, identified by their entrainment to short light pulses (threshold, 0.1-1 ms) were silent (nine cells) or had a low and regular level of activity (2.1 ± 0.4 Hz; 11 cells) that was not synchronized with respiration. These and nine surrounding neurons with similar characteristics were unaffected by adding up to 10% CO(2) to the breathing mixture. In conclusion, RO serotonergic neurons activate breathing frequency and amplitude and potentiate the central respiratory chemoreflex but do not appear to have a central respiratory chemoreceptor function.

Topics: Animals; Autonomic Pathways; Biophysics; Carbocyanines; Carbonates; Channelrhodopsins; Choline O-Acetyltransferase; Diaphragm; Dose-Response Relationship, Drug; Electric Stimulation; Electromyography; Female; Hypercapnia; Male; Methysergide; Mice; Motor Neurons; Photic Stimulation; Raphe Nuclei; Respiration; Serotonin; Serotonin Antagonists; Statistics, Nonparametric; Transfection; Tryptophan Hydroxylase

We characterized the effect of the first basepair on the conformational dynamics of the fluorescent dye Cy3 attached to the 5' end of double-stranded DNA using gaussian-mixture adaptive umbrella sampling simulations. In the simulations, the sampling of all five dihedral angles along the linker was enhanced, so that both stacked and unstacked states were sampled. The affinity of Cy3 for a T·A basepair (with the dye attached to T) was found to be significantly less than for the other basepairs. This was verified experimentally by measuring the activation energies for cis-trans isomerization of the dye. The simulation and experimental results indicate the existence of partially unstacked conformations amenable to photoisomerization. The simulations also showed that stacking of Cy3 straightens the DNA while stabilizing the first basepair. Our findings indicate that fluorescence is modulated by Cy3-DNA interactions in a sequence-dependent manner.

Topics: Base Pairing; Carbocyanines; Computer Simulation; DNA; Fluorescence; Isomerism; Models, Molecular; Nucleic Acid Conformation; Thermodynamics

The careful normalization of array-based comparative genomic hybridization (aCGH) data is of critical importance for the accurate detection of copy number changes. The difference in labelling affinity between the two fluorophores used in aCGH-usually Cy5 and Cy3-can be observed as a bias within the intensity distributions. If left unchecked, this bias is likely to skew data interpretation during downstream analysis and lead to an increased number of false discoveries.. In this study, we have developed aCGH.Spline, a natural cubic spline interpolation method followed by linear interpolation of outlier values, which is able to remove a large portion of the dye bias from large aCGH datasets in a quick and efficient manner.. We have shown that removing this bias and reducing the experimental noise has a strong positive impact on the ability to detect accurately both copy number variation (CNV) and copy number alterations (CNA).

Topics: Carbocyanines; Comparative Genomic Hybridization; DNA Copy Number Variations; Fluorescent Dyes; Software

In this chapter, we present a novel, cost-effective microarray strategy that utilizes expression-ready plasmid DNAs to generate protein arrays on-demand and its use to validate protein-protein interactions. These expression plasmids were constructed in such a way so as to serve a dual purpose of synthesizing the protein of interest as well as capturing the synthesized protein. The microarray system is based on the high affinity binding of Escherichia coli "Tus" protein to "Ter," a 20 bp DNA sequence involved in the regulation of DNA replication. The protein expression is carried out in a cell-free protein synthesis system, with rabbit reticulocyte lysates, and the target proteins are detected either by labeled incorporated tag specific or by gene-specific antibodies. This microarray system has been successfully used for the detection of protein-protein interaction because both the target protein and the query protein can be transcribed and translated simultaneously in the microarray slides. The utility of this system for detecting protein-protein interaction is demonstrated by a few well-known examples: Jun/Fos, FRB/FKBP12, p53/MDM2, and CDK4/p16. In all these cases, the presence of protein complexes resulted in the localization of fluorophores at the specific sites of the immobilized target plasmids. Interestingly, during our interactions studies we also detected a previously unknown interaction between CDK2 and p16. Thus, this Tus-Ter based system of protein microarray can be used for the validation of known protein interactions as well as for identifying new protein-protein interactions. In addition, it can be used to examine and identify targets of nucleic acid-protein, ligand-receptor, enzyme-substrate, and drug-protein interactions.

Topics: Animals; Antibodies; Carbocyanines; Cloning, Molecular; DNA-Binding Proteins; Escherichia coli; Escherichia coli Proteins; Nucleic Acid Hybridization; Plasmids; Printing; Protein Array Analysis; Protein Binding; Quality Control

Topics: Carbocyanines; DNA; Fluorescence Resonance Energy Transfer; Imidazoles; Nylons; Photons; Pyrroles

We have developed a new technique using fluorescent silica nanotubes for simple and sensitive DNA detection. The quantum-dot-embedded silica nanotubes (QD-SNTs) were fabricated by a sol-gel reaction using anodic aluminum silica oxide (AAO) as a template. The fluorescent QD-SNTs of different colors were then immobilized with single-stranded DNA and used as nanoprobes for DNA detection. The optical and structural properties of QD-SNT nanoprobes were examined using photoluminescence spectroscopy, confocal microscopy and transmission electron microscopy (TEM). The QD-SNT nanoprobes were applied to detect dye-labeled target DNA in a solution phase. The obvious color change of the QD-SNT nanoprobes was observed visually under a simple microscope after the successful detection with target DNA. The quantitative analyses indicated that ∼ 100 attomole of target DNA in one nanoprobe can generate a distinguishable and observable color change. The detection results also demonstrated that our assay exhibited high specificity, high selectivity and very low nonspecific adsorption. Our simple DNA assay based on QD-SNT nanoprobes is expected to be quite useful for the needs of fast DNA screening and detection applications.

Topics: Adsorption; Carbocyanines; DNA; DNA Probes; DNA, Complementary; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Nanotubes; Nucleic Acid Hybridization; Particle Size; Quantum Dots; Rhodamines; Silicon Dioxide; Spectrometry, Fluorescence

Long ago inferred by biochemists, the linear diffusion of proteins along DNA has recently been observed at a single-molecule level using fluorescence microscopy. This imaging technique requires labeling the protein of interest with a fluorophore, usually an organic nanosized dye that is not supposed to impact the dynamics of the protein. Yet individual proteins can also be tracked using much larger labels, like quantum dots or beads. We investigate here the impact of such a large label on the protein diffusion along DNA. Solving a Fokker-Planck equation, we estimate the diffusion constant of a protein-label complex diffusing in a periodic potential that mimics the DNA-protein interaction, the link between the protein and the label being modeled as a Hookean spring. Our results indicate that the diffusion constant can generally be calculated by considering that the motion of the protein in the DNA potential is decoupled from the Brownian motion of the label. Our conclusions are in good agreement with the experimental results we obtained with the restriction enzyme EcoRV, assuming a rotation-coupled diffusion of the enzyme along DNA.

Topics: Carbocyanines; Deoxyribonucleases, Type II Site-Specific; Diffusion; DNA; Fluorescent Dyes; Linear Models; Models, Biological; Movement; Proteins

Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h.

Topics: Animals; Carbocyanines; DNA, Complementary; Fluorescent Dyes; Gene Expression Profiling; Male; Mice; Mice, Inbred BALB C; Oligonucleotide Array Sequence Analysis

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

Topics: Brain Neoplasms; Carbocyanines; Humans; Oligonucleotide Array Sequence Analysis; Quality Control; RNA

An optical tweezers directed parallel DNA oligonucleotide synthesis methodology is described in which controlled pore glass (CPG) beads act as solid substrates in a two-stream microfluidic reactor. The reactor contains two parallel sets of physical confinement features that retain beads in the reagent stream for synthetic reaction but allow the beads to be optically trapped and transferred between the reagent and the inert streams for sequence programming. As a demonstration, we synthesized oligonucleotides of target sequence 25-nt, one deletion and one substitution using dimethoxytrityl (DMT) nucleoside phosphoramidite chemistry. In detecting single-nucleotide mismatches, fluorescence in situ hybridization of the bead-conjugated probes showed high specificity and signal-to-noise ratios. These preliminary results suggest further possibilities of creating a novel type of versatile, sensitive and multifunctional reconfigurable one-bead one-compound (OBOC) bead array.

Topics: Carbocyanines; Equipment Design; Fluorescent Dyes; Microfluidic Analytical Techniques; Microspheres; Oligonucleotides; Optical Tweezers; Particle Size; Silanes; Spectrometry, Fluorescence

DNA microarrays have been used as powerful tools in genomics studies and single nucleotide polymorphisms analysis. However, the fluorescence detection used in most conventional DNA microarrays is still limited by its sensitivity. The aim of this study is to use a cationic surfactant, cetyl trimethylammonium bromide (CTAB), to enhance the fluorescence intensity of 6-carboxy-fluorescene (FAM)-labeled DNA probes immobilized on a DNA microarray. We show that in the presence of CTAB the immobilized FAM-labeled DNA probes is 11-fold brighter than that without exposure to CTAB. Similarly, when we hybridize FAM-labeled DNA targets to a DNA microarray and treat the surface with CTAB solution, the fluorescence intensity shows a 26-fold increase for perfect-match DNA targets. More importantly, the contrast between perfect-match and 1-mismatch DNA is also increased from 1.3-fold to 15-fold. This method offers a simple and efficient technique to enhance the detection limit of DNA microarrays.

Topics: Base Sequence; Carbocyanines; Cetrimonium; Cetrimonium Compounds; DNA Probes; Fluoresceins; Limit of Detection; Models, Molecular; Nucleic Acid Conformation; Oligonucleotide Array Sequence Analysis; Solutions; Spectrometry, Fluorescence; Surface-Active Agents

Cigarette smoke exposure is known to induce obstructive lung disease and several cardiovascular disease states in humans and also in animal models. Smoking leads to oxidative stress and inflammation that are important in triggering pulmonary and cardiovascular disease. The objective of the current study was to quantify differences in expression levels of plasma proteins of cigarette smoke -exposed and control mice, at the time of disease onset, and identify these proteins for use as potential biomarkers of the onset of smoking-induced disease. We utilized 2-D DIGE/MS to characterize these proteomic changes. 2-D DIGE of plasma samples identified 11 differentially expressed proteins in cigarette smoke -exposed mice. From these 11 proteins, 9 were downregulated and 2 were upregulated. The proteins identified are involved in vascular function, coagulation, metabolism and immune function. Among these, the alterations in fibrinogen (2.2-fold decrease), α-1-antitrypsin (1.8-fold increase) and arginase (4.5-fold decrease) are of particular interest since these have been directly linked to cardiovascular and lung pathology. Differences in expression levels of these proteins were also confirmed by immunoblotting. Thus, we observe that chronic cigarette smoke exposure in mice leads to prominent changes in the protein expression profile of blood plasma and these changes in turn can potentially serve as markers predictive of the onset and progression of cardiovascular and pulmonary disease.

Topics: Animals; Biomarkers; Blood Proteins; Blotting, Western; Carbocyanines; Case-Control Studies; Electrophoresis, Gel, Two-Dimensional; Image Processing, Computer-Assisted; Male; Mass Spectrometry; Metabolic Networks and Pathways; Mice; Mice, Inbred C57BL; Oxidative Stress; Proteome; Signal Transduction; Tobacco Smoke Pollution

We investigate interactions between graphene oxide and a Pb(2+)-dependent DNAzyme, based on which a Pb(2+) sensor with high sensitivity, selectivity and tunable dynamic range is developed.

Topics: Carbocyanines; DNA, Catalytic; Graphite; Ions; Lead; Metals; Nanostructures; Oxides; Spectrometry, Fluorescence

To digitally analyze expression levels of multiple genes in one reaction, we proposed a method termed as 'MDHB' (Multiplexed Digital-PCR coupled with Hydrogel Bead-array). The template for bead-based emulsion PCR (emPCR) was prepared by reverse transcription using sequence-tagged primers. The beads recovered from emPCR were immobilized with hydrogel to form a single-bead layer on a chip, and then decoded by gene-specific probe hybridization and Cy3-dUTP based primer extension reaction. The specificity of probe hybridization was improved by using electrophoresis to remove mismatched probes on the bead's surface. The number of positive beads reflects the abundance of expressed genes; the expression levels of target genes were normalized to a housekeeping gene and expressed as the number ratio of green beads to red beads. The discrimination limit of MDHB is 0.1% (i.e., one target molecule from 1000 background molecules), and the sensitivity of the method is below 100 cells when using the β-actin gene as the detection target. We have successfully employed MDHB to detect the relative expression levels of four colorectal cancer (CRC)-related genes (c-myc, COX-2, MMP7, and DPEP1) in 8 tissue samples and 9 stool samples from CRC patients, giving the detection rates of 100% and 77%, respectively. The results suggest that MDHB could be a potential tool for early non-invasive diagnosis of CRC.

Topics: Carbocyanines; Colorectal Neoplasms; Cyclooxygenase 2; Deoxyuracil Nucleotides; Dipeptidases; DNA Primers; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Hydrogels; Matrix Metalloproteinase 7; Polymerase Chain Reaction; Proto-Oncogene Proteins c-myc

Glioblastoma (GB) is currently characterized by low survival rates and therapies with insufficient efficacy. Here, we describe biodegradable polymers that can deliver genes to primary GB cells as well as GB tumor stem cells in vitro with low non-specific toxicity and transfection efficiencies of up to 60.6 ± 5% in normal (10%) serum conditions. We developed polymer-DNA nanoparticles that remained more stable in normal serum and could also be stored for at least 3 months in ready-to-use form with no measurable decrease in efficacy, expanding their potential in a practical or clinical setting. A subset of polymers was identified that shows a high degree of specificity to tumor cells compared with healthy astrocytes and human neural stem cells when cultured (separately or in co-culture), yielding higher transfection in GB cells while having little to no apparent effect on healthy cells.

Topics: Astrocytes; Carbocyanines; Cell Line, Tumor; Cell Survival; Cryoprotective Agents; DNA; Fetal Stem Cells; Freeze Drying; Gene Expression; Gene Transfer Techniques; Glioblastoma; Green Fluorescent Proteins; Humans; Luminescent Proteins; Nanoparticles; Neoplastic Stem Cells; Polymers; Red Fluorescent Protein; Spheroids, Cellular; Sucrose; Time Factors; Transfection

We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G⋅GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA⋅EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.

Topics: Adenosine Triphosphate; Carbocyanines; Fluorescence Resonance Energy Transfer; Kinetics; Models, Chemical; Models, Genetic; Models, Molecular; Molecular Dynamics Simulation; Peptide Elongation Factor G; Protein Biosynthesis; Ribosomal Proteins; Ribosomes; RNA, Transfer

Bird on a wire: a recent study by Rant and Burley showed that it is possible to transfer excited-state energy along double-stranded DNA by arranging fluorophore relays with base-pair precision through Dervan-polyamides in a cascade-like process. The resulting assembly acts as the largest DNA-based photonic wire yet.

Topics: Benzoxazoles; Carbocyanines; DNA; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Photons; Quinolines; Succinimides

DNA damage responses (DDR) occur during oncogenesis and therapeutic responses to DNA damaging cytotoxic drugs. Thus, a real-time method to image DNA damage in vivo would be useful to diagnose cancer and monitor its treatment. Toward this end, we have developed fluorophore- and radioisotope-labeled immunoconjugates to target a DDR signaling protein, phosphorylated histone H2A variant H2AX (γH2AX), which forms foci at sites of DNA double-strand breaks. Anti-γH2AX antibodies were modified by the addition of diethylenetriaminepentaacetic acid (DTPA) to allow (111)In labeling or the fluorophore Cy3. The cell-penetrating peptide Tat (GRKKRRQRRRPPQGYG) was also added to the immunoconjugate to aid nuclear translocation. In irradiated breast cancer cells, confocal microscopy confirmed the expected colocalization of anti-γH2AX-Tat with γH2AX foci. In comparison with nonspecific antibody conjugates, (111)In-anti-γH2AX-Tat was retained longer in cells. Anti-γH2AX-Tat probes were also used to track in vivo DNA damage, using a mouse xenograft model of human breast cancer. After local X-ray irradiation or bleomycin treatment, the anti-γH2AX-Tat probes produced fluorescent and single photon emission computed tomography signals in the tumors that were proportionate to the delivered radiation dose and the amount of γH2AX present. Taken together, our findings establish the use of radioimmunoconjugates that target γH2AX as a noninvasive imaging method to monitor DNA damage, with many potential applications in preclinical and clinical settings.

Topics: Amino Acid Sequence; Animals; Antibodies; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; DNA Damage; DNA, Neoplasm; Female; Gene Products, tat; Histones; Humans; Immunoconjugates; Indium Radioisotopes; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Sequence Data; Pentetic Acid; Transplantation, Heterologous

In recent years, the capability of synthetic biology to design large genetic circuits has dramatically increased due to rapid advances in DNA synthesis technology and development of tools for large-scale assembly of DNA fragments. Large genetic circuits require more components (parts), especially regulators such as transcription factors, sigma factors, and viral RNA polymerases to provide increased regulatory capability, and also devices such as sensors, receivers, and signaling molecules. All these parts may have a potential impact upon the host that needs to be considered when designing and fabricating circuits. DNA microarrays are a well-established technique for global monitoring of gene expression and therefore are an ideal tool for systematically assessing the impact of expressing parts of genetic circuits in host cells. Knowledge of part impact on the host enables the user to design circuits from libraries of parts taking into account their potential impact and also to possibly modify the host to better tolerate stresses induced by the engineered circuit. In this chapter, we present the complete methodology of performing microarrays from choice of array platform, experimental design, preparing samples for array hybridization, and associated data analysis including preprocessing, normalization, clustering, identifying significantly differentially expressed genes, and interpreting the data based on known biology. With these methodologies, we also include lists of bioinformatic resources and tools for performing data analysis. The aim of this chapter is to provide the reader with the information necessary to be able to systematically catalog the impact of genetic parts on the host and also to optimize the operation of fully engineered genetic circuits.

Topics: Algorithms; Carbocyanines; Fluorescent Dyes; Gene Expression Profiling; Oligonucleotide Array Sequence Analysis; Research Design; RNA; Software; Synthetic Biology

We synthesized fluorescent ferritin nanoparticles (FFNPs) through bacterial expression of the hybrid gene consisting of human ferritin heavy chain (hFTN-H), spacer (glycine-rich peptide), and enhanced green (or red) fluorescent protein [eGFP (or DsRed)] genes. The self-assembly activity of hFTN-H that leads to the formation of nanoparticles (12 nm in diameter), the conformational flexibility of the C-terminus of hFTN-H, and the glycine-rich spacer enabled eGFPs (or DsReds) to be well displayed on the surface of each ferritin nanoparticle, resulting in the construction of green (or red) FFNPs [gFFNPs (or rFFNPs)]. As compared to eGFP (or DsRed) alone, it is notable that the developed FFNPs showed significantly amplified fluorescence intensity and also enhanced stability. DNA aptamers were chemically conjugated to gFFNP via each eGFP's cysteine residue that was newly introduced through site-directed mutagenesis (Ser175Cys). The DNA-aptamer-conjugated gFFNPs were used as a fluorescent reporter probe in the aptamer-based "sandwich" assay of a cancer marker [i.e., platelet-derived growth factor B-chain homodimer (PDGF-BB)] in phosphate-buffered saline buffer or diluted human serum. This is a simple two-step assay without any additional steps for signal amplification, showing that compared to the same aptamer-based assays using eGFP alone or Cy3, the detection signals, affinity of the reporter probe to the cancer marker, and assay sensitivity were significantly enhanced; i.e., the limit of detection was lowered to the 100 fM level. Although the PDGF-BB assay is reported here as a proof-of-concept, the developed FFNPs can be applied in general to any aptamer-based sandwich assays.

Topics: Aptamers, Nucleotide; Becaplermin; Carbocyanines; Ferritins; Green Fluorescent Proteins; Humans; Nanoparticles; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Recombinant Fusion Proteins; Spectrometry, Fluorescence

A conductive nanoporous antimony-doped tin oxide (ATO) powder has been prepared using the sol-gel method that contains three-dimensionally interconnected pores within the metal oxide and highly tunable pore sizes on the nanoscale. It is demonstrated that these porous materials possess the capability of hosting a tetrahedral-shaped DNA nanostructure of defined dimensions with high affinity. The tunability of pore size enables the porous substrate to selectively absorb the DNA nanostructures into the metal oxide cavities or exclude them from entering the surface layer. Both confocal fluorescence microscopy and solution FRET experiments revealed that the DNA nanostructures maintained their integrity upon the size-selective incorporation into the cavities of the porous materials. As DNA nanostructures can serve as a stable three-dimensional nanoscaffold for the coordination of electron transfer mediators, this work opens up new possibilities of incorporating functionalized DNA architectures as guest molecules to nanoporous conductive metal oxides for applications such as photovoltaics, sensors, and solar fuel cells.

Topics: Antimony; Carbocyanines; DNA; Electric Conductivity; Models, Molecular; Nanopores; Nucleic Acid Conformation; Tin Compounds

Development of novel approaches for quantitative analysis of gene expression in intact tumour cells could provide new methods for the detection of cervical cancer. Molecular beacons (MBs) are single-stranded oligonucleotide probes that form stem-and-loop structures. Survivin, a member of the 'inhibitor of apoptosis' family of proteins, is highly expressed in cervical cancer. The aim of this study was to determine the feasibility of MBs targeting wild-type survivin in the diagnosis of cervical cancer.. MBs with oligonucleotide sequences complementing survivin mRNA and covalently linked with FITC or Cy3 were designed and synthesized. The specificity and sensitivity of survivin MBs were examined in human cervical cancer cell lines and smears from cervical cancer patients, and confirmed by reverse transcriptase polymerase chain reaction (RT-PCR), immunocytochemistry, Western blotting and the thinprep cytological test (TCT).. Both survivin MB-FITC and MB-Cy3 produced a strong fluorescent signal in cervical cancer cells, and the intensity was consistent with the results from RT-PCR, Western blotting and immunocytochemical staining. In the initial clinical cohort study, the sensitivity of survivin MBs was 61.37% (27/44) and the specificity was 72.72% (34/44); the sensitivities of Western blotting and TCT were 76.1% (32/42) and 68.19% (30/44), respectively. No significant difference in sensitivity was observed between MBs, Western blotting and TCT for different tumour grades and International Federation of Gynecology and Obstetrics (FIGO) stages.. Survivin MBs are specific and sensitive molecular probes for the detection of cervical cancer cells. They have great potential in the early detection and follow-up of patients with cervical cancer.

Topics: Adult; Carbocyanines; Cell Line, Tumor; Cohort Studies; DNA, Single-Stranded; Feasibility Studies; Female; Fluorescein-5-isothiocyanate; Humans; Inhibitor of Apoptosis Proteins; Inverted Repeat Sequences; Middle Aged; Molecular Imaging; Neoplasm Grading; Neoplasm Proteins; Neoplasm Staging; Oligonucleotide Probes; Sensitivity and Specificity; Survivin; Uterine Cervical Neoplasms; Vaginal Smears

The coupling between cell-cell and cell-matrix adhesion systems is known to affect the stability of the adhesive status of cells, as well as tissue cohesion. In this work, we perform quantitative assays of integrin-cadherin cross talk in controlled and reproducible conditions. This is achieved by plating cells on microprinted fibronectin patterns of different sizes, and simulating the formation of an intercellular contact with a microbead coated with E-cadherin extracellular domains and brought to the cell membrane. Using an optical trap, we measure the average rigidity modulus of the E-cadherin bead-cell contact as a function of the contact incubation time and of the cell spreading area. For a given incubation time, this rigidity modulus decreases by three orders of magnitude as the cell-matrix contact area, A, increases from 100 to 700 μm(2). In a similar way, the dynamics of formation of the bead-cell contact gets slower as this area increases. This is clear evidence for a strong negative feedback from cell-fibronectin onto cell-cell adhesive contacts, for which we discuss some possible mechanisms.

Topics: Animals; Biomechanical Phenomena; Cadherins; Carbocyanines; Cell Adhesion; Cell Communication; Egtazic Acid; Elastic Modulus; Feedback, Physiological; Fibronectins; Green Fluorescent Proteins; Integrins; Mice; Microspheres; Oligopeptides; Polylysine; Protein Binding; Recombinant Fusion Proteins

Fluorescent imaging of cytoskeletal structures permits studies of both organization within the cell and dynamic reorganization of the cytoskeleton itself. Traditional fluorescent labels of microtubules, part of the cytoskeleton, have been used to study microtubule localization, structure, and dynamics, both in vivo and in vitro. However, shortcomings of existing labels make imaging of microtubules with high precision light microscopy difficult. In this paper, we report a new fluorescent labeling technique for microtubules, which involves a GTP analog modified with a bright, organic fluorophore (TAMRA, Cy3, or Cy5). This fluorescent GTP binds to a specific site, the exchangeable site, on tubulin in solution with a dissociation constant of 1.0±0.4 µM. Furthermore, the label becomes permanently incorporated into the microtubule lattice once tubulin polymerizes. We show that this label is usable as a single molecule fluorescence probe with nanometer precision and expect it to be useful for modern subdiffraction optical microscopy of microtubules and the cytoskeleton.

Topics: Animals; Binding Sites; Carbocyanines; Cattle; Fluorescent Dyes; Guanosine Triphosphate; Microtubules; Rhodamines; Staining and Labeling; Tubulin

Cy3 and Cy5 are among the most commonly used oligonucleotide labeling molecules. Studies of nucleic acid structure and dynamics use these dyes, and they are ubiquitous in microarray experiments. They are sensitive to their environment and have higher quantum yield when bound to DNA. The fluorescent intensity of terminal cyanine dyes is also known to be significantly dependent on the base sequence of the oligonucleotide. We have developed a very precise and high-throughput method to evaluate the sequence dependence of oligonucleotide labeling dyes using microarrays and have applied the method to Cy3 and Cy5. We used light-directed in-situ synthesis of terminally-labeled microarrays to determine the fluorescence intensity of each dye on all 1024 possible 5'-labeled 5-mers. Their intensity is sensitive to all five bases. Their fluorescence is higher with 5' guanines, and adenines in subsequent positions. Cytosine suppresses fluorescence. Intensity falls by half over the range of all 5-mers for Cy3, and two-thirds for Cy5. Labeling with 5'-biotin-streptavidin-Cy3/-Cy5 gives a completely different sequence dependence and greatly reduces fluorescence compared with direct terminal labeling.

Topics: Base Sequence; Biotin; Carbocyanines; DNA; Genomics; Oligodeoxyribonucleotides; Oligonucleotide Array Sequence Analysis; Organophosphorus Compounds; Spectrometry, Fluorescence

In this paper, we report the role of surfactants in minimizing nonspecific protein adsorption in liquid crystal (LC)-based immunoassays in which LC is used as a readout system. Among all surfactants tested, only nonionic surfactant such as Tween 20 can effectively reduce the nonspecific protein adsorption, while maintaining the selectivity of the LC-based immunoassay. We also show that to minimize nonspecific protein adsorption, Tween 20 can be added directly into the antibody solution to a final concentration of 0.8 mM. After the addition of Tween 20, better correlations between the antibody concentrations and the interference colors of LCs can therefore be obtained. For example, when Cy3 antibiotin was used, black, yellow, red, and green interference colors correspond to a concentration of 5, 25, 50, and 100 μg/mL, respectively. This feature gives LC immunoassay a unique advantage over the fluorescence-based immunoassay.

Topics: Adsorption; Antibodies; Biotin; Carbocyanines; Dimethylpolysiloxanes; Immunoassay; Liquid Crystals; Microfluidic Analytical Techniques; Polysorbates; Proteins; Surface-Active Agents

Breast cancer presents greatest challenge in health care in today's world. The key to ultimately successful treatment of breast cancer disease is an early and accurate diagnosis. Current breast cancer treatments are often associated with severe side effects. Driven by the need, we report the design of novel hybrid nanomaterial using gold nano popcorn-attached single wall carbon nanotube for targeted diagnosis and selective photothermal treatment. Targeted SK-BR-3 human breast cancer cell sensing have been performed in 10 cancer cells/mL level, using surface enhanced Raman scattering of single walls carbon nanotube's D and G bands. Our data show that S6 aptamer attached hybrid nanomaterial based SERS assay is highly sensitive to targeted human breast cancer SK-BR-3 cell line and it will be able to distinguish it from other non targeted MDA-MB breast cancer cell line and HaCaT normal skin cell line. Our results also show that 10 min of photothermal therapy treatment by 1.5 W/cm(2) power, 785 nm laser is enough to kill cancer cells very effectively using S6 aptamer attached hybrid nanomaterials. Possible mechanisms for targeted sensing and operating principle for highly efficient photothermal therapy have been discussed. Our experimental results reported here open up a new possibility for using aptamers modified hybrid nanomaterial for reliable diagnosis and targeted therapy of cancer cell lines quickly.

Topics: Aptamers, Nucleotide; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Female; Gold; Humans; Metal Nanoparticles; Nanostructures; Nanotubes, Carbon; Phototherapy; Spectrum Analysis, Raman

The conformational states of Escherichia coli Rep helicase undergoing ATP hydrolysis while bound to a partial-duplex DNA (pdDNA) were studied using single-molecule FRET. Crystallographic studies showed that Rep bound to single-stranded DNA can exist in open and closed conformations that differ in the orientation of the 2B subdomain. FRET measurements between eight Rep mutants donor-labeled at different residues and pdDNA acceptor-labeled at the junction were conducted at each of the four nucleotide states. The positions of donor-labeled residues, based on crystal structure, and FRET measurements between these donor molecules and the acceptor fluorophore at the DNA junction were used to predict the most likely position for the DNA junction using a triangulation algorithm. These predicted junction positions are compared with the crystal structure to determine whether the open or closed conformation is more consistent with the FRET data. Our data revealed that there are two distinct Rep-pdDNA conformations in the ATPγS and ADP states, an unexpected finding. The primary conformation is similar to that observed in nucleotide-free and ADP.Pi states, and the secondary conformation is a novel conformation where the duplex DNA and 2B subdomain moved as a unit by 13 Å relative to the rest of the protein. The primary conformation found in all nucleotide states is consistent with the closed conformation of the crystal structure however; the secondary conformation is a new conformation that has not been observed before. We discuss the possible implications of this newly observed conformation.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Algorithms; Carbocyanines; DNA; DNA Helicases; Escherichia coli; Escherichia coli Proteins; Fluorescence Resonance Energy Transfer; Hydrolysis; Models, Molecular; Nanoparticles; Protein Structure, Secondary

Cytoplasmic microtubules (MTs) continuously grow and shorten at their free plus ends, a behavior that allows them to capture membrane organelles destined for MT minus end-directed transport. In Xenopus melanophores, the capture of pigment granules (melanosomes) involves the +TIP CLIP-170, which is enriched at growing MT plus ends. Here we used Xenopus melanophores to test whether signals that stimulate minus end MT transport also enhance CLIP-170-dependent binding of melanosomes to MT tips. We found that these signals significantly (>twofold) increased the number of growing MT plus ends and their density at the cell periphery, thereby enhancing the likelihood of interaction with dispersed melanosomes. Computational simulations showed that local and global increases in the density of CLIP-170-decorated MT plus ends could reduce the half-time of melanosome aggregation by ~50%. We conclude that pigment granule aggregation signals in melanophores stimulate MT minus end-directed transport by the increasing number of growing MT plus ends decorated with CLIP-170 and redistributing these ends to more efficiently capture melanosomes throughout the cytoplasm.

Topics: Animals; Carbocyanines; Cells, Cultured; Centrosome; Computer Simulation; Cyclic AMP-Dependent Protein Kinases; Fluorescent Dyes; Isoquinolines; Kinetics; Melanophores; Melanosomes; Melatonin; Microscopy, Fluorescence; Microtubule-Associated Proteins; Microtubules; Models, Biological; Neoplasm Proteins; Protein Multimerization; Protein Stability; Sulfonamides; Xenopus

Cyanine fluorophores are commonly used in single-molecule FRET experiments with nucleic acids. We have previously shown that indocarbocyanine fluorophores attached to the 5'-termini of DNA and RNA via three-carbon atom linkers stack on the ends of the helix, orienting their transition moments. We now investigate the orientation of sulfoindocarbocyanine fluorophores tethered to the 5'-termini of DNA via 13-atom linkers. Fluorescence lifetime measurements of sulfoindocarbocyanine 3 attached to double-stranded DNA indicate that the fluorophore is extensively stacked onto the terminal basepair at 15 °C, with properties that depend on the terminal sequence. In single molecules of duplex DNA, FRET efficiency between sulfoindocarbocyanine 3 and 5 attached in this manner is modulated with helix length, indicative of fluorophore orientation and consistent with stacked fluorophores that can undergo lateral motion. We conclude that terminal stacking is an intrinsic property of the cyanine fluorophores irrespective of the length of the tether and the presence or absence of sulfonyl groups. However, compared to short-tether indocarbocyanine, the mean rotational relationship between the two fluorophores is changed by ∼60° for the long-tether sulfoindocarbocyanine fluorophores. This is consistent with the transition moments becoming approximately aligned with the long axis of the terminal basepair for the long-linker species.

Topics: Base Pairing; Carbocyanines; DNA; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Models, Molecular

In order to operate in a coordinated fashion, multisubunit enzymes use cooperative interactions intrinsic to their enzymatic cycle, but this process remains poorly understood. Accordingly, ATP number distributions in various hydrolyzed states have been obtained for single copies of the mammalian double-ring multisubunit chaperonin TRiC/CCT in free solution using the emission from chaperonin-bound fluorescent nucleotides and closed-loop feedback trapping provided by an Anti-Brownian ELectrokinetic trap. Observations of the 16-subunit complexes as ADP molecules are dissociating shows a peak in the bound ADP number distribution at 8 ADP, whose height falls over time with little shift in the position of the peak, indicating a highly cooperative ADP release process which would be difficult to observe by ensemble-averaged methods. When AlFx is added to produce ATP hydrolysis transition state mimics (ADP·AlFx) locked to the complex, the peak at 8 nucleotides dominates for all but the lowest incubation concentrations. Although ensemble averages of the single-molecule data show agreement with standard cooperativity models, surprisingly, the observed number distributions depart from standard models, illustrating the value of these single-molecule observations in constraining the mechanism of cooperativity. While a complete alternative microscopic model cannot be defined at present, the addition of subunit-occupancy-dependent cooperativity in hydrolysis yields distributions consistent with the data.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Allosteric Regulation; Animals; Biophysical Phenomena; Carbocyanines; Chaperonin Containing TCP-1; Enzymes; Fluorescent Dyes; Hydrolysis; Indoles; Kinetics; Models, Biological; Protein Subunits; Solutions

Using the principle of self-assembly, a fluorescence-based photonic network is constructed with one input and two spatially and spectrally distinct outputs. A hexagonal DNA nanoassembly is used as a scaffold to host both the input and output dyes. The use of DNA to host functional groups enables spatial resolution on the level of single base pairs, well below the wavelength of light. Communication between the input and output dyes is achieved through excitation energy transfer. Output selection is achieved by the addition of a mediator dye intercalating between the DNA base pairs transferring the excitation energy from input to output through energy hopping. This creates a tool for selective excitation energy transfer on the nanometer scale with spectral and spatial control. The ability to direct excitation energy in a controlled way on the nanometer scale is important for the incorporation of photochemical processes in nanotechnology.

Topics: Carbocyanines; Computer Simulation; DNA; Fluorescein; Fluorescence Resonance Energy Transfer; Nanotechnology

An ultrasensitive protein assay method was developed based on silver nanoparticle (AgNP) hybrid probes and metal-enhanced fluorescence. Two aptamer based silver nanoparticles, Aptamer/Oligomer-A/Cy3-modified AgNPs (Tag-A) and Aptamer/Oligomer-B/Cy3-modified AgNPs (Tag-B) were hybridized to form a silver nanoparticle aggregate that produced a red shift and broadening of the Localized Surface Plasmon Resonance (LSPR) peak. The enhanced fluorescence resulted from the increased content of Cy3 molecules and their emission resonance coupled to the broadened localized surface plasmon (LSP) of AgNP aggregate. The separation distance between Cy3 and AgNPs was 8 nm which was the most optimal for metal enhanced fluorescence and the separation distance between adjacent AgNPs was about 16 nm and this was controlled by the lengths of oligomer-A and oligomer-B. The protein array was prepared by covalently immobilizing capture antibodies on aldehyde-coated slide. After addition of protein IgE sample, two kinds of aptamer-modified AgNPs (Tag-A and Tag-B) were employed to specifically recognize IgE and form the AgNP aggregate on the arrays based on their hybridization. The detection property of the aptamer-modified AgNP aggregate was compared to two other modified aptamer-based probes, aptamer-modified Cy3 and Tag-A. The modified AgNP hybrid probe (Tag-A and Tag-B) showed remarkable superiority in both sensitivity and detection limit due to the formed AgNP aggregate. The new hybrid probe also produced a wider linear range from 0.49 to 1000 ng/mL with the detection limit reduced to 40 pg/mL (211 fM). The presented method showed that the newly designed strategy of combining aptamer-based nanomaterials to form aggregates results in a highly sensitive optical detection method based on localized surface plasmon.

Topics: Animals; Aptamers, Nucleotide; Carbocyanines; Goats; Immunoglobulin E; Metal Nanoparticles; Silver; Spectrophotometry, Ultraviolet; Surface Plasmon Resonance

We have systematically studied the fluorescence resonance energy transfer (FRET) efficiency between the photoluminescent graphene oxide (GO) and Cy3.5 dye by controlling the donor-acceptor distance with a double stranded DNA and demonstrated that the GO serves as an acceptor rather than a donor in this FRET system.

Topics: Carbocyanines; DNA; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Graphite; Luminescence; Microscopy, Atomic Force; Nucleic Acid Hybridization; Oxides

The subretinal space, which borders the retinal pigment epithelium (RPE), photoreceptors, and Müller cells, is an ideal location to deliver genetic vectors, morpholino oligos, and nanopharmaceuticals. Unfortunately, materials injected into the space tend to stay localized, and degenerative changes secondary to retinal detachment limit its usefulness. Furthermore, such injection requires penetration of the sclera, RPE/choroid, or the retina itself. Here, we developed a strategy in Xenopus to utilize the continuity of the brain ventricle and optic vesicle lumen during embryogenesis as a means to access the subretinal space.. Wild-type and transgenic embryos expressing green fluorescent protein under the rod-opsin promoter were used for optic vesicle and brain ventricle injections. For injection directly into the optic vesicle, embryos were laid on one side in clay troughs. For brain ventricle injections, embryos were placed standing in foxholes cored from agarose dishes. Linear arrays with each embryo positioned dorsal side toward the micromanipulator facilitated high throughput injections. Twenty-five micrometer micropipettes, which were positioned with a micromanipulator or by hand, were used to pressure inject ~1.0 nl of test solution (brilliant blue, India ink, fluorescein isothiocyanate dextran, or 0.04 µm of latex polystyrene microspheres [FluoSpheres®]). FluroSpheres® were particularly useful in confirming successful injections in living embryos. Anesthetized embryos and tadpoles were fixed in 4% paraformaldehyde and cryoprotected for frozen sections, or dehydrated in ethanol and embedded in methacrylate resin compatible with the microspheres.. Direct optic vesicle injections resulted in filling of the brain ventricle, contralateral optic vesicle, and central canal. Stages 24 and 25 gave the most consistent results. However, even with experience, the success rate was only ~25%. Targeting the vesicle was even more difficult beyond stage 26 due to the flattening of the lumen. In contrast, brain ventricle injections were easier to perform and had a ~90% success rate. The most consistent results were obtained in targeting the diencephalic ventricle, which is located along the midline, and protrudes anteriorly just under the frontal ectoderm and prosencephalon. An anterior midline approach conveniently accessed the ventricle without disturbing the optic vesicles. Beyond stage 30, optic vesicle filling did not occur, presumably due to closure of the connection between the ventricular system and the optic vesicles. Securing the embryos in an upright position in the agarose foxholes allowed convenient access to the frontal cephalic region. On methacrylate sections, the RPE-neural retina interphase was intact and labeled with the microspheres. As development continued, no distortion or malformation of the orbital structures was detected. In green fluorescent protein (GFP), transgenic embryos allowed to develop to stage 41, retinal FluoSpheres® labeling and photoreceptor GFP expression could be observed through the pupil. On cryosections, it was found that the FluoSpheres® extended from the diencephalon along the embryonic optic nerve to the ventral subretinal area. GFP expression was restricted to rod photoreceptors. The microspheres were restricted to the subretinal region, except focally at the lip of the optic cup, where they were present within the retina; this was presumably due to incomplete formation of the peripheral zonulae adherens. Embryos showed normal anatomic relationships, and formation of eye and lens appeared to take place normally with lamination of the retina into its ganglion cell and the inner and outer nuclear layers.. Diencephalic ventricular injection before stage 31 provides an efficient strategy to introduce molecules into the embryonic Xenopus subretinal space with minimal to the developing eye or retina.

Topics: Animals; Carbocyanines; Cerebral Ventricles; Dextrans; Diencephalon; Embryo, Nonmammalian; Fluorescein-5-isothiocyanate; Gene Transfer Techniques; Microspheres; Retina; Xanthenes; Xenopus laevis

We have developed a strategy for the detection of single protein molecules, which uses single-pair fluorescence resonance energy transfer (spFRET) as the readout modality and provides exquisite analytical sensitivity and reduced assay turn-around-time by eliminating various sample pre-processing steps. The single-protein detection assay uses two independent aptamer recognition events to form an assembly conducive to intramolecular hybridization of oligonucleotide complements that are tethered to the aptamers. This hybridization brings a donor-acceptor pair within the Förster distance to create a fluorescence signature indicative of the presence of the protein-aptamer(s) association complex. As an example of spFRET, we demonstrate the technique for the analysis of serum thrombin. The assay requires co-association of two distinct epitope-binding aptamers, each of which is labeled with a donor or acceptor fluorescent dye (Cy3 or Cy5, respectively) to produce a FRET response. The FRET response between Cy3 and Cy5 was monitored by single-molecule photon-burst detection, which provides high analytical sensitivity when the number of single-molecule events is plotted versus the target concentration. We are able to identify thrombin with high efficiency based on photon burst events transduced in the Cy5 detection channel. We also demonstrate that the technique can discriminate thrombin molecules from its analogue prothrombin. The analytical sensitivity was >200-fold better than an ensemble measurement.

Topics: Aptamers, Nucleotide; Base Sequence; Biosensing Techniques; Carbocyanines; DNA; Fluorescence Resonance Energy Transfer; Humans; Limit of Detection; Nucleic Acid Denaturation; Nucleic Acid Hybridization; Polymerase Chain Reaction; Proteins; Thrombin; Transition Temperature

A single-stranded DNA binding protein (SSB), labeled with a fluorophore, interacts with single-stranded DNA (ssDNA), giving a 6-fold increase in fluorescence. The labeled protein is the adduct of the G26C mutant of the homotetrameric SSB from Escherichia coli and a diethylaminocoumarin {N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide}. This adduct can be used to assay production of ssDNA during separation of double-stranded DNA by helicases. To use this probe effectively, as well as to investigate the interaction between ssDNA and SSB, the fluorescent SSB has been used to develop the kinetic mechanism by which the protein and ssDNA associate and dissociate. Under conditions where approximately 70 base lengths of ssDNA wrap around the tetramer, initial association is relatively simple and rapid, possibly diffusion-controlled. The kinetics are similar for a 70-base length of ssDNA, which binds one tetramer, and poly(dT), which could bind several. Under some conditions (high SSB and/or low ionic strength), a second tetramer binds to each 70-base length, but at a rate 2 orders of magnitude slower than the rate of binding of the first tetramer. Dissociation kinetics are complex and greatly accelerated by the presence of free wild-type SSB. The main route of dissociation of the fluorescent SSB x ssDNA complex is via association first with an additional SSB and then dissociation. Comparison of binding data with different lengths of ssDNA gave no evidence of cooperativity between tetramers. Analytical ultracentrifugation was used to determine the dissociation constant for labeled SSB(2) x dT(70) to be 1.1 microM at a high ionic strength (200 mM NaCl). Shorter lengths of ssDNA were tested for binding: only when the length is reduced to 20 bases is the affinity significantly reduced.

Topics: Carbocyanines; Coumarins; DNA Helicases; DNA-Binding Proteins; DNA, Bacterial; DNA, Single-Stranded; Escherichia coli Proteins; Fluorescent Dyes; Protein Binding; Spectrometry, Fluorescence

Topics: Adenosine Triphosphate; Carbocyanines; Fluorescence Resonance Energy Transfer; Humans; Ligands; Mutant Proteins; Myosin Heavy Chains; Myosin Type V; Quantum Dots; Time Factors

The hexahistidine (His(6))/nickel(II)-nitrilotriacetic acid (Ni(2+)-NTA) system is widely used for affinity purification of recombinant proteins. The NTA group has many other applications, including the attachment of chromophores, fluorophores, or nanogold to His(6) proteins. Here we explore several applications of the NTA derivative, (Ni(2+)-NTA)(2)-Cy3. This molecule binds our two model His(6) proteins, N-ethylmaleimide sensitive factor (NSF) and O(6)-alklyguanine-DNA alkyltransferase (AGT), with moderate affinity (K approximately 1.5 x 10(6) M(-1)) and no effect on their activity. Its high specificity makes (Ni(2+)-NTA)(2)-Cy3 ideal for detecting His(6) proteins in complex mixtures of other proteins, allowing (Ni(2+)-NTA)(2)-Cy3 to be used as a probe in crude cell extracts and as a His(6)-specific gel stain. (Ni(2+)-NTA)(2)-Cy3 binding is reversible in 10mM ethylenediaminetetraacetic acid (EDTA) or 500 mM imidazole, but in their absence it exchanges slowly (k(exchange) approximately 5 x 10(-6) s(-1) with 0.2 microM labeled protein in the presence of 1 microM His(6) peptide). Labeling with (Ni(2+)-NTA)(2)-Cy3 allows characterization of hydrodynamic properties by fluorescence anisotropy or analytical ultracentrifugation under conditions that prevent direct detection of protein (e.g., high ADP absorbance). In addition, fluorescence resonance energy transfer (FRET) between (Ni(2+)-NTA)(2)-Cy3-labeled proteins and suitable donors/acceptors provides a convenient assay for binding interactions and for measurements of donor-acceptor distances.

Topics: Alkyl and Aryl Transferases; Carbocyanines; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Histidine; N-Ethylmaleimide-Sensitive Proteins; Nitrilotriacetic Acid; Oligopeptides; Organometallic Compounds; Protein Interaction Domains and Motifs; Spectrometry, Fluorescence; Ultracentrifugation

A simple bifunctional colorimetric/fluorescent sensing assay is demonstrated for the detection of HIV-1 specific antibodies. This assay makes use of a short peptide sequence coupled to an environmentally sensitive dye that absorbs and emits in the visible portion of the spectrum. The core peptide sequence is derived from the highly antigenic six-residue epitope of the HIV-1 p17 protein and is situated adjacent to a terminal cysteine residue which enables site-specific fluorescent labeling with Cy3 cyanine dye. Interaction of the Cy3-labeled p17 peptide with monoclonal anti-p17 antibody resulted in an up to 4-fold increase in dye absorption and greater than 5-fold increase in fluorescent emission, yielding a limit of detection as low as 73 pM for the target antibody. This initial study demonstrates both proof-of-concept for this approach and suggests that the resulting sensor could potentially be used as a rapid screening method for HIV-1 infection while requiring minimal equipment and reagents. The potential for utilizing this assay in simple field-portable point-of-care and diagnostic devices is discussed.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibody Specificity; Carbocyanines; Cattle; Colorimetry; Epitopes; Fluorescent Dyes; HIV-1; Limit of Detection; Molecular Sequence Data; Peptides; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet

Fluorescence enhancement from a multilayered substrate fabricated with Ag and Al(2)O(3) was investigated using fluorescein, rhodamine B, Cy3, and Cy5 as fluorophores. The change in the fluorescence enhancement with Al(2)O(3) had two peaks and one valley in the range from 0 to 300 nm of Al(2)O(3) thickness, and such peaks and valley were found to appear periodically. Moreover, the reflection of the excitation light from the multilayered substrate was investigated. The reflection of the excitation light periodically changed depending on the Al(2)O(3) thickness as well, and the maximum reflection was observed near the Al(2)O(3) thickness of the peak fluorescence enhancement. It was found that the periodic changes of the fluorescence enhancement and the reflection of the excitation light could be explained, for the most part, with the integral multiples of the lambda/4 derived by a simple interference theory.

Topics: Aluminum Oxide; Carbocyanines; Fluorescein; Fluorescence; Fluorescent Dyes; Radiographic Image Enhancement; Rhodamines

We have developed an instrument for spectral cross-talk-free dual-color fluorescence cross-correlation spectroscopy (FCCS), which provides a readout modality for the study of enzyme activity in application areas such as high-throughput screening. Two spectrally distinct (approximately 250 nm) fluorophores, Cy3 and IRD800, were excited simultaneously using two different excitation sources: one poised at 532 nm and the other at 780 nm. The fluorescence information was processed on two different color channels monitored with single-photon avalanche diodes (SPADs) that could transduce events at the single-molecule level. The system provided no color cross-talk (cross-excitation and/or cross-emission) and/or fluorescence resonance energy transfer (FRET), significantly improving data quality. To provide evidence of cross-talk-free operation, the system was evaluated using bright microspheres (lambda(abs) = 532 nm, lambda(em) = 560 nm) and quantum dots (lambda(abs) = 532 nm, lambda(em) = 810 nm). Experimental results indicated that no color leakage from the microspheres or quantum dots into inappropriate color channels was observed. To demonstrate the utility of the system, the enzymatic activity of APE1, which is responsible for nicking the phosphodiester backbone in DNA on the 5' side of an apurinic/apyrimidinic site, was monitored by FCCS using a double-stranded DNA substrate dual labeled with Cy3 and IRD800. Activity of APE1 was also monitored in the presence of an inhibitor (7-nitroindole-2-carboxylic acid) of the enzyme using this cross-talk-free FCCS platform. In all cases, no spectral leakage from single-molecule events into inappropriate color channels was observed.

Topics: Base Sequence; Carbocyanines; Color; DNA; DNA-(Apurinic or Apyrimidinic Site) Lyase; Enzyme Assays; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Indoles; Reproducibility of Results; Spectrometry, Fluorescence; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Time Factors

In a nonviral gene delivery system, localization of a plasmid DNA in the nucleus is a prerequisite for expression of a desired therapeutic protein encoded in the plasmid DNA. In this study, a reducible polymer-based gene delivery system for improved intracellular trafficking and nuclear translocation of plasmid DNA is introduced. The system is consisted of two components, a plasmid DNA having repeated binding sequence for a karyophilic protein, NFkappaB, and a reducible polymer. A reducible poly(amido ethylenimine), poly(TETA-CBA), was synthesized by a Michael-type addition polymerization between cystamine bisacrylamide and triethyl tetramine. The polymer forming tight complexes with plasmid DNA could be degraded in the reductive cytosol to release the plasmid DNA. The triggered release mechanism in the cytosol could facilitate the interaction between cytosolic NFkappaB and the plasmid DNA having repeated NFkappaB biding motif. Upon activation of NFkappaB by interleukin-1beta (IL-1beta), most of the plasmid distributed in the cytoplasm was localized within the nucleus, resulting in significantly higher gene transfection efficiency than controls with nondegradable PEI. The current study suggests an alternative way of improving transfection efficiency by taking advantage of endogenous transport machinery for intracellular trafficking and nuclear translocation of a plasmid DNA.

Topics: Acrylic Resins; Active Transport, Cell Nucleus; Animals; Base Sequence; Buthionine Sulfoximine; Carbocyanines; Cell Nucleus; DNA; Mice; NIH 3T3 Cells; Oxidation-Reduction; Plasmids; Transfection

A simple molecular combing method for analysis of biochemical reactions, called the moving droplet method, has been developed. In this method, small droplets containing DNA molecules run down a sloped glass substrate, and this creates a moving interface among the air, droplet, and substrate that stretches the DNA molecules. This method requires a much smaller volume of sample solution than other established combing methods, allowing wider application in various fields. Using this method, lambdaDNA molecules were stretched and absorbed to a glass substrate, and single-molecule analysis of DNA synthesis by DNA polymerases was performed.

Topics: Animals; Carbocyanines; DNA-Directed DNA Polymerase; DNA, Viral; Fluorescent Dyes; Glass; Rats

Aptamers are short single-stranded DNA or RNA sequences that are selected in vitro based on their high affinity to a target molecule. Dye-binding aptamers are promising tools for real-time detection of not only DNA or RNA sequences but also proteins of interest both in vitro and in vivo. In this study, we aimed to isolate an RNA aptamer to Cy3, a widely used, membrane-permeant, and nontoxic fluorescent cyanine dye. Extensive selection of affinity RNA molecules to Cy3 yielded a unique sequence aptamer named Cy3_apt. The selected Cy3_apt was 83 nucleotides long and successfully shortened to 49 nucleotides long with increased affinity to Cy3 by multiple base changes. The shortest Cy3_apt is composed of two separate hairpin modules that are required for the affinity to Cy3 as monitored by the surface plasmon resonance (SPR) assay. Also, the fluorescence of Cy3 increased on binding to Cy3_apt. The two modules of Cy3_apt, when detached from each other, functioned as a binary aptamer probe. We demonstrate that the binary Cy3_apt probe is applicable to the detection of target oligonucleotides or RNA-RNA interaction by tagging with target sequences. This binary probe consists of two folded modules, referred to as a folded binary probe.

Topics: Aptamers, Nucleotide; Base Sequence; Carbocyanines; Fluorescent Dyes; Molecular Sequence Data; Nucleic Acid Conformation; RNA; SELEX Aptamer Technique; Surface Plasmon Resonance

Molecular beacons (MBs) have the potential to provide a powerful tool for rapid RNA detection in living cells, as well as monitoring the dynamics of RNA expression in response to external stimuli. To exploit this potential, it is necessary to distinguish true signal from background signal due to non-specific interactions. Here, we show that, when cyanine-dye labeled 2'-deoxy and 2'-O-methyl oligonucleotide probes are inside living cells for >5 h, most of their signals co-localize with mitochondrial staining. These probes include random-sequence MB, dye-labeled single-strand linear oligonucleotide and dye-labeled double-stranded oligonucleotide. Using carbonyl cyanide m-chlorophenyl hydrazone treatment, we found that the non-specific accumulation of oligonucleotide probes at mitochondria was driven by mitochondrial membrane potential. We further demonstrated that the dye-labeled oligonucleotide probes were likely on/near the surface of mitochondria but not inside mitochondrial inner membrane. Interestingly, oligonucleotides probes labeled respectively with Alexa Fluor 488 and Alexa Fluor 546 did not accumulate at mitochondria, suggesting that the non-specific interaction between dye-labeled ODN probes and mitochondria is dye-specific. These results may help design and optimize fluorescence imaging probes for long-time RNA detection and monitoring in living cells.

Topics: Carbocyanines; Cells, Cultured; Fluorescent Dyes; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Oligonucleotide Probes

Photosensitive probes are powerful tools to study cellular processes with high temporal and spatial resolution. However, most synthetic fluorophores suited for biomolecular imaging have not been converted yet to appropriate photosensitive analogues. Here we describe a generally applicable strategy for the generation of photoactivatable and photoconvertible fluorescent probes that can be selectively coupled to SNAP-tag fusion proteins in living cells. Photoactivatable versions of fluorescein and Cy3 as well as a photoconvertible Cy5-Cy3 probe were prepared and coupled to selected proteins on the cell surface, in the cytosol, and in the nucleus of cells. In proof-of-principle experiments, the photoactivatable Cy3 probe was used to characterize the mobility of a lipid-anchored cell surface protein and of a G protein coupled receptor (GPCR). This work establishes a generally applicable strategy for the generation of a large variety of different photosensitive fluorophores with tailor-made properties for biomolecular imaging.

Topics: Carbocyanines; Cell Membrane; Fluorescein; Fluorescent Dyes; Microscopy, Confocal; Microscopy, Fluorescence; Photochemistry; Receptors, G-Protein-Coupled; Recombinant Fusion Proteins; Spectrometry, Fluorescence

We describe a procedure for the labeling of membranous vesicular purified subcellular fractions, to image them, typically by confocal laser scanning microscopy. Being intracellular organelles, these fractions, once purified cannot be attached to glass slides as for cells. Fractions are labeled "in batch" without prior embedding or freezing. Each labeling step performed by passages of resuspension/centrifugation is followed by washings. Then samples are dispersed on the glass slides. Mammalian retinal rod outer segment disks, intact brain stem myelin vesicles, and brain synaptosomes were chosen, as these subcellular fractions can be purified by well established procedures. These fractions were immunolabeled with specific antibodies. Moreover, by the earlier procedure, we show that the mitochondrial vital membrane potential probe MitoTracker Deep Red 633 stains myelin vesicles and rod disks before fixation, consistently with our previous reports of a respiring capacity of these membranes. Therefore, the technique seems adequate to become an instrument to study the structure and the function of these and other subcellular fractions.

Topics: Animals; Carbocyanines; Cattle; Cell Fractionation; Cytoplasmic Vesicles; Fluorescent Dyes; Immunochemistry; Mice; Microscopy, Confocal; Myelin Sheath; Neostriatum; Prosencephalon; Rod Cell Outer Segment; Staining and Labeling; Subcellular Fractions; Synaptosomes

We present a new application of multispectral analysis for subcellular measurement of multiple proteins in formalin-fixed paraffin embedded tissue and cells. Typically, the targets of interest are present in the same or spatially overlapping cellular compartments. Such co-localization can complicate analysis and interpretation of the images obtained using traditional fluorescence, especially when spectrally overlapping labels are present. The spectral properties of currently available fluorescent dyes set an upper limit to the number of molecules that can be detected simultaneously with traditional fluorescence. By exciting a set of fluorophores at the same wavelength and unmixing their emission signals from background autofluorescence, we were able to image three targets in a single channel. This parallel imaging approach provides significant advantages for multiplexed analysis of tissues and cells.

Topics: Algorithms; Animals; Biomarkers; Breast; Breast Neoplasms; Carbocyanines; Cyclic AMP Response Element-Binding Protein; Female; Humans; Image Processing, Computer-Assisted; Lung; Male; Mice; Microscopy, Fluorescence; Microscopy, Fluorescence, Multiphoton; Prostate; Proteins; Signal Processing, Computer-Assisted; Tissue Array Analysis

Fluorescence resonance energy transfer is utilized to engineer donor photophysics for facile signal amplification and selective fluorescence recovery from high background. This is generalized such that many different fluorophores can be used in optical modulation schemes to drastically improve fluorescence imaging sensitivity. Dynamic, simultaneous, and direct excitation of the acceptor brightens and optically modulates higher energy donor emission. The externally imposed modulation waveform enables selective donor fluorescence extraction through demodulation. By incorporating an acceptor with significant, spectrally shifted, dark-state population, necessary excitation intensities are quite low and agree well with simulated enhancements. Enhancement versus modulation frequency directly yields dark-state lifetimes in a simple ensemble measurement. Using the long-lived Cy5 dark state in conjunction with Cy3 donors, we demonstrate image extraction from a large background to yield >>10-fold sensitivity improvements through synchronously amplified fluorescence image recovery (SAFIRe).

Topics: Base Sequence; Carbocyanines; DNA; Fluorescence Resonance Energy Transfer; Inverted Repeat Sequences; Molecular Imaging; Optical Phenomena

Gene therapy is a promising therapeutic concept for a large number of incurable diseases. Lipid/DNA complexes (lipoplexes) are used to deliver genes into cells. However, while large efforts have been made to investigate the fate of lipoplexes once inside the cell, the rate of intracellular dissociation is still largely unknown. Analysis of the dissociation rates of DNA from lipid/DNA complexes is crucial for the evaluation of a gene delivery system's efficiency. This study introduces a new fluorescence resonance energy transfer (FRET) approach for the intracellular dissociation analysis of lipid/DNA complexes. Here, the labeling of both complex components, DNA as well as lipid, reveals whether DNA is still associated with the lipid or has dissociated. In this study the kinetic properties of complex dissociation were consistently measured with flow cytometry and fluorescence microscopy, and indicated that most complexes were dissociated after 24h in A-10 cells.

Topics: Animals; Carbocyanines; Cells, Cultured; DNA; Flow Cytometry; Fluorescence Resonance Energy Transfer; Lipids; Microscopy, Fluorescence; Muscle, Smooth; Rats

According to previously published ultrastructural studies, oligodendrocytes in white matter exhibit gap junctions with astrocytes, but not among each other, while in vitro oligodendrocytes form functional gap junctions. We have studied functional coupling among oligodendrocytes in acute slices of postnatal mouse corpus callosum. By whole-cell patch clamp we dialyzed oligodendrocytes with biocytin, a gap junction-permeable tracer. On average 61 cells were positive for biocytin detected by labeling with streptavidin-Cy3. About 77% of the coupled cells stained positively for the oligodendrocyte marker protein CNPase, 9% for the astrocyte marker GFAP and 14% were negative for both CNPase and GFAP. In the latter population, the majority expressed Olig2 and some NG2, markers for oligodendrocyte precursors. Oligodendrocytes are known to express Cx47, Cx32 and Cx29, astrocytes Cx43 and Cx30. In Cx47-deficient mice, the number of coupled cells was reduced by 80%. Deletion of Cx32 or Cx29 alone did not significantly reduce the number of coupled cells, but coupling was absent in Cx32/Cx47-double-deficient mice. Cx47-ablation completely abolished coupling of oligodendrocytes to astrocytes. In Cx43-deficient animals, oligodendrocyte-astrocyte coupling was still present, but coupling to oligodendrocyte precursors was not observed. In Cx43/Cx30-double deficient mice, oligodendrocyte-to-astrocyte coupling was almost absent. Uncoupled oligodendrocytes showed a higher input resistance. We conclude that oligodendrocytes in white matter form a functional syncytium predominantly among each other dependent on Cx47 and Cx32 expression, while astrocytic connexins expression can promote the size of this network.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Antigens; Astrocytes; Basic Helix-Loop-Helix Transcription Factors; Carbocyanines; Connexin 30; Connexins; Corpus Callosum; Gap Junction beta-1 Protein; Gap Junctions; Glial Fibrillary Acidic Protein; In Vitro Techniques; Lysine; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Tissue Proteins; Oligodendrocyte Transcription Factor 2; Oligodendroglia; Patch-Clamp Techniques; Proteoglycans; Stem Cells; Streptavidin

To investigate the feasibility of molecular beacons (MBs) for screening urine samples from patients with suspected bladder cancer. Our previous study showed that MBs could detect bladder cancer cells and cells shed in the urine from patients with bladder cancer.. Samples from 35 patients with bladder cancer and 35 healthy adults were initially evaluated. Cyanine 3-labeled MBs linked to a survivin mRNA probe were used to detect exfoliative cells in urine. Exfoliative cytology, enzyme-linked immunosorbent assay, and Western blotting of the tissues were used to confirm the MB results. We then evaluated the urine samples from 187 patients with suspected bladder cancer. All 187 patients also underwent cystoscopy.. In the initial cohort evaluated, MBs detected cancerous cells in 28 (80%) of the 35 patients with confirmed bladder cancer. Survivin protein was detected by Western blotting in 25 (71.4%) of the 35 patients. The sensitivity and specificity of enzyme-linked immunosorbent assay was 54.3% (20 of 35) and 68.6% (24 of 35), respectively. In a large group of patients with suspected bladder cancer, the sensitivity of MBs was 77.3% (85 of 110) and the specificity was 76.6% (59 of 77) compared with the cystoscopy data. Differences in the protein levels between the tumor grades and stages were not significant.. Our results have demonstrated that it is feasible to detect survivin mRNA in the exfoliated cells in urine using MBs. With further development, MBs could be used in a noninvasive clinical diagnostic procedure for the early detection of bladder cancer and postoperative follow-up.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Carbocyanines; Carcinoma, Transitional Cell; Feasibility Studies; Female; Humans; Inhibitor of Apoptosis Proteins; Male; Microtubule-Associated Proteins; Middle Aged; Molecular Diagnostic Techniques; Sensitivity and Specificity; Survivin; Urinary Bladder Neoplasms; Urine; Young Adult

In traditional biochemical experiments, the behavior of individual proteins is obscured by ensemble averaging. To better understand the behavior of proteins that bind to and/or translocate on DNA, we have developed instrumentation that uses optical trapping, microfluidic solution delivery, and fluorescent microscopy to visualize either individual proteins or assemblies of proteins acting on single molecules of DNA. The general experimental design involves attaching a single DNA molecule to a polystyrene microsphere that is then used as a microscopic handle to manipulate individual DNA molecules with a laser trap. Visualization is achieved by fluorescently labeling either the DNA or the protein of interest, followed by direct imaging using high-sensitivity fluorescence microscopy. We describe the sample preparation and instrumentation used to visualize the interaction of individual proteins with single molecules of DNA. As examples, we describe the application of these methods to the study of proteins involved in recombination-mediated DNA repair, a process essential for the maintenance of genomic integrity.

Topics: Antibodies; Carbocyanines; DNA; Exodeoxyribonuclease V; Fluorescent Dyes; Humans; Microfluidic Analytical Techniques; Microscopy, Fluorescence; Nanoparticles; Optical Tweezers; Proteins; Rad51 Recombinase; Rec A Recombinases

Topics: Azo Compounds; Base Sequence; Carbocyanines; DNA; Fluorescent Dyes; Oligonucleotide Probes; Spectrometry, Fluorescence; Spectrophotometry

Treatment with monoclonal antibody (mAbs) is a viable therapeutic option in cancer. Recently, these mAbs such as cetuximab, herceptin, etc., have been used as targeting agents to selectively deliver chemotherapeutics to cancerous cells. However, mechanisms of nanoparticles-mAbs interactions with the target cells and its effect on intracellular trafficking and mechanism are currently unknown. In this paper, we demonstrate that the distinct patterning and dynamics of anti-EGFR (epidermal growth factor receptor) antibody cetuximab (C225)- induced EGFR internalization in pancreatic cancer cells with variable receptor expression is altered upon nanoconjugation. Nanoconjugation uniformly enhanced C225-induced EGFR endocytosis in PANC-1, AsPC-1, and MiaPaca-2 cells, influenced its compartmentalization and regulated the involvement of dynamin-2 in the endocytic processes. Receptor endocytosis and its intracellular trafficking were monitored by confocal microscopy and transmission electron microscopy. The role of dynamin-2 in EGFR endocytosis was determined after overexpressing either wild-type dynamin-2 or mutant dynamin-2 in pancreatic cancer cells followed by tracking the receptor-antibody complex internalization by confocal microscopy. Significantly, these findings demonstrate that the nanoconjugation cannot be construed as an innocuous reaction involved in attaching the targeting agent to the nanoparticle, instead it may distinctly alter the cellular processes at the molecular level, at least antibody induced receptor endocytosis. This information is critical for successful design of a nanoparticle-based targeted drug delivery system for future clinical translation.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Biological Transport; Carbocyanines; Cell Line, Tumor; Cetuximab; Dynamin II; Endocytosis; Endosomes; ErbB Receptors; Gold; Golgi Apparatus; Humans; Lysosomes; Metal Nanoparticles; Microscopy, Confocal; Microscopy, Electron, Transmission; Models, Biological; Mutation; Pancreatic Neoplasms

We present an efficient method to synthesize three new asymmetric trimethine cyanine dyes containing only one carboxylic acid group for bioconjugation. Two of them have better protein labeling performance than other conventional cyanine dyes due to their particular structure design.

Topics: Animals; Carbocyanines; Cattle; Fluorescent Dyes; Molecular Structure; Serum Albumin, Bovine; Spectrometry, Fluorescence

The use of highly active beta-agonists as growth promoters is not appropriate because of the potential hazard for human and animal health. To investigate the residue level of these beta-agonists, hapten microarrays were employed for clenbuterol (CLB), ractopamine (RAC) and salbutamol (SAL) residue analysis. CLB, RAC and SAL conjugates were immobilized on the slides, which were precoated by agarose film to construct hapten microarrays, and then the corresponding monoclonal antibodies of these beta-agonists and the standards or samples were introduced for indirect competitive immunoassay. Finally, Cy3-labeled secondary antibody was employed to indicate the antigen-antibody complex. The fluorescence intensity of each spot was imaged and recorded, and the calibration curve of each analyte was obtained by plot fluorescence intensity against different standard concentrations. Compared to the ELISA, the hapten microarray method was more sensitive, which got the detection limits 0.09 microg/L for CLB, 0.50 microg/L for RAC, and 0.01 microg/L for SAL. What's more, with the recovery rate between 96.5% and 106.4%, and the coefficient of variation below 10%, the proposed hapten microarray method was shown to be both quantitative and reproducible.

Topics: Adrenergic beta-Agonists; Animals; Antibodies, Immobilized; Calibration; Carbocyanines; Cattle; Environmental Pollutants; Haptens; Immunoassay; Microarray Analysis; Microscopy, Confocal; Serum Albumin, Bovine; Spectrometry, Fluorescence

Microarray technology has received considerable attention for rapid analysis of biomolecular interactions and high-throughput screening to identify binding partners. An efficient and selective immobilization technique of substances on the surface is essential for successful construction of microarrays. Although a variety of immobilization methods have been exploited to prepare microarrays over the past decade, a superior technique needs to be developed for diverse applications. Recently, an efficient and simple method that relies on selective reactions between the hydrazide conjugated to substances and the epoxide derivatized on the solid surface was developed to fabricate chemical microarrays. Reactions between hydrazides with epoxides are highly selective in that they take place even in the presence of other potent nucleophiles such as amines and thiols. This technique is utilized to immobilize various substances such as small molecules, carbohydrates, and peptides to glass surfaces. The microarrays constructed by this immobilization method are used to evaluate protein binding to carbohydrates, peptides, and small molecules. In addition, the microarrays are also employed to determine binding affinities between proteins and binding partners as well as profiling of enzyme activities.

Topics: Animals; Carbocyanines; Carbohydrate Metabolism; Enzyme Assays; Epoxy Compounds; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Glass; Hydrazines; Immobilized Proteins; Microarray Analysis; Microtechnology; N-Acetyllactosamine Synthase; Peptides; Substrate Specificity; Surface Properties

To compare the fluorescence intensity and duration of qdots streptavidin conjugate (QDs-SA) with Cy3 as the molecular probe of β-amyloid precursor protein (APP), and to provide evidence for early molecular imaging and diagnosis of Alzheimer's disease (AD).. With the help of laser scanning confocal microscope and flow cytometry, the flurescence probe based on the QDs-SA was used to detect APP in HEK293 cells stably transfected pcDNA3.1/APP, and to compare with conventional fluroimmunoassay Cy3.. The immunofluorescence staining detection indicated APP expression was mainly located in the plasma membrane. The mean fluorescence intensity of QDs-SA (34.2336±4.2455) was greater than that of Cy3 (21.6023±3.0102)under the confocal fluorescence microscope (P<0.05). After persistent exciting for 12 min, the fluorescence intensity of APP stained by QDs-SA decreased by 27.87%. The other stained by Cy3 decreased by 79.60%. The positive rate of APP staining had no significant difference between the QDs-SA(54.4700±3.4433)% and Cy3 (54.3800±8.5229)% by flow cytometry, but the mean fluorescence intensity had statistical significance(P<0.05). The QDs-SA (1 045.4167±47.3623) was significantly higher than the mean fluorescence intensity of Cy3 (658.5467±55.0591).. QDs-SA fluorescence probes can effectively recognize APP and are sensitive and exceptionally photostable, suggesting that QDs-SA fluorescence probes could be a potential method in APP detection and offer a novel way for the diagnosis of Alzheimer's disease.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Carbocyanines; Fluorescent Dyes; Gene Transfer Techniques; HEK293 Cells; Humans; Molecular Imaging; Quantum Dots

We describe the self-assembly of a DNA crystal that contains two tensegrity triangle molecules per asymmetric unit. We have used X-ray crystallography to determine its crystal structure. In addition, we have demonstrated control over the colors of the crystals by attaching either Cy3 dye (pink) or Cy5 dye (blue-green) to the components of the crystal, yielding crystals of corresponding colors. Attaching the pair of dyes to the pair of molecules yields a purple crystal.

Topics: Base Sequence; Carbocyanines; Crystallography, X-Ray; DNA; Models, Molecular; Molecular Sequence Data

REIC/Dkk-3 was first identified as a down-regulated gene in a number of human immortalized cells and human tumor-derived cell lines. Overexpression of the REIC/Dkk-3 gene using an adenovirus vector (Ad-REIC) has showed a potent selective therapeutic effect on various human cancers through induction of ER stress. Furthermore, we recently showed that Ad-REIC has an indirect host-mediated anti-tumor activity by induction of IL-7. However, the physiological function of REIC/Dkk-3 is still unclear. As a first step to study the possible receptor(s) for secreted REIC/Dkk-3, we analyzed the internalization of Cy3-labeled recombinant REIC/Dkk-3 protein. Among the cell lines screened, mouse induced pluripotent stem (iPS) cells showed a unique pattern of internalization. The internalization was observed in peripheral cells of spherical colonies formed spontaneously, but not in undifferentiated iPS cells. When we analyzed embryoid bodies (EBs) derived from iPS cells, REIC/Dkk-3 protein was internalized specifically by differentiated cells located at the periphery of EBs. Interestingly, Dkk-1 was internalized by undifferentiated cells at the center of the EBs. When developmental tissue was analyzed, internalization of REIC/Dkk-3 protein was strictly limited to extra-embryonic tissue, such as the trophectoderm layer of 4.5 days post-coitus (dpc) blastocysts and the chorionic membrane at 16.5 dpc. The mechanism of the internalization was confirmed to be endocytosis. These findings will contribute to knowledge on the interaction of REIC/Dkk-3 with a possible receptor(s).

Topics: Adaptor Proteins, Signal Transducing; Animals; Carbocyanines; Cell Line; Chemokines; Embryoid Bodies; Endocytosis; Flow Cytometry; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Mice; Pluripotent Stem Cells; Recombinant Proteins

To improve the sensitivity of fluorescence detection in biochip, a new kind of substrates was developed by agarose coating on silica opal film. In this study, silica opal film was fabricated on glass substrate using the vertical deposition technique. It can provide stronger fluorescence signals and thus improve the detection sensitivity. After coating with agarose, the hybrid film could provide a 3D support for immobilizing sample. Comparing with agarose-coated glass substrate, the agarose-coated opal substrates could selectively enhance particular fluorescence signals with high sensitivity when the stop band of the silica opal film in the agarose-coated opal substrate overlapped the fluorescence emission wavelength. A DNA hybridization experiment demonstrated that fluorescence intensity of special type of agarose-coated opal substrates was about four times that of agarose-coated glass substrate. These results indicate that the optimized agarose-coated opal substrate can be used for improving the sensitivity of fluorescence detection with high quality and selectivity.

Topics: Carbocyanines; Drug Stability; Glass; Materials Testing; Minerals; Oligonucleotide Array Sequence Analysis; Sensitivity and Specificity; Sepharose; Silicon Dioxide; Spectrometry, Fluorescence

Topics: Awards and Prizes; Carbocyanines; Cells, Cultured; Coated Pits, Cell-Membrane; Fluorescent Dyes; Image Processing, Computer-Assisted; Imaging, Three-Dimensional; Microscopy, Fluorescence; Microtubules; Ultraviolet Rays

Combining the inherent scaffolding provided by DNA structure with spatial control over fluorophore positioning allows the creation of DNA-based photonic wires with the capacity to transfer excitation energy over distances greater than 150 Å. We demonstrate hybrid multifluorophore DNA-photonic wires that both self-assemble around semiconductor quantum dots (QDs) and exploit their unique photophysical properties. In this architecture, the QDs function as both central nanoscaffolds and ultraviolet energy harvesting donors that drive Förster resonance energy transfer (FRET) cascades through the DNA wires with emissions that approach the near-infrared. To assemble the wires, DNA fragments labeled with a series of increasingly red-shifted acceptor-dyes were hybridized in a predetermined linear arrangement to a complementary DNA template that was chemoselectively modified with a hexahistidine-appended peptide. The peptide portion facilitated metal-affinity coordination of multiple hybridized DNA-dye structures to a central QD completing the final nanocrystal-DNA photonic wire structure. We assembled several such hybrid structures where labeled-acceptor dyes were excited by the QDs and arranged to interact with each other via consecutive FRET processes. The inherently facile reconfiguration properties of this design allowed testing of alternate formats including the addition of an intercalating dye located in the template DNA or placement of multiple identical dye acceptors that engaged in homoFRET. Lastly, a photonic structure linking the central QD with multiple copies of DNA hybridized with 4-sequentially arranged acceptor dyes and demonstrating 4-consecutive energy transfer steps was examined. Step-by-step monitoring of energy transfer with both steady-state and time-resolved spectroscopy allowed efficiencies to be tracked through the structures and suggested that acceptor dye quantum yields are the predominant limiting factor. Integrating such DNA-based photonic structures with QDs can help create a new generation of biophotonic wire assemblies with widespread potential in nanotechnology.

Topics: Carbocyanines; DNA; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Histidine; Oligopeptides; Photochemical Processes; Photons; Quantum Dots

Topics: Carbocyanines; Cytological Techniques; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Models, Molecular; Molecular Probe Techniques; Quantum Dots; Sequence Analysis, DNA

We report a layer-by-layer approach to the assembly of ultrathin and erodible DNA-containing films on the surfaces of polymer microparticles. DNA-containing multilayered films were fabricated layer-by-layer on the surfaces of polystyrene microspheres (approximately 6 microm) by iterative and alternating cycles of particle suspension, centrifugation and resuspension in solutions of plasmid DNA and a hydrolytically degradable polyamine. Film growth occurred in a stepwise manner, as demonstrated by characterization of the zeta potentials and fluorescence intensities of film-coated particles during film assembly. Characterization of film-coated particles by confocal fluorescence microscopy and scanning electron microscopy revealed the multilayered particle coatings to be smooth, uniform and free of large-scale physical defects. Film-coated microparticles sustained the release of transcriptionally active DNA into solution for approximately three days when incubated in physiologically relevant media. Previous studies have demonstrated that the adsorption of DNA onto the surfaces of cationic microparticles can be used to target the delivery of DNA to antigen-presenting cells. As a first step toward the application of this layer-by-layer approach to the development of methods for the delivery of DNA to antigen-presenting cells, we demonstrated that film-coated microparticles could be used to transport DNA into macrophage cells in vitro using a model mouse macrophage cell line. Our results suggest the basis of a general approach that could, with further development, prove useful for the delivery of DNA-encoded antigens to macrophages, or other antigen-presenting cells, and provide new materials-based methods for the formulation and delivery of DNA vaccines.

Topics: Animals; Antigen-Presenting Cells; Biotechnology; Carbocyanines; Cell Line; Chlorocebus aethiops; Coated Materials, Biocompatible; COS Cells; DNA; Electrolytes; Fluorescent Dyes; Gene Transfer Techniques; Gold; Green Fluorescent Proteins; Macrophages; Mice; Microspheres; Models, Chemical; Molecular Structure; Molecular Weight; Particle Size; Plasmids; Polymers; Polystyrenes; Rotation; Temperature; Time Factors; Water

We report on the role of dye-nucleobase interactions on the photophysical properties of the indocarbocyanine Cy3. The fluorescence efficiency and lifetime ofCy3 increase in the presence of all four nucleoside monophosphates. This behavior correlates with an increase in the activation energy for photoisomerization and a approximately 4 nm red shift in the fluorescence spectrum. Changes are more dramatic for the purines (dAMP, dGMP) than the pyrimidines(dCMP, dTMP), and for the nonsulfonated cyanine(DiIC2(3)) than the sulfonated dye (Cy3-SE). These results are consistent with a model in which Cy3-nucleoside pi-pi interactions decrease the efficiency of photoisomerization,increasing the efficiency of fluorescence.

Topics: Absorption; Carbocyanines; DNA; Fluorescence; Fluorescent Dyes; Nucleotides; Spectrometry, Fluorescence; Stereoisomerism

Bevacizumab is a potent recombinant humanized monoclonal antibody directed against vascular endothelial growth factor (VEGF). The purpose of this study was to evaluate the therapeutic effect of subconjunctival injection of bevacizumab on corneal neovascularization (NV) in different rabbit models.. Several rabbit models of corneal NV were used, including (1) a corneal micropocket assay with VEGF pellet, (2) a corneal micropocket assay with basic fibroblast growth factor (b-FGF) pellets, (3) mechanical limbal injury-induced corneal NV, and (4) an alkali-induced model of corneal NV. Subconjunctival injections of bevacizumab (0.25-2.5 mg) were applied twice per week for 2 to 8 weeks. Digital photographs of the cornea were analyzed to determine the length of corneal NV and the area of cornea covered by NV as a percentage of the total corneal area. Immunohistochemical staining with anti-human IgG antibody labeled with Cy3 was used to determine the detection of intracorneal distribution of bevacizumab after injection.. Subconjunctival injection of bevacizumab caused significant inhibition of corneal NV formation as measured by length or surface area in all animal models (P<0.05). No significant ocular complications were found. Staining of bevacizumab was found in the corneal stroma for 3 to at least 14 days in the different rabbit models.. Subconjunctival injection of bevacizumab is effective in inhibiting corneal NV in several rabbit models. Bevacizumab may diffuse into the corneal stroma and persist for a few days after injection. It may be useful in preventing corneal NV in the acute phase of various kinds of corneal inflammation.

Topics: Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bevacizumab; Blotting, Western; Carbocyanines; Conjunctiva; Corneal Neovascularization; Disease Models, Animal; Endothelium, Vascular; Female; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Injections; Platelet Endothelial Cell Adhesion Molecule-1; Rabbits; Vascular Endothelial Growth Factor A

The signal transduction pathway leading from the insulin receptor to stimulate the fusion of vesicles containing the glucose transporter GLUT4 with the plasma membrane in adipocytes and muscle cells is not completely understood. Current evidence suggests that in addition to the Rab GTPase-activating protein AS160, at least one other substrate of Akt (also called protein kinase B), which is as yet unidentified, is required. Sec8 is a component of the exocyst complex that has been previously implicated in GLUT4 trafficking. In the present study, we report that insulin stimulates the phosphorylation of Sec8 on Ser-32 in 3T3-L1 adipocytes. On the basis of the sequence around Ser-32 and the finding that phosphorylation is inhibited by the PI3K (phosphoinositide 3-kinase) inhibitor wortmannin, it is likely that Akt is the kinase for Ser-32. We examined the possible role of Ser-32 phosphorylation in the insulin-stimulated trafficking of GLUT4, as well as the TfR (transferrin receptor), to the plasma membrane by determining the effects of overexpression of the non-phosphorylatable S32A mutant of Sec8 and the phosphomimetic S32E mutant of Sec8. Substantial overexpression of both mutants had no effect on the amount of GLUT4 or TfR at the cell surface in either the untreated or insulin-treated states. These results indicate that insulin-stimulated phosphorylation of Sec8 is not part of the mechanism by which insulin enhances the fusion of vesicles with the plasma membrane.

Topics: 3T3-L1 Cells; Adipocytes; Animals; Carbocyanines; Carrier Proteins; Cells, Cultured; Culture Media, Serum-Free; Electroporation; Epitopes; Exocytosis; Fluorescent Antibody Technique, Direct; Fluorescent Dyes; Genes, Reporter; Glucose Transporter Type 4; Green Fluorescent Proteins; Hemagglutinins; Hypoglycemic Agents; Insulin; Membrane Proteins; Mice; Phosphorylation; Plasmids; Time Factors

Single molecule fluorescent microscopy is a method for the analysis of the dynamics of biological macromolecules by detecting the fluorescence signal produced by fluorophores associated with the macromolecule. Two fluorophores located in a close proximity may result in Förster resonance energy transfer (FRET), which can be detected at the single molecule level and the efficiency of energy transfer calculated. In most cases, the experimentally observed distribution of FRET efficiency exhibits a significant width corresponding to 0.07-0.2 (on a scale of 0-1). Here, we present a general approach describing the analysis of experimental data for a DNA/RNA duplex. We have found that for a 15 bp duplex with Cy3 and Cy5 fluorophores attached to the opposite ends of the helix, the width of the energy transfer distribution is mainly determined by the photon shot noise and the orientation factor, whereas the variation of inter-dye distances plays a minor role.

Topics: Algorithms; Carbocyanines; Computer Simulation; DNA; Fluorescence; Fluorescence Resonance Energy Transfer; Models, Molecular; Photons; RNA

In Streptococcus uberis, the fluoroquinolone antibiotic ciprofloxacin induces a mutagenic response that is distinct from the SOS paradigm. Two-dimensional differential gel electrophoresis was employed to investigate the effect of ciprofloxacin exposure on the proteome of S. uberis. Twenty-four protein spots exhibiting differential expression (p < 0.05) were identified as enzymes with potential role in oxidative stress, NADH generation and nucleotide biosynthesis. We suggest that these metabolic changes provide S. uberis means to stimulate mutagenesis and adaptation.

Topics: Base Sequence; Carbocyanines; Ciprofloxacin; Electrophoresis, Gel, Two-Dimensional; Image Processing, Computer-Assisted; Isoelectric Focusing; Molecular Sequence Data; Mutagenesis; Nucleotides; Oxidative Stress; Proteomics; Reproducibility of Results; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Streptococcus

We describe an optimized procedure for replacing the dihydrouridine residues of charged tRNAs with Cy3 and Cy5 dyes linked to a hydrazide group, and demonstrate that the labeled molecules are functional in ribosomal activities including 30S initiation complex formation, EF-Tu-dependent binding to the ribosome, translocation, and polypeptide synthesis. This procedure should be straightforwardly generalizable to the incorporation of other hydrazide-linked fluorophores into tRNA or other dihydrouridine-containing RNAs. In addition, we use a rapid turnover FRET experiment, measuring energy transfer between Cy5-labeled tRNA(fMet) and Cy3-labeled fMetPhe-tRNA(Phe), to obtain direct evidence supporting the hypothesis that the early steps of translocation involve movements of the flexible 3'-single-stranded regions of the tRNAs, with the considerable increase in the distance separating the two tRNA tertiary cores occurring later in the process.

Topics: Carbocyanines; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Methods; Nucleic Acid Conformation; Peptide Elongation Factor Tu; Peptides; Poly U; Protein Biosynthesis; Ribosome Subunits, Small; RNA, Fungal; RNA, Transfer; RNA, Transfer, Amino Acyl; RNA, Transfer, Met; Uridine

Determining the long-range haplotypes in a diploid individual is a major technical challenge. Here we report a method of molecular haplotyping by directly imaging multiple polymorphic sites on individual human DNA molecules simultaneously. We demonstrate the utility of this technology by accurately determining the haplotypes consisting of up to 16 single-nucleotide polymorphisms in genomic regions up to 50 kilobases.

Topics: Carbocyanines; Chromosomes, Human, Pair 17; Chromosomes, Human, Pair 7; Chromosomes, Human, Pair 8; DNA; DNA Primers; DNA-Directed DNA Polymerase; DNA, Single-Stranded; Exodeoxyribonucleases; Genetic Techniques; Haplotypes; Heterozygote; Homozygote; Humans; Microscopy, Fluorescence; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Sequence Analysis, DNA; Staining and Labeling; Tissue Plasminogen Activator

Methods for the in-depth study of the physics of microscale actuation of microfluidics environments by flagellated bacteria 'teamsters' have been developed. These methods, which include single and multi-colour fluorescent labelling and electron microscopy allow for the analysis of the effect that individual flagellar filaments have on bacterially driven microstructures, and allow for the investigation of the interaction and coordination of flagellar filaments of neighbouring bacteria on densely packed monolayers of bacteria, 'bacterial carpets'. We show that the flagella of bacteria that are immobilized on a surface often interact with each other, and that the flagella of these bacteria do not often form multi-flagella bundles that are aligned with the cell body.

Topics: Carbocyanines; Flagella; Fluorescent Dyes; Microfluidics; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Movement; Organic Chemicals; Salmonella typhimurium; Serratia marcescens

We report here a novel, water-soluble, nonfluorescent dye that efficiently quenches fluorescence from a broad range of visible and near-infrared (NIR) fluorophores in Förster resonance energy transfer (FRET) systems. A model FRET-based caspase-3 assay system was used to test the performance of the quencher dye. Fluorogenic caspase-3 substrates were prepared by conjugating the quencher, IRDye QC-1, to a GDEVDGAK peptide in combination with fluorescein (emission maximum approximately 540 nm), Cy3 (approximately 570 nm), Cy5 (approximately 670 nm), IRDye 680 (approximately 700 nm), IRDye 700DX (approximately 690 nm), or IRDye 800CW (approximately 790 nm). The Förster distance R(0) values are calculated as 41 to 65A for these dye/quencher pairs. The fluorescence quenching efficiencies of these peptides were determined by measuring the fluorescence change on complete cleavage by recombinant caspase-3 and ranged from 97.5% to 98.8%. The fold increase in fluorescence on caspase cleavage of the fluorogenic substrates ranged from 40 to 83 depending on the dye/quencher pair. Because IRDye QC-1 effectively quenches both the NIR fluorophores (e.g., IRDye 700DX, IRDye 680, IRDye 800CW) and the visible fluorophores (e.g., fluorescein, Cy3, Cy5), it should find broad applicability in FRET assays using a wide variety of fluorescent dyes.

Topics: Carbocyanines; Caspase 3; Fluorescein; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Jurkat Cells; Spectroscopy, Near-Infrared

Progress toward clinical application of biodegradable fluorescent calcium phosphate (CP) nanoparticles as a bioimaging agent requires detailed knowledge of chromophore interaction with CP. As readouts of this cargo-matrix interaction, we determined the principle photophysical properties of Cy3 encapsulated in CP nanparticles (CPNPs) using steady-state and time-resolved fluorescence spectroscopy. Fluorescence correlation spectroscopy (FCS)-determined diffusion coefficients and associated hydrodynamic radii confirmed the presence of highly monodisperse CPNPs with radii ranging from 7 to 10 nm. Single CP nanoparticles were 20 times brighter than free dye molecules because of a CP-induced 5-fold increase in quantum efficiency and encapsulation of four dye molecules per particle. Solvatochromic shifts resulting from hydrogen bonding between free dye and solvent or restricted intramolecular mobility by solvent viscosity were absent when Cy3 was encapsulated in CP. Encapsulation-mediated increases in radiative decay rates and decreases in nonradiative decay rates resulting in longer fluorescence lifetimes of Cy3 were attributed to solvent and CP-related local refractive indices and restricted flexibility of dye by rigid CP. Enhanced brightness of CPNPs enabled imaging of single nanoparticles under epifluorescence using both standard and total internal reflection fluorescence (TIRF) modes with camera exposure times on the order of tens of milliseconds. These enhanced photophysical properties together with excellent biocompatibility make CPNPs ideal for bioimaging applications ranging from single-molecule tracking to in vivo tumor detection and offer the possibility of timed codelivery of drugs to control cell function.

Topics: Calcium Phosphates; Carbocyanines; Nanoparticles; Photochemistry; Spectrometry, Fluorescence

Discrete chromatin domains (ChrD), containing an average of approximately 1 Mbp DNA, represent the basic structural units for the regulation of DNA organization and replication in situ. In this study, a bio-computational approach is employed to simultaneously measure the translational motion of large populations of ChrD in the cell nucleus of living cells. Both movement and configurational changes are strikingly higher in early S-phase replicating ChrD compared to those that replicate in mid and late S-phase. The chromatin dynamics was not sensitive to transcription inhibition by alpha-amanitin but was significantly reduced by actinomycin D treatment. Since a majority of active genes replicate in early S-phase, our results suggest a correlation between levels of chromatin dynamics and chromatin poised for active transcription. Analysis of ChrD colocalization with transcription sites and cDNA with ChrD and transcription sites further supports this proposal.

Topics: Alpha-Amanitin; Carbocyanines; Cell Nucleus; Chromatin; Chromosome Positioning; Dactinomycin; Deoxyuracil Nucleotides; DNA Replication; Gene Expression; HeLa Cells; Humans; In Situ Hybridization, Fluorescence; Kinetics; Microinjections; Microscopy, Fluorescence; S Phase; Time Factors

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs. The array is produced in our laboratory for use by researchers studying gene expression in pine and other conifer species. PtGen2 was developed as a result of our gene discovery efforts in loblolly pine, and is comprised of sequences identified primarily from root tissues, but also from needle and stem. PtGen2 has been tested by hybridizing different Cy-dye labeled conifer target cDNAs, using both amplified and non-amplified indirect labeling methods, and also tested with a number of hybridization and washing conditions. This video focuses on the handling and processing of slides before and after pre-hybridization, as well as after hybridization, using some modifications to procedures developed previously. Also included, in text form only, are the protocols used for the generation, labeling and clean up of target cDNA s, as well as information on software used for downstream data processing. PtGen2 is printed with a proprietary print buffer that contains high concentrations of salt that can be difficult to remove completely. The slides are washed first in a warm SDS solution prior to pre-hybridization. After pre-hybridization, the slides are washed vigorously in several changes of water to complete removal of remaining salts. LifterSlips are then cleaned and positioned on the slides and labeled cDNA is carefully loaded onto the microarray by way of capillary action which provides for even distribution of the sample across the slide, and reduces the chance of bubble incorporation. Hybridization of targets to the array is done at 48 degrees C in high humidity conditions. After hybridization, a series of standard washes are done at 53 degrees C and room temperature for extended times. Processing PtGen2 slides using this technique reduces salt and SDS-derived artifacts often seen when the array is processed less rigorously. Hybridizing targets derived from several different conifer RNA sources, this processing protocol yielded fewer artifacts, reduced background, and provided better consistency among different experimental groups of arrays.

Topics: Carbocyanines; DNA, Complementary; DNA, Plant; Fluorescent Dyes; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Pinus taeda; RNA, Plant

The pattern of homoeologous metaphase I (MI) pairing has been fully characterized in durum wheat x Aegilops cylindrica hybrids (2n = 4x = 28, ABC(c)D(c)) by an in situ hybridization procedure that has permitted individual discrimination of every wheat and wild constituent genome. One of the three hybrid genotypes examined carried the ph1c mutation. In all cases, MI associations between chromosomes of both species represented around two-third of total. Main results from the analysis are as follows (a) the A genome chromosomes are involved in wheat-wild MI pairing more frequently than the B genome partners, irrespective of the alien genome considered; (b) both durum wheat genomes pair preferentially with the D(c) genome of jointed goatgrass. These findings are discussed in relation to the potential of genetic transference between wheat crops and this weedy relative. It can also be highlighted that inactivation of Ph1 provoked a relatively higher promotion of MI associations involving B genome.

Topics: Avidin; Biotinylation; Carbocyanines; Chromosomes, Plant; Crosses, Genetic; DNA, Plant; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Gene Transfer Techniques; Genome, Plant; Genotype; In Situ Hybridization; Meiosis; Metaphase; Poaceae; Polyploidy; Triticum

To visualize native or non-engineered RNA in live cells with single-molecule sensitivity, we developed multiply labeled tetravalent RNA imaging probes (MTRIPs). When delivered with streptolysin O into living human epithelial cancer cells and primary chicken fibroblasts, MTRIPs allowed the accurate imaging of native mRNAs and a non-engineered viral RNA, of RNA co-localization with known RNA-binding proteins, and of RNA dynamics and interactions with stress granules.

Topics: Actin-Related Protein 2; Actins; Animals; Avian Proteins; Bacterial Proteins; Biotin; Carbocyanines; Cell Line, Tumor; Cell Membrane Permeability; Cell Survival; Cells, Cultured; Chick Embryo; DNA-Binding Proteins; Fibroblasts; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Microscopy, Confocal; Microscopy, Fluorescence; Molecular Probe Techniques; Oligonucleotide Probes; Poly(A)-Binding Proteins; Respiratory Syncytial Virus, Human; RNA; RNA-Binding Proteins; RNA, Messenger; Streptavidin; Streptolysins; T-Cell Intracellular Antigen-1

A macromolecular nucleoprotein complex in retrovirus-infected cells, termed the preintegration complex, is responsible for the concerted integration of linear viral DNA genome into host chromosomes. Isolation of sufficient quantities of the cytoplasmic preintegration complexes for biochemical and biophysical analysis is difficult. We investigated the architecture of HIV-1 nucleoprotein complexes involved in the concerted integration pathway in vitro. HIV-1 integrase (IN) non-covalently juxtaposes two viral DNA termini forming the synaptic complex, a transient intermediate in the integration pathway, and shares properties associated with the preintegration complex. IN slowly processes two nucleotides from the 3' OH ends and performs the concerted insertion of two viral DNA ends into target DNA. IN remains associated with the concerted integration product, termed the strand transfer complex. The synaptic complex and strand transfer complex can be isolated by native agarose gel electrophoresis. In-gel fluorescence resonance energy transfer measurements demonstrated that the energy transfer efficiencies between the juxtaposed Cy3 and Cy5 5'-end labeled viral DNA ends in the synaptic complex (0.68+/-0.09) was significantly different from that observed in the strand transfer complex (0.07+/-0.02). The calculated distances were 46+/-3 A and 83+/-5 A, respectively. DNaseI footprint analysis of the complexes revealed that IN protects U5 and U3 DNA sequences up to approximately 32 bp from the end, suggesting two IN dimers were bound per terminus. Enhanced DNaseI cleavages were observed at nucleotide positions 6 and 9 from the terminus on U3 but not on U5, suggesting independent assembly events. Protein-protein cross-linking of IN within these complexes revealed the presence of dimers, tetramers, and a larger multimer (>120 kDa). Our results suggest a new model where two IN dimers individually assemble on U3 and U5 ends before the non-covalent juxtaposition of two viral DNA ends, producing the synaptic complex.

Topics: Antibodies, Viral; Base Sequence; Carbocyanines; Chromosome Pairing; Cross-Linking Reagents; Deoxyribonuclease I; DNA Footprinting; DNA, Viral; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; HIV-1; Integrases; Models, Biological; Protein Multimerization; Protein Subunits; Spectrometry, Fluorescence; Substrate Specificity; Terminal Repeat Sequences; Virus Integration

A DNA microarray scanner was used as a digital fluorescence microscope to simplify the diagnosis of autoimmune bullous diseases. Frozen sections of skin biopsies were taken from 3 patients with bullous pemphigoid and 1 patient each with lichen planus pemphigoides, linear immunoglobulin (Ig) A disease, and dermatitis herpetiformis. After incubation with cyanine-labeled antibodies, the tissues were scanned at 5-mum resolution using an instrument originally designed to study gene expression. The microarray scanner's large field of view, unlike that of fluorescence microscopy, allowed a view of the entire specimen, considerably easing the orientation of tissue. All images were diagnostic and included a linear pattern along the basement membrane zone (BMZ) using anti-IgG and anti-C3 in all cases of bullous pemphigoid, a linear pattern of IgG along the BMZ in lichen planus pemphigoides, and a linear pattern of IgA along the BMZ in linear IgA dermatosis. IgA deposition along dermal papillary tips was seen in dermatitis herpetiformis, but a granular pattern was indiscernible at the 5-mum resolution. The advantages of the microarray scanner over standard fluorescence microscopy include speed, technical ease, large field of view, potential for visualizing multiple antibodies simultaneously in a tissue, and convenience of digital image archiving.

Topics: Autoimmune Diseases; Basement Membrane; Biopsy; Carbocyanines; Complement C3; Dermatitis Herpetiformis; Equipment Design; Fluorescent Antibody Technique; Fluorescent Dyes; Humans; Immunoglobulin A; Immunoglobulin G; Lichen Planus; Microscopy, Fluorescence; Oligonucleotide Array Sequence Analysis; Pemphigoid, Bullous; Predictive Value of Tests; Skin; Skin Diseases, Vesiculobullous

Gastric cancer is the second most common fatal malignancy in the world. Proteomics studies of clinical tumor samples have led to the identification of specific protein markers of gastric cancer detection and better understanding the carcinogenesis of gastric cancer. Gastric cancer tissue of epithelial origin and adjacent normal mucosa were examined in pair by fluorescence 2-D differential in-gel electrophoresis proteomics analysis utilizing 2-D PAGE protein separation. Intensity changes of 33 spots were detected with statistical significance. Twenty-two out of the 33 spots were identified by MALDI-TOF MS or MS/MS. Of the 9 up-regulated proteins, 7 were identified, including heat shock protein 60 (HSP60), mutant desmin, effector cell proteinase receptor 1 splice form 1b, hypothetical protein, unnamed protein product, and manganese superoxide dismutase (MnSOD), a protein similar to alpha-actin. Of the 20 down-regulated proteins, 16 were identified, including selenium binding protein 1, fibrinogen gamma, HSP27, tubulin alpha 6, zinc finger protein 160, prostaglandin F synthase, and eukaryotic translation elongation factor 1 alpha 1. Our results suggest that MnSOD may be a potential serum marker for molecular diagnosis of gastric carcinoma, and DIGE is a useful technique for screening differentially expressed proteins in cancer tissues.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Biopsy; Carbocyanines; Electrophoresis, Gel, Two-Dimensional; Female; Fluorescent Dyes; Humans; Male; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Proteomics; Spectrometry, Fluorescence; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stomach Neoplasms; Tandem Mass Spectrometry

Although fluorescence is the prevailing labeling technique in biosensing applications, sensitivity improvement is still a striving challenge. We show that coating standard microscope slides with nanoroughened silver films provides a high fluorescence signal enhancement due to plasmonic interactions. As a proof of concept, we applied these films with tailored plasmonic properties to DNA microarrays. Using common optical scanning devices, we achieved signal amplifications of more than 40-fold.

Topics: Biosensing Techniques; Carbocyanines; Fluorescence; Fluorescent Dyes; Microscopy, Atomic Force; Nanotechnology; Normal Distribution; Oligonucleotide Array Sequence Analysis; Rhodamines; Sensitivity and Specificity; Silver; Surface Plasmon Resonance; Surface Properties

Endothelial cells (ECs) constantly exposed to shear flow increase nitric oxide production via the activation of endothelial nitric oxide synthase. Nitric oxide-mediated S-nitrosylation has recently been identified as an important post-translational modification that may alter signalling and/or protein function. S-nitrosylation of endothelial proteins after shear flow treatment has not been fully explored. In this study, the CyDye switch method was utilized to examine S-nitrosylated proteins in ECs after exposure to shear flow.. Human umbilical vein ECs were subjected to shear flow for 30 min, and S-nitrosylated proteins were detected by the CyDye switch method. In principle, free thiols in proteins become blocked by alkylation, the S-nitrosylated bond is reduced by ascorbate, and then CyDye labels proteins. Proteins that separately labelled with Cy3 or Cy5 were mixed and subjected to two-dimensional gel electrophoresis for further analysis. More than 100 S-nitrosoproteins were detected in static and shear-treated ECs. Among these, 12 major proteins of heterogeneous function showed a significant increase in S-nitrosylation following shear flow. The S-nitrosylated residues in tropomyosin and vimentin, which were localized in the hydrophobic motif of each protein, were identified as Cys170 and Cys328, respectively.. Post-translational S-nitrosylation of proteins in ECs can be detected by a reliable CyDye switch method. This flow-induced S-nitrosylation of endothelial proteins may be essential for the adaptation and remodelling of ECs under flow conditions.

Topics: Adaptation, Physiological; Amino Acid Motifs; Amino Acid Sequence; Carbocyanines; Cells, Cultured; Cysteine; Electrophoresis, Gel, Two-Dimensional; Endothelial Cells; Enzyme Activation; Fluorescent Dyes; Humans; Hydrophobic and Hydrophilic Interactions; Mass Spectrometry; Molecular Sequence Data; Nitric Oxide; Nitric Oxide Synthase Type III; Protein Processing, Post-Translational; Proteins; Proteomics; Stress, Mechanical; Tropomyosin; Vimentin

Single nucleotide polymorphisms (SNPs) are indispensable in such applications as association mapping and construction of high-density genetic maps. These applications usually require genotyping of thousands of SNPs in a large number of individuals. Although a number of SNP genotyping assays are available, most of them are designed for SNP genotyping in diploid individuals. Here, we demonstrate that the Illumina GoldenGate assay could be used for SNP genotyping of homozygous tetraploid and hexaploid wheat lines. Genotyping reactions could be carried out directly on genomic DNA without the necessity of preliminary PCR amplification. A total of 53 tetraploid and 38 hexaploid homozygous wheat lines were genotyped at 96 SNP loci. The genotyping error rate estimated after removal of low-quality data was 0 and 1% for tetraploid and hexaploid wheat, respectively. Developed SNP genotyping assays were shown to be useful for genotyping wheat cultivars. This study demonstrated that the GoldenGate assay is a very efficient tool for high-throughput genotyping of polyploid wheat, opening new possibilities for the analysis of genetic variation in wheat and dissection of genetic basis of complex traits using association mapping approach.

Topics: Algorithms; Alleles; Carbocyanines; Chromosomes, Plant; Cluster Analysis; Data Interpretation, Statistical; DNA, Plant; Fluorescent Dyes; Gene Frequency; Genome, Plant; Genotype; Homozygote; Oligonucleotide Array Sequence Analysis; Polymorphism, Single Nucleotide; Polyploidy; Sequence Analysis, DNA; Time Factors; Triticum

Axonal transport is responsible for the movement of signals and cargo between nerve termini and cell bodies. Pathogens also exploit this pathway to enter and exit the central nervous system. In this study, we characterised the binding, endocytosis and axonal transport of an adenovirus (CAV-2) that preferentially infects neurons. Using biochemical, cell biology, genetic, ultrastructural and live-cell imaging approaches, we show that interaction with the neuronal membrane correlates with coxsackievirus and adenovirus receptor (CAR) surface expression, followed by endocytosis involving clathrin. In axons, long-range CAV-2 motility was bidirectional with a bias for retrograde transport in nonacidic Rab7-positive organelles. Unexpectedly, we found that CAR was associated with CAV-2 vesicles that also transported cargo as functionally distinct as tetanus toxin, neurotrophins, and their receptors. These results suggest that a single axonal transport carrier is capable of transporting functionally distinct cargoes that target different membrane compartments in the soma. We propose that CAV-2 transport is dictated by an innate trafficking of CAR, suggesting an unsuspected function for this adhesion protein during neuronal homeostasis.

Topics: Adenoviridae; Animals; Axonal Transport; Axons; Carbocyanines; Cells, Cultured; Clathrin-Coated Vesicles; Coated Pits, Cell-Membrane; Constitutive Androstane Receptor; Endocytosis; Endosomes; Fluorescent Dyes; Ganglia, Spinal; Hydrogen-Ion Concentration; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Motor Neurons; Nerve Tissue Proteins; Rats; Receptors, Cytoplasmic and Nuclear; Sciatic Nerve; Transcription Factors; Vesicular Transport Proteins

Single molecule (SM) techniques are relatively new additions to the field of biophysics that allow one to manipulate individual molecules and study their behavior. To make these studies more relevant to what actually happens in the cell, one needs to move beyond the studies of individual molecules in isolation and study many different molecules working in concert. This presents a technical challenge as most SM experiments measure only one observable as a function of time, whereas complex biomolecular systems require multidimensional SM analysis. Förster resonance energy transfer (FRET) is one of the most common single molecule approaches and can report on the real time distance changes. However, FRET requires two fluorophores which will ultimately limit the degree of multiplexing in future SM applications. It will be useful if a single fluorophore can be used to provide equivalent information. In this communication, we show that fluorescence lifetime analysis of a single Cy3 fluorophore attached to the promoter region of the DNA can be used to reveal transient reaction intermediates during transcription initiation by T7 RNA polymerase. This work represents the first demonstration of real-time biochemical reactions observed via single molecule fluorescence lifetime trajectories of immobilized molecules.

Topics: Carbocyanines; DNA; DNA-Directed RNA Polymerases; Fluorescence; Nucleic Acid Denaturation; Photons; Promoter Regions, Genetic; Time Factors; Transcription, Genetic; Viral Proteins

Single nucleotide polymorphisms (SNPs) are important markers which can be used in association studies searching for susceptible genes of complex diseases. High-throughput methods are needed for SNP genotyping in a large number of samples. In this study, we applied polyacrylamide gel-based microarray combined with dual-color hybridization for association study of four BDNF polymorphisms with autism. All the SNPs in both patients and controls could be analyzed quickly and correctly. Among four SNPs, only C270T polymorphism showed significant differences in the frequency of the allele (chi(2) = 7.809, p = 0.005) and genotype (chi(2) = 7.800, p = 0.020). In the haplotype association analysis, there was significant difference in global haplotype distribution between the groups (chi(2) = 28.19, p = 3.44e-005). We suggest that BDNF has a possible role in the pathogenesis of autism. The study also show that the polyacrylamide gel-based microarray combined with dual-color hybridization is a rapid, simple and high-throughput method for SNPs genotyping, and can be used for association study of susceptible gene with disorders in large samples.

Topics: Alleles; Autistic Disorder; Brain-Derived Neurotrophic Factor; Carbocyanines; Child; Child, Preschool; Electrophoresis, Gel, Pulsed-Field; Female; Fluorescent Dyes; Genetic Association Studies; Genotype; Haplotypes; Humans; Male; Oligonucleotide Array Sequence Analysis; Polymorphism, Single Nucleotide

Among various factors affecting operation of oligonucleotide microchips, the variations in concentration and in homogeneous distribution of immobilized probes over the cells are one of the most important. The labeling of immobilized probes ensures the complete current monitoring on the probe distribution and is reliable and convenient. Using hydrogel-based oligonucleotide microchips, the applicability of Cy3-labeled immobilized probes for quality control and signal normalization after hybridization with Cy5-labeled target DNA was investigated. This study showed that proper signal normalization should be different in thermodynamic conditions and in transient regime with hybridization far from saturation. This kinetic effect holds for both hydrogel-based and surface oligonucleotide microchips. Besides proving basic features, the technique was assessed on a sampling batch of 50 microchips developed for identifying mutations responsible for rifampicin and isoniazid resistance of Mycobacterium tuberculosis.

Topics: Carbocyanines; DNA, Bacterial; Fluorescent Dyes; Gene Expression Profiling; Mycobacterium tuberculosis; Oligonucleotide Array Sequence Analysis

We have studied myoblasts from a patient with a severe autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD) caused by an arginine 545 to cystein point mutation (p.R545C) in the carboxy-terminal domain of the lamin A/C gene. This mutation has pleiotropic cellular effects on these myoblasts as demonstrated by nuclear structural defects, exhibiting lobulations which increase with cell passages in culture. The organization of both lamin A/C and its inner nuclear membrane partner emerin are altered, eventually showing a honeycomb pattern upon immunofluorescence microscopy. In addition, the distribution of histone H3 trimethylated at lysine 27 and of phosphorylated RNA polymerase II, markers of inactive and active chromatin domains, respectively, are altered suggesting an impact on gene expression. Patient myoblasts also presented a high index of senescence in ex vivo culture. Moreover, our data show for the first time in an AD-EDMD context that the 20S core particle of the proteasome was inactivated. With cell passages, the 20S core protein progressively accumulated into discrete nuclear foci that largely colocalized with promyelocytic leukemia (PML) bodies while p21 accumulated throughout the nuclear compartment. Proteasome inactivation has been linked to normal cellular ageing. Our data indicate that it may also contribute to premature senescence in AD-EDMD patient myoblasts. Finally, when transferred to low-serum medium, patient myoblasts were deficient in ex vivo differentiation, as assessed by the absence of myotube formation and myogenin induction. Altogether, these data suggest that the LMNA mutation p.R545C impairs both proliferation and differentiation capacities of myoblasts as part of the pathogenesis of AD-EDMD.

Topics: Aging; Amino Acid Substitution; Antibodies; Bisbenzimidazole; Carbocyanines; Case-Control Studies; Cell Culture Techniques; Cell Differentiation; Cell Nucleus; Cells, Cultured; Cysteine; Female; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; Lamin Type A; Male; Muscular Dystrophy, Emery-Dreifuss; Mutation, Missense; Myoblasts; Point Mutation; Xanthenes

Recent structural data have revealed two distinct conformations of the ribosome during initiation. We employed single-molecule fluorescence methods to probe the dynamic relation of these ribosomal conformations in real time. In the absence of initiation factors, the ribosome assembles in two distinct conformations. The initiation factors guide progression of the ribosome to the conformation that can enter the elongation cycle. In particular, IF2 both accelerates the rate of subunit joining and actively promotes the transition to the elongation-competent conformation. Blocking GTP hydrolysis by IF2 results in 70S complexes formed in the conformation unable to enter elongation. We observe that rapid GTP hydrolysis by IF2 drives the transition to the elongation-competent conformation, thus committing the ribosome to enter the elongation cycle.

Topics: Carbocyanines; Eukaryotic Initiation Factor-2; Fluorescence Resonance Energy Transfer; Guanosine Triphosphate; Hydrolysis; Kinetics; Models, Molecular; Peptide Chain Initiation, Translational; Peptide Initiation Factors; Prokaryotic Initiation Factor-2; Protein Binding; Protein Conformation; Ribosomes; RNA, Messenger; RNA, Transfer

Rad51 is a DNA recombinase functioning in the repair of DNA double-strand breaks and the generation of genetic diversity by homologous recombination (HR). In the presence of ATP, Rad51 self-assembles into an extended polymer on single-stranded DNA to catalyze strand exchange. Inappropriate HR causes genomic instability, and it is normally prevented by remodeling enzymes that antagonize the activities of Rad51 nucleoprotein filaments. In yeast, the Srs2 helicase/translocase suppresses HR by clearing Rad51 polymers from single-stranded DNA. We have examined the mechanism of disassembly of Rad51 nucleoprotein filaments by Srs2 and find that a physical interaction between Rad51 and the C-terminal region of Srs2 triggers ATP hydrolysis within the Rad51 filament, causing Rad51 to dissociate from DNA. This allosteric mechanism explains the biological specialization of Srs2 as a DNA motor protein that antagonizes HR.

Topics: Adenosine Triphosphate; Carbocyanines; DNA Helicases; DNA Repair; DNA, Single-Stranded; Electrophoresis, Polyacrylamide Gel; Fluorescence; Hydrolysis; Kinetics; Mutation; Oligonucleotides; Protein Binding; Rad51 Recombinase; Recombination, Genetic; Saccharomyces cerevisiae Proteins

The Na(+)/K(+)-ATPase, a membrane-associated ion pump, uses energy from the hydrolysis of ATP to pump 3 Na(+) ions out of and 2 K(+) into cells. The dependence of ATP hydrolysis on ATP concentration was measured using a fluorescence coupled-enzyme assay. The dependence on concentration of nucleotide association with the ATPase was examined using ADP and ATP-induced quenching of the fluorescence of ATPase labeled with Cy3-maleimide (Cy3-ATPase) or Alexa Fluor 546 carboxylic acid, succinimidyl ester (AF-ATPase). The kinetics of ATP hydrolysis in the presence of Na(+) and K(+) exhibited negative cooperativity with a Hill coefficient (n(H)) of 0.66 and a half-maximal concentration (K(0.5)) of 61 microM; in the absence of K(+), n(H) was 0.58 and K(0.5) was 13 microM. Nucleotide-induced fluorescence quenching exhibited negative cooperativity with an n(H) of 0.3-0.5. These results suggest that negative cooperativity observed in ATP hydrolysis is attributable to negative cooperativity in nucleotide association to the ATPase. Interaction between AF-ATPase and ATP labeled with Alexa Fluor 647 (AF-ATP) showed significant Förster resonance energy transfer (FRET). These results indicate that the ATPase exists as oligoprotomeric complexes in this preparation, and that this aggregation has significant effects on enzyme function.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Carbocyanines; Ducks; Fluorescence; Fluorescent Dyes; Potassium; Quinolinium Compounds; Salt Gland; Sodium; Sodium-Potassium-Exchanging ATPase

Characterizing the structural dynamics of the translating ribosome remains a major goal in the study of protein synthesis. Deacylation of peptidyl-tRNA during translation elongation triggers fluctuations of the pretranslocation ribosomal complex between two global conformational states. Elongation factor G-mediated control of the resulting dynamic conformational equilibrium helps to coordinate ribosome and tRNA movements during elongation and is thus a crucial mechanistic feature of translation. Beyond elongation, deacylation of peptidyl-tRNA also occurs during translation termination, and this deacylated tRNA persists during ribosome recycling. Here we report that specific regulation of the analogous conformational equilibrium by translation release and ribosome recycling factors has a critical role in the termination and recycling mechanisms. Our results support the view that specific regulation of the global state of the ribosome is a fundamental characteristic of all translation factors and a unifying theme throughout protein synthesis.

Topics: Carbocyanines; Electrophoresis, Polyacrylamide Gel; Fluorescence; Kinetics; Models, Biological; Peptide Chain Termination, Translational; Peptide Termination Factors; Protein Binding; Ribosomal Proteins; Ribosomes; RNA, Messenger

Topics: Carbocyanines; Cryoelectron Microscopy; DNA; Fluorescence; Gold; Metal Nanoparticles; Nucleic Acid Denaturation

Cyanine dyes are extensively used as fluorescent probes in molecular biology, biochemical and biophysical applications. We investigated the fluorescent properties of Cy3 covalently attached to the 5' terminus of DNA oligonucleotides, and demonstrated that its fluorescence efficiency and lifetime depend strongly on DNA sequence. DNA sequence determines the extent and nature of the interactions between the dye and the DNA bases, which are responsible for the unusual enhancement in fluorescence observed for a large number of oligonucleotides. Results are discussed in terms of a photoisomerization mechanism that deactivates the excited state and thus competes with fluorescence. The efficiency of isomerization decreases when Cy3-DNA interactions prevent rotation around the double bonds, resulting in an increase in the lifetime of the singlet excited state. We have shown that the ability of Cy3 to interact with DNA depends on the flexibility of the oligonucleotide and the presence of purines in the chain.

Topics: Base Sequence; Carbocyanines; DNA, Single-Stranded; Fluorescence

In the field of nanotechnology, quantum dots (QDs) are a novel class of inorganic fluorochromes composed of nanometre-scale crystals made of a semiconductor material. Given the remarkable optical properties that they possess, they have been proposed as an ideal material for use in fluorescent in-situ hybridization (FISH). That is, they are resistant to photobleaching and they excite at a wide range of wavelengths but emit light in a very narrow band that can be controlled by particle size and thus have the potential for multiplexing experiments. The principal aim of this study was to compare the potential of QDs against traditional organic fluorochromes in both indirect (i.e. QD-conjugated streptavidin) and direct (i.e. synthesis of QD-labelled FISH probes) detection methods. In general, the indirect experiments met with a degree of success, with FISH applications demonstrated for chromosome painting, BAC mapping and use of oligonucleotide probes on human and avian chromosomes/nuclei. Many of the reported properties of QDs (e.g. brightness, 'blinking' and resistance to photobleaching) were observed. On the other hand, signals were more frequently observed where the chromatin was less condensed (e.g. around the periphery of the chromosome or in the interphase nucleus) and significant bleed-through to other filters was apparent (despite the reported narrow emission spectra). Most importantly, experimental success was intermittent (sometimes even in identical, parallel experiments) making attempts to improve reliability difficult. Experimentation with direct labelling showed evidence of the generation of QD-DNA constructs but no successful FISH experiments. We conclude that QDs are not, in their current form, suitable materials for FISH because of the lack of reproducibility of the experiments; we speculate why this might be the case and look forward to the possibility of nanotechnology forming the basis of future molecular cytogenetic applications.

Topics: Animals; Biotin; Biotinylation; Carbocyanines; Cell Nucleus; Cells, Cultured; Chickens; Chromosome Painting; Chromosomes; Chromosomes, Artificial, Bacterial; Chromosomes, Human, Pair 12; Clone Cells; Digoxigenin; DNA; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Humans; Hybridization, Genetic; In Situ Hybridization, Fluorescence; Indicators and Reagents; Indoles; Lymphocytes; Male; Metaphase; Microscopy, Fluorescence; Nanotechnology; Oligonucleotide Probes; Photobleaching; Quantum Dots; Semiconductors; Spermatozoa; Streptavidin; Xanthenes

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.

Topics: Animals; Carbocyanines; DNA, Complementary; Fluorescent Dyes; Gene Expression Profiling; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Zebrafish

An encounter between a DNA-translocating enzyme and a DNA-bound protein must occur frequently in the cell, but little is known about its outcome. Here we developed a multicolor single-molecule fluorescence approach to simultaneously monitor single-stranded DNA (ssDNA) translocation by a helicase and the fate of another protein bound to the same DNA. Distance-dependent fluorescence quenching by the iron-sulfur cluster of the archaeal XPD (Rad3) helicase was used as a calibrated proximity signal. Despite the similar equilibrium DNA-binding properties, the two cognate ssDNA-binding proteins RPA1 and RPA2 differentially affected XPD translocation. RPA1 competed with XPD for ssDNA access. In contrast, RPA2 did not interfere with XPD-ssDNA binding but markedly slowed down XPD translocation. Mechanistic models of bypassing DNA-bound proteins by the Rad3 family helicases and their biological implications are discussed.

Topics: Adenosine Triphosphate; Archaeal Proteins; Binding Sites; Carbocyanines; DNA; DNA Repair; Fluorescent Dyes; Kinetics; Models, Molecular; Nucleic Acid Conformation; Protein Conformation; Replication Protein A; Signal Processing, Computer-Assisted; Spectrometry, Fluorescence; Xeroderma Pigmentosum Group D Protein

The aim of the current study is to test the ability to label and detect murine embryonic stem cell-derived cardiovascular progenitor cells (ES-CPC) with cardiac magnetic resonance (CMR) using the novel contrast agent Gadofluorine M-Cy3 (GdFM-Cy3).. Cell therapy shows great promise for the treatment of cardiovascular disease. An important limitation to previous clinical studies is the inability to accurately identify transplanted cells. GdFM-Cy3 is a lipophilic paramagnetic contrast agent that contains a perfluorinated side chain and an amphiphilic character that allows for micelle formation in an aqueous solution. Previous studies reported that it is easily taken up and stored within the cytosol of mesenchymal stem cells, thereby allowing for paramagnetic cell labeling. Investigators in our laboratory have recently developed techniques for the robust generation of ES-CPC. We reasoned that GdFM-Cy3 would be a promising agent for the in vivo detection of these cells after cardiac cell transplantation.. ES-CPC were labeled with GdFM-Cy3 by incubation. In vitro studies were performed to assess the impact of GdFM-Cy3 on cell function and survival. A total of 500,000 GdFM-Cy3-labeled ES-CPC or control ES-CPC were injected into the myocardium of mice with and without myocardial infarction. Mice were imaged (9.4-T) before and over a 2-week time interval after stem cell transplantation. Mice were then euthanized, and their hearts were sectioned for fluorescence microscopy.. In vitro studies demonstrated that GdFM-Cy3 was easily transfectable, nontoxic, stayed within cells after labeling, and could be visualized using CMR and fluorescence microscopy. In vivo studies confirmed the efficacy of the agent for the detection of cells transplanted into the hearts of mice after myocardial infarction. A correspondence between CMR and histology was observed.. The results of the current study suggest that it is possible to identify and potentially track GdFM-Cy3-labeled ES-CPC in murine infarct models via CMR.

Topics: Animals; Carbocyanines; Cell Line; Cell Proliferation; Cell Survival; Contrast Media; Disease Models, Animal; Embryonic Stem Cells; Female; Fluorescent Dyes; Fluorocarbons; Magnetic Resonance Imaging; Mesenchymal Stem Cell Transplantation; Mice; Mice, SCID; Microscopy, Fluorescence; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Organometallic Compounds; Staining and Labeling; Time Factors

Topics: Animals; Carbocyanines; Cell Proliferation; Cell Survival; Contrast Media; Fluorescent Dyes; Fluorocarbons; Humans; Magnetic Resonance Imaging; Mice; Microscopy, Fluorescence; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Organometallic Compounds; Staining and Labeling; Stem Cell Transplantation; Stem Cells; Time Factors

Several pathophysiological conditions, including nephrotic syndrome, are characterized by increased renal activity of the epithelial Na(+) channel (ENaC). We recently identified plasmin in nephrotic urine as a stimulator of ENaC activity and undertook this study to investigate the mechanism by which plasmin stimulates ENaC activity. Cy3-labeled plasmin was found to bind to the surface of the mouse cortical collecting duct cell line, M-1. Binding depended on a glycosylphosphatidylinositol (GPI)-anchored protein. Biotin-label transfer showed that plasmin interacted with the GPI-anchored protein prostasin on M-1 cells and that plasmin cleaved prostasin. Prostasin activates ENaC by cleavage of the gamma-subunit, which releases an inhibitory peptide from the extracellular domain. Removal of GPI-anchored proteins from the M-1 cells with phosphatidylinositol-specific phospholipase C (PI-PLC) inhibited plasmin-stimulated ENaC current in monolayers of M-1 cells at low plasmin concentration (1-4 microg/ml). At a high plasmin concentration of 30 microg/ml, there was no difference between cell layers treated with or without PI-PLC. Knockdown of prostasin attenuated binding of plasmin to M1 cells and blocked plasmin-stimulated ENaC current in single M-1 cells, as measured by whole-cell patch clamp. In M-1 cells expressing heterologous FLAG-tagged prostasin, gammaENaC and prostasin were colocalized. A monoclonal antibody directed against the inhibitory peptide of gammaENaC produced specific immunofluorescence labeling of M-1 cells. Pretreatment with plasmin abolished labeling of M-1 cells in a prostasin-dependent way. We conclude that, at low concentrations, plasmin interacts with GPI-anchored prostasin, which leads to cleavage of the gamma-subunit and activation of ENaC, while at higher concentrations, plasmin directly activates ENaC.

Topics: Animals; Biotinylation; Carbocyanines; Cell Line; Disease Models, Animal; Epithelial Sodium Channels; Fibrinolysin; Fluorescent Antibody Technique; Fluorescent Dyes; Ion Channel Gating; Kidney Tubules, Collecting; Membrane Potentials; Mice; Nephrotic Syndrome; Oligopeptides; Patch-Clamp Techniques; Peptides; Phosphoinositide Phospholipase C; Protein Binding; Rats; Recombinant Fusion Proteins; RNA Interference; Serine Endopeptidases; Time Factors

Using stoichiometrically controlled 1:1 functionalization of gold nanoparticles with fluorescent dye molecules in which the dye molecule is held away from the particle surface by a rigid DNA spacer allows precise determination of the distance-dependent effect of the metal nanoparticles on fluorescence intensity. Two dyes were studied, Cy3 and Cy5, with two sizes of nanoparticles, 5 and 10 nm. The larger the particle, the more quenching of the photoluminescence (PL) intensity, due to increased overlap of the dye's emission spectrum with the Au surface plasmon resonance. Fluorescence is quenched significantly for distances somewhat larger than the particle diameter, in good agreement with the predictions of an electrodynamics model based on interacting dipoles. The distance dependence of surface energy transfer behavior, i.e. quenching efficiency, is proportional to 1/d(4), which involves no consideration of the size of the particle and the spectral overlap of the dye and AuNp. This surface energy transfer model is found qualitatively and agrees with the electrodynamic model, though the exponent is greater than 4 for the smaller nanoparticles (5 nm), and smaller than 4 for the larger nanoparticles (10 nm).

Topics: Carbocyanines; Computer Simulation; DNA; Fluorescent Dyes; Gold; Metal Nanoparticles; Nucleic Acid Hybridization; Spectrophotometry, Ultraviolet

This paper describes the patterning of DNA arrays on plastic surfaces using an elastomeric, two-dimensional microcapillary system (muCS). Fluidic structures were realized through hot-embossing lithography using Versaflex CL30. Like elastomers based on poly(dimethylsiloxane), this thermoplastic block copolymer is able to seal a surface in a reversible manner, making it possible to confine DNA probes with a level of control that is unparalleled using standard microspotting techniques. We focus on muCSs that support arrays comprising up to 2 x 48 spots, each being 45 mum in diameter. Substrates were fabricated from two hard thermoplastic materials, poly(methylmethacrylate) and a polycyclic olefin (e.g., Zeonor 1060R), which were both activated with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride and N-hydroxysuccinimide to mediate covalent attachment of DNA molecules. The approach was exemplified by using 0.25-32 muM solutions of amino-modified oligonucleotides labeled with either Cy3 or Cy5 fluorescent dye in phosphate-buffered saline, allowing for a direct and sensitive characterization of the printed arrays. Solutions were incubated for durations of 1 to >48 h at 22, 30, and 40 degrees C to probe the conditions for obtaining uniform spots of high fluorescence intensity. The length (l) and depth (d) of microfluidic supply channels were both important with respect to depletion as well as evaporation of the solvent. While selective activation of the substrate proved helpful to limit unproductive loss of oligonucleotides along trajectories, incubation of solution in a humid environment was necessary to prevent uncontrolled drying of the liquid, keeping the immobilization process intact over extended periods of time. When combined, these strategies effectively promoted the formation of high-quality DNA arrays, making it possible to arrange multiple probes in parallel with a high degree of uniformity. Moreover, we show that resultant arrays are compatible with standard hybridization protocols, which allowed for reliable discrimination of individual strands when exposed to a specific ssDNA target molecule.

Topics: Base Sequence; Carbocyanines; DNA; DNA, Single-Stranded; Dose-Response Relationship, Drug; Equipment Design; Hydrolysis; Microfluidic Analytical Techniques; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Oligonucleotides; Optics and Photonics; Plastics; Surface Properties

We show that plasmonic nanoresonators composed of two gold nanoparticles change not only the intensity but also the spectral shape of the emission of fluorescent molecules. The plasmonic resonance frequency can be tuned by varying the distance between the nanoparticles, which allows us to selectively favor transitions of a fluorescent molecule to a specific vibrational ground state. Experimental data from correlated scattering and fluorescence microscopy agree well with calculations in the framework of generalized Mie theory. Our results show that the widely used description of a dye molecule near a metal surface as a mere two-level system is inadequate.

Topics: Antibodies; Carbocyanines; Digoxigenin; Fluorescent Dyes; Gold; Metal Nanoparticles; Serum Albumin, Bovine; Spectrometry, Fluorescence; Surface Plasmon Resonance

We present an Anti-Brownian Electrokinetic trap (ABEL trap) capable of trapping individual fluorescently labeled protein molecules in aqueous buffer. The ABEL trap operates by tracking the Brownian motion of a single fluorescent particle in solution, and applying a time-dependent electric field designed to induce an electrokinetic drift that cancels the Brownian motion. The trapping strength of the ABEL trap is limited by the latency of the feedback loop. In previous versions of the trap, this latency was set by the finite frame rate of the camera used for video-tracking. In the present system, the motion of the particle is tracked entirely in hardware (without a camera or image-processing software) using a rapidly rotating laser focus and lock-in detection. The feedback latency is set by the finite rate of arrival of photons. We demonstrate trapping of individual molecules of the protein GroEL in buffer, and we show confinement of single fluorophores of the dye Cy3 in water.

Topics: Buffers; Carbocyanines; Chaperonin 60; Electrochemistry; Fluorescent Dyes; Image Processing, Computer-Assisted; Kinetics; Microfluidics; Microscopy, Video; Motion; Movement; Particle Size; Proteins; Time Factors; Water

Macrophages are believed to play a crucial role in atherogenesis and atherosclerotic plaque progression, mainly through their role in the accumulation of large amounts of cholesteryl ester and foam cell formation after the uptake into the arterial intima of oxidized LDL (oxLDL) particles known to be proatherogenic. The aim of this study was to use a differential proteomic approach to identify the response of human monocyte-derived macrophages after treatment with oxLDL for 24 h. Mass spectrometry analysis (MALDI-TOF) of 2D-DIGE gels made it possible to identify 9 intracellular and 3 secreted proteins that were up-regulated, 11 intracellular and 1 secreted proteins that were down-regulated, and 2 secreted proteins that were induced. This methodological approach not only confirmed the differential expression levels of proteins known to be regulated by oxLDL in macrophages, such as catalase and pyruvate kinase, but also identified oxLDL modulation of other proteins for the first time, including heat shock proteins (HSP) and Actin cytoskeletal proteins. Semiquantitative Western blot confirmed their role. The HSPs identified included heat shock cognate 71 kDa protein (Hsc70), 75 kDa glucose-regulated protein (GRP75), heat shock 70 kDa protein (Hsp70), and 60 kDa (Hsp60) proteins. These highly conserved intracellular protein chaperones, commonly seen in atherosclerotic plaques, appear to participate in protection against cellular stress. Interestingly, oxLDL also modulated several F-Actin capping proteins involved in Actin polymerization and motility: gelsolin, CapG, and CapZ. In conclusion, we have demonstrated the effects of oxLDL in the modulation of several proteins in human macrophages and established a functional profile of the human macrophage during the atherosclerotic process.

Topics: Actins; CapZ Actin Capping Protein; Carbocyanines; Catalase; Cells, Cultured; Cytoskeletal Proteins; Electrophoresis, Gel, Two-Dimensional; Fluorescent Dyes; Gelsolin; Heat-Shock Proteins; Humans; Intracellular Space; Lipoproteins, LDL; Macrophages; Microfilament Proteins; Nuclear Proteins; Proteome; Pyruvate Kinase; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The hepatitis C virus (HCV) RNA replication complex (RC), which is composed of viral nonstructural (NS) proteins and host cellular proteins, replicates the viral RNA genome in association with intracellular membranes. Two viral NS proteins, NS3 and NS5A, are essential elements of the RC. Here, by using immunoprecipitation and fluorescence resonance energy transfer assays, we demonstrated that NS3 and NS5A interact with tubulin and actin. Furthermore, immunofluorescence microscopy and electron microscopy revealed that HCV RCs were aligned along microtubules and actin filaments in both HCV replicon cells and HCV-infected cells. In addition, the movement of RCs was inhibited when microtubules or actin filaments were depolymerized by colchicine and cytochalasin B, respectively. Based on our observations, we propose that microtubules and actin filaments provide the tracks for the movement of HCV RCs to other regions in the cell, and the molecular interactions between RCs and microtubules, or RCs and actin filaments, are mediated by NS3 and NS5A.

Topics: Actin Cytoskeleton; Antibodies, Monoclonal; Carbocyanines; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Colchicine; Cytochalasin B; Fluorescein-5-isothiocyanate; Fluorescence Resonance Energy Transfer; Fluorescent Antibody Technique, Direct; Fluorescent Dyes; Hepacivirus; Humans; Indoles; Kidney; Liver Neoplasms; Microtubules; Replicon; RNA Helicases; RNA, Viral; Serine Endopeptidases; Tubulin; Tubulin Modulators; Viral Nonstructural Proteins; Virus Replication

Aiming at identification of novel peptides that can be employed for effective targeting of malignant gliomas, we used a 12-mer peptide phage display library and cultured human malignant glioma cells for phage selection. Several common phage clones emerged after 4 rounds of biopanning against the U87MG glioblastoma cell line. The most abundant phage clone VTW, expressing a sequence of VTWTPQAWFQWV, bound to U87MG cells 700-fold more efficiently than the original unselected library. The VTW phage also bound strongly to other human glioma cell lines, including H4, SW1088 and SW1783, but very weakly to normal human astrocytes and SV40-immortalized human astroglial cells. When compared to other non-glial tumor cells, the phage showed 400- to 1400-fold higher binding efficiency for U87MG cells. After linked to positively charged lysine peptides, the VTW peptide became water soluble and was able to deliver biologically active, hydrophilic beta-galactosidase into U87MG cells, with up to 90% of the cells being stained intensively blue. This peptide carrier did not show obvious protein delivery activities in the human astrocytes. Our results provide a proof of principle to the concept that peptides identified through phage display technology can be used to develop protein carriers that are capable of mediating intracellular delivery of hydrophilic macromolecules in a tumor cell-specific manner.

Topics: Amino Acid Sequence; Astrocytes; beta-Galactosidase; Carbocyanines; Cell Line, Tumor; Drug Carriers; Endocytosis; Glioblastoma; Humans; Molecular Sequence Data; Neuroglia; Peptide Library; Protein Binding; Proteins; Receptors, Cell Surface; Transfection

Nasopharyngeal carcinoma (NPC), one of the most common cancers in Southeast Asia, is commonly diagnosed late due to its deep location and vague symptoms. To identify biomarkers for improving NPC diagnosis, we established a proteomic platform for detecting aberrant serum proteins in nude mice bearing NPC xenografts. We first removed the three most abundant proteins from serum samples of tumor-bearing and control mice, and then labeled the samples with different fluorescent cyanine (Cy) dyes. The labeled serum proteins were then mixed equally and fractionated with ion-exchange chromatography followed by SDS-PAGE. Differentially expressed proteins were identified by in-gel tryptic digestion and MALDI-TOF MS. We identified peroxiredoxin 2 (Prx-II) and carbonic anhydrase 2 (CA-II) as being elevated in the xenograft mouse model compared to controls. Western blot analysis confirmed up-regulation of Prx-II and CA-II in plasma from five NPC patients, and ELISA showed that plasma Prx-II levels were significantly higher in NPC patients (n = 84) versus healthy controls (n = 90) (3.03 +/- 4.47 versus 1.90 +/- 2.74 microg/mL, p = 0.047). In conclusion, Cy dye labeling combined with three-dimensional fractionation is a feasible strategy for identifying differentially expressed serum proteins in an NPC xenograft model, and Prx-II may represent a potential NPC biomarker.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Biomarkers, Tumor; Carbocyanines; Carbonic Anhydrase II; Child; Disease Models, Animal; Female; Fluorescent Dyes; Humans; Male; Mice; Mice, Nude; Middle Aged; Nasopharyngeal Neoplasms; Neoplasm Transplantation; Peroxiredoxins; Proteomics; Transplantation, Heterologous; Up-Regulation

We have found that the efficiency of fluorescence resonance energy transfer between Cy3 and Cy5 terminally attached to the 5' ends of a DNA duplex is significantly affected by the relative orientation of the two fluorophores. The cyanine fluorophores are predominantly stacked on the ends of the helix in the manner of an additional base pair, and thus their relative orientation depends on the length of the helix. Observed fluorescence resonance energy transfer (FRET) efficiency depends on the length of the helix, as well as its helical periodicity. By changing the helical geometry from B form double-stranded DNA to A form hybrid RNA/DNA, a marked phase shift occurs in the modulation of FRET efficiency with helix length. Both curves are well explained by the standard geometry of B and A form helices. The observed modulation for both polymers is less than that calculated for a fully rigid attachment of the fluorophores. However, a model involving lateral mobility of the fluorophores on the ends of the helix explains the observed experimental data. This has been further modified to take account of a minor fraction of unstacked fluorophore observed by fluorescent lifetime measurements. Our data unequivocally establish that Förster transfer obeys the orientation dependence as expected for a dipole-dipole interaction.

Topics: Carbocyanines; DNA; Fluorescence Resonance Energy Transfer; Models, Chemical; Nucleic Acid Conformation; Nucleic Acid Heteroduplexes

Protein S-nitrosation is a reversible post-translation modification critical for redox-sensitive cell signaling that is typically studied using the Biotin Switch method. This method and subsequent modifications usually require avidin binding or Western blot analysis to detect biotin labeled proteins. We describe here a modification of the Biotin Switch assay that eliminates the need for Western blot or avidin enrichment protocols and allows direct comparison of the S-nitrosation state proteins from two different samples in the same gel lane or on the same 2D gel. This S-FLOS method offers detection, identification and quantification of S-nitrosated proteins, with the potential for site-specific identification of nitrosation events.

Topics: Animals; Biotin; Brain Chemistry; Carbocyanines; Databases, Protein; Fluorescence; Maleimides; Mice; Nitric Oxide; Nitric Oxide Synthase; Nitrosation; Oxidation-Reduction; Protein Processing, Post-Translational; Proteins; Proteomics; S-Nitrosothiols; Silver Staining; Tandem Mass Spectrometry

Covalent heterodimers of the Cy3 and Cy5 fluorophores have been prepared from commercially available starting materials and characterized at the single-molecule level. This system behaves as a discrete molecular photoswitch, in which photoexcitation of the Cy5 results in fluorescence emission or, with a much lower probability, causes the Cy5 to enter into a long-lived, but metastable, dark state. Photoinduced recovery of the emissive Cy5 is achieved by very low intensity excitation (5 W cm(-2)) of the Cy3 fluorophore at a shorter wavelength. A similar system consisting of proximal, but not covalently linked, Cy3 and Cy5 has found application in stochastic optical reconstruction microscopy (STORM), a single-molecule localization-based technique for super-resolution imaging that requires photoswitching. The covalent Cy3-Cy5 heterodimers described herein eliminate the need for probabilistic methods of situating the Cy3 and Cy5 in close proximity to enable photoswitching. As proof of principle, these heterodimers have been applied to super-resolution imaging of the tubular stalk structures of live Caulobacter crescentus bacterial cells.

Topics: Carbocyanines; Caulobacter crescentus; Dimerization; Photochemistry; Spectrometry, Mass, Electrospray Ionization

Proteomic analysis has the potential to yield vast amounts of data. The available proteomic methods have been hampered by methodological errors in quantification due to large gel-to-gel variations. The inclusion of an internal standard greatly reduces this variation, and therefore the purpose of this investigation was: 1) to develop a sample preparation protocol for human skeletal muscle for two-dimensional differentiated gel electrophoresis (DIGE) and 2) to investigate the repeatability of one particular system, the Ettan DIGE. To test repeatability, nine aliquots from the same homogenate were labelled with three different CyDye(trade mark) dyes (Cy2, Cy3, Cy5). Samples were run on 18 x 24 cm gels, scanned with a Typhoon 9410 laser scanner and analysed in the DeCyder software. When selecting spots appearing only in triplicate (n = 1314), the mean error was 1.7 % (SD: 10.5 %; 95 % CI: 1.1-2.4 %). When setting the significance level to 99 %, no false-positive changes in protein volume ratios were detected. In the protocol presented here, only 0.5 mg tissue was used and separation of >2500 distinct protein spots in the pH range 3-11 and MW 10-200 kDa. Changes in protein abundance of <20 % could be detected. The method is especially useful when comparing muscle proteins between different conditions; for example, healthy and diseased tissue, before and after treatment or different exercise protocols.

Topics: Algorithms; Blotting, Western; Carbocyanines; Electrophoresis, Gel, Two-Dimensional; Humans; Hydrogen-Ion Concentration; Male; Muscle Proteins; Muscle, Skeletal; Principal Component Analysis; Reproducibility of Results

Rat mammary gland proteomes at day 21 (prepubertal) and day 50 (late puberty) were compared by 2D difference gel electrophoresis. Two-hundred fifty-one spots were significantly different ( p < 0.05) in abundance. Peptide mass fingerprint analysis of a subset of these proteins identified two significantly over-represented classes including structural and blood proteins (increased), and metabolism-relevant proteins (reduced) in day 50 relative to day 21 glands. This is a first report of mammary gland proteome differences at these important breast cancer-relevant time-points.

Topics: Aging; Animals; Carbocyanines; Electrophoresis, Gel, Two-Dimensional; Female; Fluorescent Dyes; Mammary Glands, Animal; Peptide Mapping; Proteome; Rats; Rats, Sprague-Dawley; Sexual Maturation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

We have examined the self-assembled membrane-bound aggregates of two annexin V (A5) dye conjugates and compared them to those from native A5. Native A5 and FITC-labeled A5 (A5-FITC) both formed discrete well-defined crystalline monolayer domains of p6 symmetry. However, A5-FITC also showed additional domains with a corrugated appearance not observed in native A5. In contrast, Cy3-labeled A5 (A5-Cy3) showed a mixture of crystalline monolayer and irregular multilayered domains, with the ratio of the two types varying significantly from sample to sample, and also required a much longer incubation time than native A5 and A5-FITC. When A5-FITC and A5-Cy3 were co-incubated on the same bilayer, well-defined crystalline monolayer domains containing both A5-FITC and A5-Cy3 were consistently observed at a much shorter incubation time than that of pure A5-Cy3 alone, indicating that A5-FITC facilitates the inclusion of A5-Cy3. These results suggest that dye labels can affect A5 2D self-assembly and crystal formation on membrane surfaces.

Topics: Annexin A5; Carbocyanines; Fluorescein-5-isothiocyanate; Fluorescence; Fluorescent Dyes; Incubators; Lipid Bilayers; Membranes, Artificial; Microscopy, Atomic Force; Microscopy, Fluorescence; Optics and Photonics; Protein Binding; Protein Structure, Tertiary; Temperature

Mycophenolic acid (MPA) is a potent inhibitor of the inosine monophosphate dehydrogenase and used as an immunosuppressive drug in transplantation. MPA inhibits proliferation of T- and B-lymphocytes by guanosine depletion. Since fibroblasts rely on the de novo synthesis of guanosine nucleotides, it is assumed that MPA interacts with fibroblasts causing an increased frequency of wound healing problems. We show a downregulation of the cytoskeletal proteins vinculin, actin and tubulin in fibroblasts exposed to pharmacological doses of MPA using microarray technology, real-time polymerase chain reaction (PCR) and Western blot. This reduction in RNA and protein content is accompanied by a substantial rearrangement of the cytoskeleton in MPA-treated fibroblasts as documented by immunofluorescence. The dysfunctional fibroblast growth was validated by scratch test documenting impaired migrational capacity. In contrast, cell adhesion was increased in MPA-treated fibroblasts. The results of the cultured human fibroblasts were applied to skin biopsies of renal transplant recipients. Skin biopsies of patients treated with MPA expressed less vinculin, actin and tubulin as compared to control biopsies that could explain potential wound healing problems posttransplantation. The perspective of MPA-induced cytoskeletal dysfunction may go beyond wound healing disturbances and may have beneficial effects on (renal) allografts with respect to scarring.

Topics: Biopsy; Carbocyanines; Cell Adhesion; Cell Movement; Cell Proliferation; Cells, Cultured; Dermatologic Surgical Procedures; Dose-Response Relationship, Drug; Fibroblasts; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; Immunohistochemistry; Immunosuppressive Agents; Indoles; Mycophenolic Acid; Phalloidine; Rhodamines; Skin

When confronted with environmental stress, cells either activate defence mechanisms to survive, or initiate apoptosis, depending on the type of stress. Certain types of stress, such as hypoxia, heatshock and arsenite (type 1 stress), induce cells to assemble cytoplasmic stress granules (SGs), a major adaptive defence mechanism. SGs are multimolecular aggregates of stalled translation pre-initiation complexes that prevent the accumulation of mis-folded proteins. Type 2 stress, which includes X-rays and genotoxic drugs, induce apoptosis through the stress-activated p38 and JNK MAPK (SAPK) pathways. A functional relationship between the SG and SAPK responses is unknown. Here, we report that SG formation negatively regulates the SAPK apoptotic response, and that the signalling scaffold protein RACK1 functions as a mediator between the two responses. RACK1 binds to the stress-responsive MTK1 MAPKKK and facilitates its activation by type 2 stress; however, under conditions of type 1 stress, RACK1 is sequestered into SGs. Thus, type 1 conditions suppress activation of the MTK1-SAPK pathway and apoptosis induced by type 2 stress. These findings may be relevant to the problem of hypoxia-induced resistance to cancer chemotherapy.

Topics: Animals; Annexin A5; Apoptosis; Arsenites; Carbocyanines; Cell Line; Chlorocebus aethiops; COS Cells; Cytoplasmic Granules; Enzyme Inhibitors; Fluorescent Dyes; GTP-Binding Proteins; HeLa Cells; Humans; Indoles; Kidney; Mitogen-Activated Protein Kinases; Models, Biological; Neoplasm Proteins; Oxidative Stress; Receptors for Activated C Kinase; Receptors, Cell Surface; Sodium Compounds; Sulfhydryl Reagents; Time Factors; Transfection

A high-throughput, fluorescence-based helicase assay using molecular beacons is described. The assay is tested using the NS3 helicase encoded by the hepatitis C virus (HCV) and is shown to accurately monitor helicase action on both DNA and RNA. In the assay, a ssDNA oligonucleotide molecular beacon, featuring a fluorescent moiety attached to one end and a quencher attached to the other, is annealed to a second longer DNA or RNA oligonucleotide. Upon strand separation by a helicase and ATP, the beacon strand forms an intramolecular hairpin that brings the tethered fluorescent and quencher molecules into juxtaposition, quenching fluorescence. Unlike currently available real-time helicase assays, the molecular beacon-based helicase assay is irreversible. As such, it does not require the addition of extra DNA strands to prevent products from re-annealing. Several variants of the new assay are described and experimentally verified using both Cy3 and Cy5 beacons, including one based on a sequence from the HCV genome. The HCV genome-based molecular beacon helicase assay is used to demonstrate how such an assay can be used in high-throughput screens and to analyze HCV helicase inhibitors.

Topics: Base Sequence; Carbocyanines; DNA; Fluorescence Resonance Energy Transfer; Genome, Viral; Hepacivirus; Molecular Probe Techniques; Molecular Sequence Data; Nucleic Acid Conformation; RNA; Viral Nonstructural Proteins

Non-biological signal (or noise) has been the bane of microarray analysis. Hybridization effects related to probe-sequence composition and DNA dye-probe interactions have been observed in differential methylation hybridization (DMH) microarray experiments as well as other effects inherent to the DMH protocol.. We suggest two models to correct for non-biologically relevant probe signal with an overarching focus on probe-sequence composition. The estimated effects are evaluated and the strengths of the models are considered in the context of DMH analyses.. The majority of estimated parameters were statistically significant in all considered models. Model selection for signal correction is based on interpretation of the estimated values and their biological significance.

Topics: Base Sequence; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; DNA Methylation; DNA Probes; Female; Fluorescent Dyes; Gene Expression Profiling; Humans; Methylation; Models, Genetic; Models, Statistical; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Sequence Analysis, DNA; Signal Transduction

DNA array analysis of dorsal root ganglion (DRG) using a rat model with nerve root constriction.. To determine the molecular changes in the DRG adjacent to the injured nerve root in a lumbar radiculopathy model.. DNA array analysis in lumbar radiculopathy model has so far focused on the spinal dorsal horn. The molecular changes in the DRG adjacent to the injured nerve root in lumbar radiculopathy remain to be determined.. Bilateral L5 DRGs were removed from 12 Sprague-Dawley rats on days 2, 7, 14, and 21 after nerve root ligation and on day 7 from 3 rats with sham operation. The aRNAs from the DRGs with nerve root ligation were labeled with Cy5 dye and those from the opposite side DRG (control) were labeled with Cy3 dye, and then hybridized to a 7793-spot Panorama Micro Array. It was considered to be significantly upregulated, when an average expression ratio of Cy5 to Cy3 was 2 or more. Genes upregulated were classified into early phase group (upregulated on day 2), midphase group (upregulated on days 7 and 14), and continuous group (upregulated from day 2 to 21). Seventeen genes were subjected to validation analysis with real-time quantitative PCR.. There were 16 upregulated genes in the early phase group, 56 genes in the midphase group, and 17 genes in the continuous group. Functional categorization revealed dominantly upregulated gene categories in each group; transcription/translation in the early phase group, enzyme/metabolism in the midphase group, and structure in the continuous group. Validation analysis of 17 genes demonstrated mean relative expression of 2.0 or more in all but 1 gene in the DRGs with nerve root ligation and none of them in the DRGs with sham operation.. The genes identified in this study, especially those involved in pain signaling and inflammation, serve as potential targets for molecular-based therapy for lumbar radiculopathy.

Topics: Animals; Carbocyanines; Disease Models, Animal; Fluorescent Dyes; Ganglia, Spinal; Gene Expression; Gene Expression Profiling; Lumbar Vertebrae; Male; Oligonucleotide Array Sequence Analysis; Pain; Radiculopathy; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spinal Nerve Roots; Time Factors; Up-Regulation

We studied the effect of fluorescently labeling proteins on protein-ligand reactions. Unlabeled ligands (streptavidin-binding peptides and rabbit immunoglobulin G (IgG) as antigen targets) are immobilized on epoxy-functionalized glass slides. Unlabeled and Cy3-labeled protein probes from the same batch (streptavidin and goat antibodies) subsequently react with the surface-immobilized targets. By monitoring in situ the surface mass density change using an oblique-incidence reflectivity difference scanning microscope (a label-free detector), we measured k(on) and k(off) for streptavidin-peptide reactions and antibody-antigen reaction. We found that (1) equilibrium dissociation constants, defined as K(D) = k(off)/k(on), for streptavidin-peptide reactions increases by a factor of 3-4 when the solution-phase streptavidin is labeled with Cy3 dye and (2) K(D) for reactions of solution-phase goat anti-rabbit antibodies with rabbit IgG targets also change significantly when the goat antibodies are labeled with Cy3 dye.

Topics: Animals; Carbocyanines; Epoxy Compounds; Glass; Immunoglobulin G; Kinetics; Ligands; Oligopeptides; Rabbits; Streptavidin; Surface Properties; Time Factors

The reverse transcriptase (RT) of human immunodeficiency virus (HIV) catalyzes a series of reactions to convert single-stranded viral RNA into double-stranded DNA for host cell integration. This process requires a variety of enzymatic activities, including DNA polymerization, RNA cleavage, strand transfer, and strand displacement synthesis. We used single-molecule fluorescence resonance energy transfer to probe the interactions between RT and nucleic acid substrates in real time. RT was observed to slide on nucleic acid duplexes, rapidly shuttling between opposite termini of the duplex. Upon reaching the DNA 3' terminus, RT can spontaneously flip into a polymerization orientation. Sliding kinetics were regulated by cognate nucleotides and anti-HIV drugs, which stabilized and destabilized the polymerization mode, respectively. These long-range translocation activities facilitate multiple stages of the reverse transcription pathway, including normal DNA polymerization and strand displacement synthesis.

Topics: Binding Sites; Carbocyanines; DNA Primers; DNA, Viral; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; HIV Reverse Transcriptase; HIV-1; Kinetics; Models, Molecular; Nevirapine; Nucleic Acid Hybridization; Nucleotides; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Reverse Transcriptase Inhibitors; Reverse Transcription; Ribonuclease H; RNA, Viral

Despite all remarkable progress in gel-based proteomics in recent years, there is still need to further improve quantification by decreasing the detection limits and increasing the dynamic range. These criteria are achieved best by fluorescent dyes that specifically stain the proteins either by adsorption after gel electrophoresis (in-gel staining) or covalent coupling prior to gel electrophoresis (in-solution staining). Here we report a multiplex analysis of protein samples using maleimide-activated cyanine-based (Cy3 and Cy5) and rhodamine-based dyes (Dy505, Dy535, and Dy635) to permanently label all thiol-groups of cysteine-containing proteins. The detection limits in SDS-PAGE were about 10 ng per band and even 2 ng for BSA due to its high content of cysteine residues. Thus only 5 microg protein of a mouse brain homogenate were analyzed by 2-DE. Both cyanine- and rhodamine-based dyes also stained proteins that did not contain cysteines, probably by reaction with amino groups. This side reactivity did not limit the method and might even extend its general use to proteins missing cysteine residues, but at a lower sensitivity. The dynamic range was more than two orders of magnitude in SDS-PAGE and the Dy-fluorophores did not alter the mobility of the tested proteins. Thus, a mixture of Dy505-, Dy555-, and Dy635-labeled Escherichia coli lysates were separated by 2-DE in a single gel and the three spot patterns relatively quantified.

Topics: Animals; Brain Chemistry; Carbocyanines; Cysteine; Electrophoresis, Gel, Two-Dimensional; Escherichia coli; Escherichia coli Proteins; Fluorescent Dyes; Mice; Proteins; Proteomics; Rhodamines; Sensitivity and Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in approximately 100 kilobase pair (kb) segments. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected--a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position. The SMRT array can detect alterations as small as 50 kb in size. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100 ng) this allows profiling of precious samples such as those produced by microdissection. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

Topics: Carbocyanines; Chromosomes, Artificial, Bacterial; Comparative Genomic Hybridization; DNA; Genome, Human; Humans; Oligonucleotide Array Sequence Analysis

The chloride channel, ClC-2 is expressed ubiquitously and participates in multiple physiological processes. In particular, ClC-2 has been implicated in the regulation of neuronal chloride ion homeostasis and mutations in ClC-2 are associated with idiopathic generalized epilepsy. Despite the physiological and pathophysiological significance of this channel, its regulation remains incompletely understood. The functional expression of ClC-2 at the cell surface has been shown to be enhanced by depletion of cellular ATP, implicating its possible role in cellular energy sensing. In the present study, biochemical assays of cell surface expression suggest that this gain of function reflects, in part, an increase in channel number due to the reduction in ClC-2 internalization by endocytosis. Cell surface expression of the disease-causing mutant: G715E, thought to lack wild-type nucleotide binding affinity, is similarly affected, suggesting that ATP-depletion modifies the function of proteins in the endocytic pathway rather than ClC-2 directly. Using a combination of immunofluorescence and biochemical studies, we confirmed that ClC-2 is internalized via dynamin-dependent endocytosis and that the change in surface expression evoked by ATP depletion is partially mimicked by inhibition of dynamin function using a dynamin dominant-negative mutant (DynK44A). Furthermore, trafficking via the early endosomal compartment occurs in part through rab5-associated vesicles and recycling of ClC-2 to the cell surface occurs through a rab11 dependent pathway. In summary, we have determined that the internalization of ClC-2 by endocytosis is inhibited by metabolic stress, highlighting the importance for understanding the molecular mechanisms mediating the endosomal trafficking of this channel.

Topics: Actins; Adenosine Triphosphate; Animals; Antibodies, Monoclonal; Antimetabolites; Biotinylation; Carbocyanines; Chloride Channels; Chlorocebus aethiops; CLC-2 Chloride Channels; COS Cells; Deoxyglucose; DNA, Complementary; Dynamins; Endocytosis; Endosomes; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Green Fluorescent Proteins; Humans; Mutation; Proteins; rab GTP-Binding Proteins; rab5 GTP-Binding Proteins; Rats; Rotenone; Transfection; Uncoupling Agents

Transforming growth factor beta 1 (TGF-beta1) plays a key role in connective tissue remodeling, scarring, and fibrosis. The effects of mechanical forces on TGF-beta1 and collagen deposition are not well understood. We tested the hypothesis that brief (10 min) static tissue stretch attenuates TGF-beta1-mediated new collagen deposition in response to injury. We used two different models: (1) an ex vivo model in which excised mouse subcutaneous tissue (N = 44 animals) was kept in organ culture for 4 days and either stretched (20% strain for 10 min 1 day after excision) or not stretched; culture media was assayed by ELISA for TGF-beta1; (2) an in vivo model in which mice (N = 22 animals) underwent unilateral subcutaneous microsurgical injury on the back, then were randomized to stretch (20-30% strain for 10 min twice a day for 7 days) or no stretch; subcutaneous tissues of the back were immunohistochemically stained for Type-1 procollagen. In the ex vivo model, TGF-beta1 protein was lower in stretched versus non-stretched tissue (repeated measures ANOVA, P < 0.01). In the in vivo model, microinjury resulted in a significant increase in Type-1 procollagen in the absence of stretch (P < 0.001), but not in the presence of stretch (P = 0.21). Thus, brief tissue stretch attenuated the increase in both soluble TGF-beta1 (ex vivo) and Type-1 procollagen (in vivo) following tissue injury. These results have potential relevance to the mechanisms of treatments applying brief mechanical stretch to tissues (e.g., physical therapy, respiratory therapy, mechanical ventilation, massage, yoga, acupuncture).

Topics: Animals; Carbocyanines; Collagen Type I; Culture Media; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Immunohistochemistry; Lactate Dehydrogenases; Male; Mice; Mice, Inbred C57BL; Microsurgery; Models, Biological; Organ Culture Techniques; Procollagen; Solubility; Stress, Mechanical; Subcutaneous Tissue; Time Factors; Transforming Growth Factor beta1

Transforming growth factor-beta (TGFbeta) controls expression of plasminogen activator inhibitor type 1 (PAI-1), which regulates degradation of extracellular matrix proteins in fibrotic diseases. The TGFbeta receptor-specific Smad 3 has been implicated in the PAI-1 expression. The mechanism by which non-Smad signaling contributes to this process is not known. We studied the cross-talk between Smad 3 and PI 3 kinase/Akt signaling in TGFbeta-induced PAI-1 expression in renal mesangial cells. Inhibition of PI 3 kinase and Akt kinase blocked TGFbeta- and Smad 3-mediated expression of PAI-1. In contrast, constitutively active PI 3 kinase and Akt kinase increased PAI-1 expression, similar to TGFbeta. Inhibition of PI 3 kinase and Akt kinase had no effect on TGFbeta-induced Smad 3 phosphorylation and its translocation to the nucleus. Notably, inhibition of PI 3 kinase-dependent Akt kinase abrogated TGFbeta-induced PAI-1 transcription, without affecting binding of Smad 3 to the PAI-1 Smad binding DNA element. However, PI 3 kinase inhibition and dominant negative Akt kinase antagonized the association of the transcriptional coactivator CBP with Smad 3 in response to TGFbeta, resulting in inhibition of Smad 3 acetylation. Together our findings identify TGFbeta-induced PI 3 kinase/Akt signaling as a critical regulator of Smad 3-CBP interaction and Smad 3 acetylation, which cause increased PAI-1 expression.

Topics: Adenoviridae; Animals; Carbocyanines; Cells, Cultured; Chromones; CREB-Binding Protein; Culture Media, Serum-Free; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Fluorescent Dyes; Gene Expression Regulation; Kidney Glomerulus; Mesangial Cells; Microscopy, Fluorescence; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Plasminogen Activator Inhibitor 1; Precipitin Tests; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Smad3 Protein; Time Factors; Transforming Growth Factor beta

The application of single-molecule fluorescence techniques to complex biological systems places demands on the performance of single fluorophores. We present an enzymatic oxygen scavenging system for improved dye stability in single-molecule experiments. We compared the previously described protocatechuic acid/protocatechuate-3,4-dioxygenase system to the currently employed glucose oxidase/catalase system. Under standardized conditions, we observed lower dissolved oxygen concentrations with the protocatechuic acid/protocatechuate-3,4-dioxygenase system. Furthermore, we observed increased initial lifetimes of single Cy3, Cy5, and Alexa488 fluorophores. We further tested the effects of chemical additives in this system. We found that biological reducing agents increase both the frequency and duration of blinking events of Cy5, an effect that scales with reducing potential. We observed increased stability of Cy3 and Alexa488 in the presence of the antioxidants ascorbic acid and n-propyl gallate. This new O(2)-scavenging system should have wide application for single-molecule fluorescence experiments.

Topics: Ascorbic Acid; Carbocyanines; Catalase; Enzyme Stability; Fluorescent Dyes; Free Radical Scavengers; Glucose Oxidase; Hydroxybenzoates; Microscopy, Fluorescence; Nanotechnology; Oxygen; Photobleaching; Propyl Gallate; Protocatechuate-3,4-Dioxygenase; Reactive Oxygen Species

Plasmid DNA (pUC19 and pBR322) was sequence-specifically, covalently labelled with Cy3 fluorophores using a newly synthesised N-adenosylaziridine cofactor and the DNA methyltransferase M.TaqI. The fluorescently labelled plasmids were used for transfection of mammalian cells and their intracellular distribution was visualised by epifluorescence and confocal fluorescence microscopy. Although these prokaryotic plasmids do not contain nuclear import sequences, translocation into the nuclei was observed.

Topics: Active Transport, Cell Nucleus; Animals; Base Sequence; Carbocyanines; Cells, Cultured; DNA Modification Methylases; Humans; Molecular Probe Techniques; Plasmids; Taq Polymerase; Transfection

Semiconductor nanocrystals, also called quantum dots (QDs), are novel inorganic fluorophores which are brighter and more photostable than organic fluorophores. In the present study, highly dispersive QD-labeled oligonucleotide (TAG)(8) (QD-deoxyribonucleic acid [DNA]) conjugates were constructed via the metal-thiol bond, which can be used as fluorescence in situ hybridization (FISH) probes. FISH analysis of maize metaphase chromosomes using the QD-DNA probes showed that the probes could penetrate maize chromosomes and nuclei and solely hybridized to complementary target DNAs. Compared with the conventional organic dyes such as Cy3 and fluorescein isothiocyanate, this class of luminescent labels bound with oligonucleotides is brighter and more stable against photobleaching on the chromosomes after FISH. These results suggest that QD fluorophores may be a more stable and useful fluorescent label for FISH applications in plant chromosome mapping considering their size-tunable luminescence spectra.

Topics: Biotinylation; Carbocyanines; Cell Nucleus; Chromosomes; DNA; DNA Probes; Fluorescein-5-isothiocyanate; Genes, Plant; Hybridization, Genetic; In Situ Hybridization, Fluorescence; Metaphase; Oligonucleotides; Quantum Dots; Spectrophotometry, Ultraviolet; Zea mays

Several transcription factors are essential determinants of a cortical projection neuron identity, but their mode of action (instructive versus permissive) and downstream genetic cascades remain poorly defined. Here, we demonstrate that the proneural basic helix-loop-helix (bHLH) gene Ngn2 instructs a partial cortical identity when misexpressed in ventral telencephalic progenitors, inducing ectopic marker expression in a defined temporal sequence, including early (24 h; Nscl2), intermediate (48 h; BhlhB5), and late (72 h; NeuroD, NeuroD2, Math2, and Tbr1) target genes. Strikingly, cortical gene expression was much more rapidly induced by Ngn2 in the dorsal telencephalon (within 12 to 24 h). We identify the bHLH gene Math3 as a dorsally restricted Ngn2 transcriptional target and cofactor, which synergizes with Ngn2 to accelerate target gene transcription in the cortex. Using a novel in vivo luciferase assay, we show that Ngn2 generates only approximately 60% of the transcriptional drive in ventral versus dorsal telencephalic domains, an activity that is augmented by Math3, providing a mechanistic basis for regional differences in Ngn2 function. Cortical bHLH genes thus cooperate to control transcriptional strength, thereby temporally coordinating downstream gene expression.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Biomarkers; Carbocyanines; Cell Differentiation; Cell Lineage; Cells, Cultured; Cerebral Cortex; Electroporation; Embryo, Mammalian; Fluorescent Dyes; Gene Expression Regulation, Developmental; Green Fluorescent Proteins; Immunohistochemistry; In Situ Hybridization; Luciferases; Mice; Mice, Inbred Strains; Mice, Mutant Strains; Models, Neurological; Mutation; Nerve Tissue Proteins; Neurons; Plasmids; Telencephalon; Time Factors; Transcription Factors

Recent advances in far-field fluorescence microscopy have led to substantial improvements in image resolution, achieving a near-molecular resolution of 20 to 30 nanometers in the two lateral dimensions. Three-dimensional (3D) nanoscale-resolution imaging, however, remains a challenge. We demonstrated 3D stochastic optical reconstruction microscopy (STORM) by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photoswitchable probes enables high-precision 3D localization of each probe, and thus the construction of a 3D image, without scanning the sample. Using this approach, we achieved an image resolution of 20 to 30 nanometers in the lateral dimensions and 50 to 60 nanometers in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.

Topics: Animals; Carbocyanines; Cell Line; Chlorocebus aethiops; Clathrin; Coated Pits, Cell-Membrane; Cyclic AMP; Fluorescent Antibody Technique; Fluorescent Dyes; Image Processing, Computer-Assisted; Imaging, Three-Dimensional; Microscopy, Fluorescence; Microtubules; Nanotechnology; Quantum Dots; Stochastic Processes; Streptavidin

Acyl coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Ni-affinity chromatography. The his-tag was inserted at the C-terminus since ACBP is a small molecular size (10 kDa) protein whose structure and activity are sensitive to amino acid substitutions in the N-terminus. The his-tag had no or little effect on ACBP structure or ligand binding affinity and specificity. His-ACBP bound the naturally occurring fluorescent cis-parinaroyl-CoA with very high affinity (K(d)=2.15 nM), but exhibited no affinity for non-esterified cis-parinaric acid. To determine if the presence of the C-terminal his-tag altered ACBP interactions with other proteins, direct binding to hepatocyte nuclear factor-4alpha (HNF-4alpha), a nuclear receptor regulating transcription of genes involved in lipid metabolism, was examined. His-ACBP and HNF-4alpha were labeled with Cy5 and Cy3, respectively, and direct interaction was determined by a novel fluorescence resonance energy transfer (FRET) binding assay. FRET analysis showed that his-ACBP directly interacted with HNF-4alpha (intermolecular distance of 73 A) at high affinity (K(d)=64-111 nM) similar to native ACBP. The his-tag also had no effect on ACBPs ability to interact with and stimulate microsomal enzymes utilizing or forming fatty acyl CoA. Thus, C-terminal his-tagged-ACBP maintained very similar structural and functional features of the untagged native protein and can be used in further in vitro experiments that require pure recombinant ACBP.

Topics: Acyl Coenzyme A; Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Carbocyanines; Circular Dichroism; Cloning, Molecular; Coenzyme A Ligases; Diazepam Binding Inhibitor; Escherichia coli; Fatty Acids, Unsaturated; Fluorescence Resonance Energy Transfer; Glycerol-3-Phosphate O-Acyltransferase; Hepatocyte Nuclear Factor 4; Histidine; Mice; Molecular Sequence Data; Recombinant Proteins; Spectrophotometry, Ultraviolet

DNA methylation based techniques are important tools in both clinical diagnostics and therapeutics. But most of these methods only analyze a few CpG sites in a target region. Indeed, difference of site-specific methylation may also lead to a change of methylation density in many cases, and it has been found that the density of methylation is more important than methylation of single CpG site for gene silencing.. We have developed a novel approach for quantitative analysis of CpG methylation density on the basis of microarray-based hybridization and incorporation of Cy5-dCTP into the Cy3 labeled target DNA by using Taq DNA Polymerase on microarray. The quantification is achieved by measuring Cy5/Cy3 signal ratio which is proportional to methylation density. This methylation-sensitive technique, termed RMEAM (regional methylation elongation assay on microarray), provides several advantages over existing methods used for methylation analysis. It can determine an exact methylation density of the given region, and has potential of high throughput. We demonstrate a use of this method in determining the methylation density of the promoter region of the tumor-related gene MLH1, TERT and MGMT in colorectal carcinoma patients.. This technique allows for quantitative analysis of regional methylation density, which is the representative of all allelic methylation patterns in the sample. The results show that this technique has the characteristics of simplicity, rapidness, specificity and high-throughput.

Topics: 5' Untranslated Regions; Adaptor Proteins, Signal Transducing; Base Sequence; Carbocyanines; Colorectal Neoplasms; CpG Islands; DNA; DNA Methylation; DNA Modification Methylases; DNA Repair Enzymes; DNA, Neoplasm; Fluorescent Dyes; Humans; Molecular Sequence Data; MutL Protein Homolog 1; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Taq Polymerase; Telomerase; Tumor Suppressor Proteins

Human recombinant tau can bind to the end of isolated human paired helical filaments (PHF). The sequential binding of tau protein to PHF could result in an elongation of the previously polymerized PHF. However, we have observed that the elongation takes place in a different way on different types of PHF. We have found that there are at least three populations of PHF. For one population, tau protein is able to bind to the ends of the filament and to elongate that filament. In the second PHF population, tau protein binds but does not elongates the filament. In the third, neither tau binding nor elongation was observed.

Topics: Alzheimer Disease; Binding Sites; Carbocyanines; Fluorescent Antibody Technique; Fluorescent Dyes; Humans; Intermediate Filament Proteins; Microscopy, Electron; Microtubules; Molecular Conformation; tau Proteins; Tubulin Modulators

To further understand the biological significance of amplifications for glioma development and recurrencies, we characterized amplicon frequency and size in low-grade glioma and amplicon stability in vivo in recurring glioblastoma. We developed a 12q13-21 amplicon-specific genomic microarray and a bioinformatics amplification prediction tool to analyze amplicon frequency, size, and maintenance in 40 glioma samples including 16 glioblastoma, 10 anaplastic astrocytoma, 7 astrocytoma WHO grade 2, and 7 pilocytic astrocytoma. Whereas previous studies reported two amplified subregions, we found a more complex situation with many amplified subregions. Analyzing 40 glioma, we found that all analyzed glioblastoma and the majority of pilocytic astrocytoma, grade 2 astrocytoma, and anaplastic astrocytoma showed at least one amplified subregion, indicating a much higher amplification frequency than previously suggested. Amplifications in low-grade glioma were smaller in size and displayed clearly different distribution patterns than amplifications in glioblastoma. One glioblastoma and its recurrencies revealed an amplified subregion of 5 Mb that was stable for 6 years. Expression analysis of the amplified region revealed 10 overexpressed genes (i.e., KUB3, CTDSP2, CDK4, OS-9, DCTN2, RAB3IP, FRS2, GAS41, MDM2, and RAP1B) that were consistently overexpressed in all cases that carried this amplification. Our data indicate that amplifications on 12q13-21 (a) are more frequent than previously thought and present in low-grade tumors and (b) are maintained as extended regions over long periods of time.

Topics: Adult; Aged; Blotting, Southern; Carbocyanines; Child; Child, Preschool; Chromosomes, Artificial, Bacterial; Chromosomes, Human, Pair 12; Computational Biology; Cosmids; DNA, Neoplasm; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Glioma; Humans; In Situ Hybridization, Fluorescence; Male; Middle Aged; Oligonucleotide Array Sequence Analysis

The concurrent detection and quantification of analytes that vary widely in concentration present a principal problem in multiplexed assay systems. Combining competitive and sandwich immunoassays permits coverage of a wide concentration range, and both highly abundant molecules and analytes present in low concentration can be quantified within the same assay.. The use of different fluorescence readout channels allows the parallel use of a competitive system and a sandwich configuration. The 2 generated assay signals are combined and used to calculate the amount of analyte. The measurement range can be adjusted by varying the competitor concentration, and an extension of the assay system's dynamic range is possible.. We implemented the method in a planar protein microarray-based autoimmune assay to detect autoantibodies against 13 autoantigens and to measure the concentration of a highly abundant protein, total human IgG, in one assay. Our results for autoantibody detection and IgG quantification agreed with results obtained with commercially available assays. The use of 2 readout channels in the protein microarray-based system reduced spot-to-spot variation and intraassay variation.. By combining a direct immunoassay with a competitive system, analytes present in widely varying concentrations can be quantified within a single multiplex assay. Introducing a second readout channel for analyte quantification is an effective tool for spot-to-spot normalization and helps to lower intraassay variation.

Topics: Autoanalysis; Autoantibodies; Autoantigens; Autoimmune Diseases; Carbocyanines; Enzyme-Linked Immunosorbent Assay; Fluorescence; Fluorescent Dyes; Humans; Immunoglobulin G; Protein Array Analysis; Reproducibility of Results

Most microarray studies are made using labelling with one or two dyes which allows the hybridization of one or two samples on the same slide. In such experiments, the most frequently used dyes are Cy3 and Cy5. Recent improvements in the technology (dye-labelling, scanner and, image analysis) allow hybridization up to four samples simultaneously. The two additional dyes are Alexa488 and Alexa494. The triple-target or four-target technology is very promising, since it allows more flexibility in the design of experiments, an increase in the statistical power when comparing gene expressions induced by different conditions and a scaled down number of slides. However, there have been few methods proposed for statistical analysis of such data. Moreover the lowess correction of the global dye effect is available for only two-color experiments, and even if its application can be derived, it does not allow simultaneous correction of the raw data.. We propose a two-step normalization procedure for triple-target experiments. First the dye bleeding is evaluated and corrected if necessary. Then the signal in each channel is normalized using a generalized lowess procedure to correct a global dye bias. The normalization procedure is validated using triple-self experiments and by comparing the results of triple-target and two-color experiments. Although the focus is on triple-target microarrays, the proposed method can be used to normalize p differently labelled targets co-hybridized on a same array, for any value of p greater than 2.. The proposed normalization procedure is effective: the technical biases are reduced, the number of false positives is under control in the analysis of differentially expressed genes, and the triple-target experiments are more powerful than the corresponding two-color experiments. There is room for improving the microarray experiments by simultaneously hybridizing more than two samples.

Topics: Arabidopsis; Artifacts; Calibration; Carbocyanines; Fluorescent Dyes; Gene Expression Profiling; Genes, Plant; Information Storage and Retrieval; Oligonucleotide Array Sequence Analysis; Organic Chemicals; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Sequence Analysis, DNA

Liposomally formulated siRNA can be used for RNAi applications in vivo. Intravenous bolus administration of lipoplexed siRNA has been shown to reduce gene expression in the vascular endothelium. Here, we applied immunofluorescence staining for different endothelial markers (PECAM-1, CD34, laminin) on paraffin sections to compare the respective expression pattern with the intracellular localization of intravenously administered, fluorescently labeled siRNA (siRNA-Cy3-lipoplex). By confocal microscopy, lipoplexed siRNA-Cy3 was detected inside vascular endothelial cells in vivo, which where identified with co-staining of endothelial markers. Consequently, the finding of intracellular siRNA uptake by vascular endothelial cells correlated with RNAi based specific protein reduction in situ as revealed by PECAM-1 specific immunofluorescence staining in lung tissue sections. Therefore, by using a cell biological approach these in situ data emphasize the functional uptake of liposomal siRNA molecules in vascular endothelial cells of different mouse tissues as indicated in our previous molecular study.

Topics: Animals; Antigens, CD; Antigens, CD34; Cadherins; Carbocyanines; Endothelial Cells; Endothelium, Lymphatic; Fluorescent Antibody Technique; Gene Expression; Glycoproteins; Immunohistochemistry; Intracellular Space; Laminin; Liposomes; Liver; Lung; Membrane Transport Proteins; Mice; Mice, Inbred C57BL; Mice, Nude; Myocardium; Platelet Endothelial Cell Adhesion Molecule-1; PTEN Phosphohydrolase; Pulmonary Alveoli; RNA, Small Interfering; Vascular Endothelial Growth Factor Receptor-2

This paper describes the fabrication, the characterization and the applications of a capillary electrophoresis microchip. This hybrid device (glass/PDMS) features channels and optical waveguides integrated in one common substrate. It can be used for electrophoretic separation and fluorimetric detection of molecules. The microfluidic performance of the device is demonstrated by capillary zone and gel electrophoresis of proteins.

Topics: Carbocyanines; Electroosmosis; Electrophoresis, Capillary; Microfluidic Analytical Techniques; Optics and Photonics; Proteins; Reproducibility of Results; Streptavidin

The present study describes an empirically discovered phenomenon that might be useful for development of a sensitive and rapid methodology for quantification of telomerase activity assay with simple data acquisition and possibility for calculation of telomerase product in absolute units. The method is based on the design and application of two single-stranded telomere sensing probes consisting of dual-labeled 16-mer oligonucleotides (fluorescent Cy3/Cy3-labeled and non-fluorescent IowaBlack/BHQ-labeled) that can simultaneously hybridize on the primary product of the telomerase reaction.

Topics: Base Sequence; Carbocyanines; DNA Probes; Fluorescent Dyes; Molecular Probe Techniques; Telomerase; Telomere

We have developed a sensitive, accurate, and multiplexed microRNA (miRNA) profiling assay that is based on a highly efficient labeling method and novel microarray probe design. The probes provide both sequence and size discrimination, yielding in most cases highly specific detection of closely related mature miRNAs. Using a simple, single-vial experimental protocol, 120 ng of total RNA is directly labeled using Cy3 or Cy5, without fractionation or amplification, to produce precise and accurate measurements that span a linear dynamic range from 0.2 amol to 2 fmol of input miRNA. The results can provide quantitative estimates of the miRNA content for the tissues studied. The assay is also suitable for use with formalin-fixed paraffin-embedded clinical samples. Our method allows rapid design and validation of probes for simultaneous quantitative measurements of all human miRNA sequences in the public databases and to new miRNA sequences as they are reported.

Topics: Animals; Carbocyanines; Gene Expression Profiling; Humans; MicroRNAs; Oligonucleotide Array Sequence Analysis; RNA Probes; Sensitivity and Specificity; Tissue Distribution

Although recent advances in fluorescence-based technologies, such as protein microarrays, have made it possible to analyse more than 10,000 proteins at once, there is a bottleneck in the step of preparation of large numbers of fluorescently labelled proteins for the comprehensive analysis of protein-protein interactions. Here we describe two independent methods for high-throughput fluorescence-labelling of full-length cDNA products at their C-termini using a reconstituted translation system containing fluorescent puromycin. For the first method, release factor-free systems were used. For the second method, stop codons were excluded from cDNAs by using a common mismatch primer in mutagenic PCR. These methods yielded large numbers of labelled proteins from cDNA sets of various organisms, such as mouse, yeast and Escherichia coli.

Topics: Carbocyanines; Cell-Free System; DNA, Complementary; Escherichia coli; Escherichia coli Proteins; Fluorescent Dyes; Multienzyme Complexes; Proteasome Endopeptidase Complex; Protein Biosynthesis; Proteomics; Puromycin; Saccharomyces cerevisiae Proteins

Although nuclear transport of therapeutic genes is an essential requirement of human gene therapy, factors required for nuclear entry of DNA remain to be elucidated. Non-viral vector systems have led to numerous improvements in the efficiency of delivery of exogenous DNA into cells. However, nuclear transport of plasmid is difficult to achieve.. We examined nuclear translocation efficiency of Cy3-labeled plasmid DNA (Cy3-pDNA) delivered by the hemagglutinating virus of Japan envelope (HVJ-E) vector, Lipofectamine or microinjection. We also examined the effect of actin depolymerization on nuclear transport of Cy3-pDNA.. Cy3-pDNA reached the nucleus, particularly in the nucleolus, in 30 min after fusion-mediated delivery using the HVJ-E vector, while the DNA was retained in the cytoplasm during the observed period after the delivery by cationic liposomes. HVJ-E treatment transiently depolymerized actin filaments, and acceleration of nucleolar entry of microinjected DNA was achieved when treated with either empty HVJ-E or cytochalasin D, an inhibitor of actin depolymerization, prior to microinjection.. These results suggest that plasmid DNA can be transported rapidly from the cytoplasm to the nucleolus when actin filaments are depolymerized. Thus, the HVJ-E vector can accelerate the transport of DNA to the nucleolus by actin depolymerization.

Topics: Actins; Biological Transport; Carbocyanines; Cell Nucleolus; Cells, Cultured; Coloring Agents; DNA; Fluorescent Dyes; Genetic Vectors; Humans; Microinjections; Plasmids; Sendai virus; Viral Envelope Proteins

The effects of metal-enhanced fluorescence (MEF) have been measured for two dyes commonly used in DNA microarrays, Cy3 and Cy5. Silver island films (SIFs) grown on glass microscope slides were used as substrates for MEF DNA arrays. We examined MEF by spotting biotinylated, singly-labeled 23 bp DNAs onto avidin-coated SIF substrates. The fluorescence enhancement was found to be dependent on the DNA spotting concentration: below approximately 12.5 muM, MEF increased linearly, and at higher concentrations MEF remained at a constant maximum of 28-fold for Cy5 and 4-fold for Cy3, compared to avidin-coated glass substrates. Hybridization of singly-labeled oligonucleotides to arrayed single-stranded probes showed lower maximal MEF factors of 10-fold for Cy5 and 2.5-fold for Cy3, because of the smaller amount of immobilized fluorophores as a result of reduced surface hybridization efficiencies. We discuss how MEF can be used to increase the sensitivity of DNA arrays, especially for far red emitting fluorophores like Cy5, without significantly altering current microarray protocols.

Topics: Carbocyanines; Fluorescent Dyes; Glass; Nanoparticles; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes; Photobleaching; Silver

The antibody microarray, a high-throughput multiplex immunoassay method, has become a significant tool for quantitative proteomics studies. We describe here the strategies for optimizing the condition of antibody microarray building based on agarose-coated slides. In this study, modified glass slides were robotically printed with capture antibodies against monocyte chemoattractant protein 1 (MCP-1), then dilutions of the cytokine were applied to the arrays, and the protein was detected with biotin-labeled antibody coupled with Cy3-conjugated streptavidin. Thus a protein profiling microarray based on sandwich immunoassay has been established. Various factors in the production of antibody microarrays were analyzed: the capture antibody concentrations, shelf life of the postprinting slides, blocking buffers, and reproducibility of the system. A calibration curve with a correlation coefficient of 0.9995 was established which suggested that the matrix can retain arrayed proteins in near-quantitative fashion. The results revealed high signal uniformity and reproducibility with regard to intra-array (1.3%) and the interarray (8.7%) variation at the capture antibody concentration of 125 microg/mL. Besides, the printed arrays could be stored for at least two months without any apparent change of the performance parameters.

Topics: Antibodies; Biotin; Carbocyanines; Chemokine CCL2; Immunoassay; Protein Array Analysis; Proteomics; Sepharose; Streptavidin

In mice, the relative numbers of male and female pups per litter not only can vary but can probably change over the course of pregnancy in response to numerous environmental and physiological factors. As such, a technique is required to determine gender at several developmental stages. Here we describe a robust and accurate fluorescent in situ hybridization (FISH) procedure for determining chromosomal sex that can be applied with minimal modification to sperm, pre-and post-implantation conceptuses and recovered dead post-natal pups. Sperm was prepared for FISH analysis y using a modified microwave decondensation-denaturation technique. Preimplantation conceptuses (0.5dpc) were cultured to the morula stage before sexing. They were then acid-treated to remove the zona pellucida. Tissue homogenates from postimplantational conceptuses (8.5dpc) and stillborn pups were fixed to pre-etched slides. Specimens were hybridized with identical, commercially available DNA probes for the X (FITC) and Y (Cy3) chromosomes. Sperm ratios met the expected value of 0.5 when determined by using XY FISH. Preimplantation conceptuses pre-treated with pepsin yielded distinct fluorescence of X and Y chromosomes in morulae, whereas microwave decondensation resulted in loss of conceptuses from the slide. Both 4.0 and 8.5dpc conceptuses displayed mean sex ratios of 0.5. Post-natal FISH analysis allowed gender identification of pups that could not be sexed due to developmental abnormalities or partial cannibalism. FISH analysis of sperm and of multiple conceptuses or post-natal tissue provided a cost-effective, accurate alternative to PCR-based sex determination.

Topics: Animals; Carbocyanines; Embryo, Mammalian; Female; Fluorescein-5-isothiocyanate; Fluorescent Dyes; In Situ Hybridization, Fluorescence; Male; Mice; Polymerase Chain Reaction; Pregnancy; Sex Chromosomes; Sex Determination Analysis

A novel assay using a hybridization-based method was developed for real-time monitoring of RNA synthesis. In this work, a "broken beacon" in which the fluor and quencher were located on separate but complementary oligonucleotides was used to quantify the amount of RNA production by T7 polymerase. The relative lengths of the fluor-oligo and quencher-oligo, and their relative concentrations were optimized. The experimentally determined limit-of-detection was approximately 45 nM. The new assay was compared to the "gold-standard" radiolabel ([(32)P]NTP incorporation) assay for RNA quantification. While the broken beacon assay exhibited a higher limit of detection, it provided an accurate measure of RNA production rates. However, the broken beacon assay provided the significant analytical advantages of (i) a real-time and continuous measurement, (ii) no requirement for the use of radiolabels or gel-based analysis, and (iii) substantial time and labor savings.

Topics: Carbocyanines; DNA-Directed RNA Polymerases; Kinetics; Nucleic Acid Amplification Techniques; Nucleic Acid Hybridization; Oligonucleotide Probes; Phosphorus Radioisotopes; Reproducibility of Results; RNA

A new challenge in biointerfacial science is the development of dynamic surfaces with the ability to adjust and tune the chemical functionality at the interface between the biological and nonbiological entities. In this paper we describe fabrication of indium-tin oxide (ITO) electrodes and the design of a ligand that can be switched to enable selectively controlled interactions with DNA. Tailoring the surface composition of the ITO electrode to optimize its optical and electrical properties was also studied. The surface attachment chemistry investigated utilizes thiol-disulfide exchange chemistry. This chemistry involved the covalent attachment of a thiol-functionalized silane anchor to a hydroxyl-activated ITO electrode surface. Subsequent reaction with 2-(2-pyridinyldithio)ethanamine hydrochloride formed the disulfide bridge and provided the terminal amine group, which facilitates addition of a cross-linker. DNA was then covalently bound to the cross-linker, and hybridization with the complementary Cy3-labeled target DNA was achieved. Selective release of the attached DNA was demonstrated by both chemical and electrical reduction of the disulfide bond. The surface chemistry was then recycled, and rehybridization of the target DNA was achieved. The ability to control specific release of biomolecules has applications for the development of novel biosensor platforms and a range of medical devices.

Topics: Carbocyanines; Disulfides; DNA; Electrochemistry; Electrodes; Pyridines; Silanes; Sulfhydryl Compounds; Surface Properties; Tin Compounds

Several important photophysical properties of the cyanine dye Cy3 have been determined by laser flash photolysis. The triplet-state absorption and photoisomerization of Cy3 are distinguished by using the heavy-atom effects and oxygen-induced triplet --> triplet energy transfer. Furthermore, the triplet-state extinction coefficient and quantum yield of Cy3 are also measured via triplet-triplet energy-transfer method and comparative actinometry, respectively. It is found that the triplet --> triplet (T1-->Tn) absorptions of trans-Cy3 largely overlap the ground-state absorption of cis-Cy3. Unlike what occurred in Cy5, we have not observed the triplet-state T1-->Tn absorption of cis-Cy3 and the phosphorescence from triplet state of cis-Cy3 following a singlet excitation (S0-S1) of trans-Cy3, indicating the absence of a lowest cis-triplet state as an isomerization intermediate upon excitation in Cy3. The detailed spectra of Cy3 reported in this paper could help us interpret the complicated photophysics of cyanine dyes.

Topics: Carbocyanines; Isomerism; Light; Photochemistry

Environmental ozone can rapidly degrade cyanine 5 (Cy5), a fluorescent dye commonly used in microarray gene expression studies. Cyanine 3 (Cy3) is much less affected by atmospheric ozone. Degradation of the Cy5 signal relative to the Cy3 signal in 2-color microarrays will adversely reduce the Cy5/Cy3 ratio resulting in unreliable microarray data.. Ozone in central Arkansas typically ranges between approximately 22 ppb to approximately 46 ppb and can be as high as 60-100 ppb depending upon season, meteorological conditions, and time of day. These levels of ozone are common in many areas of the country during the summer. A carbon filter was installed in the laboratory air handling system to reduce ozone levels in the microarray laboratory. In addition, the airflow was balanced to prevent non-filtered air from entering the laboratory. These modifications reduced the ozone within the microarray laboratory to approximately 2-4 ppb. Data presented here document reductions in Cy5 signal on both in-house produced microarrays and commercial microarrays as a result of exposure to unfiltered air. Comparisons of identically hybridized microarrays exposed to either carbon-filtered or unfiltered air demonstrated the protective effect of carbon-filtration on microarray data as indicated by Cy5 and Cy3 intensities. LOWESS normalization of the data was not able to completely overcome the effect of ozone-induced reduction of Cy5 signal. Experiments were also conducted to examine the effects of high humidity on microarray quality. Modest, but significant, increases in Cy5 and Cy3 signal intensities were observed after 2 or 4 hours at 98-99% humidity compared to 42% humidity.. Simple installation of carbon filters in the laboratory air handling system resulted in low and consistent ozone levels. This allowed the accurate determination of gene expression by microarray using Cy5 and Cy3 fluorescent dyes.

Topics: Animals; Artifacts; Carbocyanines; Fluorescence; Gene Expression Profiling; Laboratories; Mice; Oligonucleotide Array Sequence Analysis; Ozone; Reproducibility of Results

Topics: Cadmium Compounds; Carbocyanines; DNA, Single-Stranded; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Nucleic Acid Hybridization; Quantum Dots; Tellurium

Topics: Carbocyanines; HT29 Cells; Humans; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; RNA, Neoplasm

Lactobacillus helveticus CNRZ32 is used by the dairy industry to modulate cheese flavor. The compilation of a draft genome sequence for this strain allowed us to identify and completely sequence 168 genes potentially important for the growth of this organism in milk or for cheese flavor development. The primary aim of this study was to investigate the expression of these genes during growth in milk and MRS medium by using microarrays. Oligonucleotide probes against each of the completely sequenced genes were compiled on maskless photolithography-based DNA microarrays. Additionally, the entire draft genome sequence was used to produce tiled microarrays in which noninterrupted sequence contigs were covered by consecutive 24-mer probes and associated mismatch probe sets. Total RNA isolated from cells grown in skim milk or in MRS to mid-log phase was used as a template to synthesize cDNA, followed by Cy3 labeling and hybridization. An analysis of data from annotated gene probes identified 42 genes that were upregulated during the growth of CNRZ32 in milk (P < 0.05), and 25 of these genes showed upregulation after applying Bonferroni's adjustment. The tiled microarrays identified numerous additional genes that were upregulated in milk versus MRS. Collectively, array data showed the growth of CNRZ32 in milk-induced genes encoding cell-envelope proteinases, oligopeptide transporters, and endopeptidases as well as enzymes for lactose and cysteine pathways, de novo synthesis, and/or salvage pathways for purines and pyrimidines and other functions. Genes for a hypothetical phosphoserine utilization pathway were also differentially expressed. Preliminary experiments indicate that cheese-derived, phosphoserine-containing peptides increase growth rates of CNRZ32 in a chemically defined medium. These results suggest that phosphoserine is used as an energy source during the growth of L. helveticus CNRZ32.

Topics: Adaptation, Physiological; Animals; Carbocyanines; Culture Media; DNA, Bacterial; DNA, Complementary; Enzymes; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Lactobacillus helveticus; Metabolic Networks and Pathways; Milk; Molecular Sequence Data; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; RNA, Bacterial; Staining and Labeling

We present what is believed to be the first microstructured polymer optical fiber (mPOF) fabricated from Topas cyclic olefin copolymer, which has attractive material and biochemical properties. This polymer allows for a novel type of fiber-optic biosensor, where localized sensor layers may be activated on the inner side of the air holes in a predetermined section of the mPOF. The concept is demonstrated using a fluorescence-based method for selective detection of fluorophore-labeled antibodies.

Topics: Alkenes; Biocompatible Materials; Biosensing Techniques; C-Reactive Protein; Carbocyanines; Fiber Optic Technology; Microscopy, Fluorescence; Optical Fibers; Optics and Photonics; Polymers; Polymethyl Methacrylate; Streptavidin; Ultraviolet Rays

Dye-specific bias effects, commonly observed in the two-color microarray platform, are normally corrected using the dye swap design. This design, however, is relatively expensive and labor-intensive. We propose a self-self hybridization design as an alternative to the dye swap design. In this design, the treated and control samples are labeled with Cy5 and Cy3 (or Cy3 and Cy5), respectively, without dye swap, along with a set of self-self hybridizations on the control sample. We compare this design with the dye swap design through investigation of mouse primary hepatocytes treated with three peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists at three dose levels. Using Agilent's Whole Mouse Genome microarray, differentially expressed genes (DEG) were determined for both the self-self hybridization and dye swap designs. The DEG concordance between the two designs was over 80% across each dose treatment and chemical. Furthermore, 90% of DEG-associated biological pathways were in common between the designs, indicating that biological interpretations would be consistent. The reduced labor and expense for the self-self hybridization design make it an efficient substitute for the dye swap design. For example, in larger toxicogenomic studies, only about half the chips are required for the self-self hybridization design compared to that needed in the dye swap design.

Topics: Animals; Carbocyanines; Fluorescent Dyes; Gene Expression Regulation; Genome; Genomics; Hepatocytes; Mice; Models, Genetic; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis

U2 and U6 snRNAs pair to form a phylogenetically conserved complex at the catalytic core of the spliceosome. Interactions with divalent metal ions, particularly Mg(II), at specific sites are essential for its folding and catalytic activity. We used a novel Förster resonance energy transfer (FRET) method between site-bound luminescent lanthanide ions and a covalently attached fluorescent dye, combined with supporting stoichiometric and mutational studies, to determine locations of site-bound Tb(III) within the human U2-U6 complex. At pH 7.2, we detected three metal-ion-binding sites in: (1) the consensus ACACAGA sequence, which forms the internal loop between helices I and III; (2) the four-way junction, which contains the conserved AGC triad; and (3) the internal loop of the U6 intra-molecular stem loop (ISL). Binding at each of these sites is supported by previous phosphorothioate substitution studies and, in the case of the ISL site, by NMR. Binding of Tb(III) at the four-way junction and the ISL sites was found to be pH-dependent, with no ion binding observed below pH 6 and 7, respectively. This pH dependence of metal ion binding suggests that the local environment may play a role in the binding of metal ions, which may impact on splicing activity.

Topics: Base Pairing; Base Sequence; Binding Sites; Carbocyanines; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Ions; Molecular Sequence Data; Mutation; Nucleic Acid Conformation; RNA, Small Nuclear; Terbium

E. coli UvrD is an SF1 helicase involved in several DNA metabolic processes. Although a UvrD dimer is needed for helicase activity, a monomer can translocate with 3' to 5' directionality along single-stranded DNA, and this ATP-dependent translocation is likely involved in RecA displacement. In order to understand how the monomeric translocase functions, we have combined fluorescence stopped-flow kinetic methods with recently developed analysis methods to determine the kinetic mechanism, including ATP coupling stoichiometry, for UvrD monomer translocation along ssDNA. Our results suggest that the macroscopic rate of UvrD monomer translocation is not limited by each ATPase cycle but rather by a slow step (pause) in each translocation cycle that occurs after four to five rapid 1 nt translocation steps, with each rapid step coupled to hydrolysis of one ATP. These results suggest a nonuniform stepping mechanism that differs from either a Brownian motor or previous structure-based inchworm mechanisms.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Carbocyanines; DNA Helicases; DNA-Binding Proteins; DNA, Bacterial; DNA, Single-Stranded; Escherichia coli; Escherichia coli Proteins; Fluorescein; Kinetics; Models, Biological; RecQ Helicases; Translocation, Genetic

In quantitative proteomics, the false discovery rate (FDR) can be defined as the number of false positives within statistically significant changes in expression. False positives accumulate during the simultaneous testing of expression changes across hundreds or thousands of protein or peptide species when univariate tests such as the Student's t test are used. Currently most researchers rely solely on the estimation of p values and a significance threshold, but this approach may result in false positives because it does not account for the multiple testing effect. For each species, a measure of significance in terms of the FDR can be calculated, producing individual q values. The q value maintains power by allowing the investigator to achieve an acceptable level of true or false positives within the calls of significance. The q value approach relies on the use of the correct statistical test for the experimental design. In this situation, a uniform p value frequency distribution when there are no differences in expression between two samples should be obtained. Here we report a bias in p value distribution in the case of a three-dye DIGE experiment where no changes in expression are occurring. The bias was shown to arise from correlation in the data from the use of a common internal standard. With a two-dye schema, where each sample has its own internal standard, such bias was removed, enabling the application of the q value to two different proteomics studies. In the case of the first study, we demonstrate that 80% of calls of significance by the more traditional method are false positives. In the second, we show that calculating the q value gives the user control over the FDR. These studies demonstrate the power and ease of use of the q value in correcting for multiple testing. This work also highlights the need for robust experimental design that includes the appropriate application of statistical procedures.

Topics: Animals; Bacterial Proteins; Carbocyanines; Cell Cycle Proteins; Data Interpretation, Statistical; Electrophoresis, Gel, Two-Dimensional; Fluorescent Dyes; Mice; Mice, Knockout; Mutation; Nuclear Proteins; Pectobacterium carotovorum; Period Circadian Proteins; Proteome; Proteomics; Transcription Factors

For biotechnological research, development, and production various analytical methods are required to determine the quality of the target product. In this context, the determination of isoforms is state-of-the-art; however, the majority of applied techniques are more qualitative than quantitative. To address this fact, we evaluated different post- and pre-electrophoretic staining dyes for their applicability on linear IPG gels using recombinant human erythropoietin as a model protein. Each evaluated dyes was able to detect all isoforms reproducibly, but CyDyes were found to be the most promising. Using CyDyes, up to three samples can be focused on the same lane under identical electrophoretic conditions, thus, a fast, reproducible, sensitive and quantitative isoform determination can be performed. To illustrate the practical relevance, quantitative CyDye technique was used for the characterization of our model protein, recombinant human Epo-Fc. This method makes it possible to determine the isoform pattern of nonpurified supernatants as well as purified proteins. We conclude that quantitative pre-electrophoretic staining IEF using CyDyes is a fast, simple, accurate method to determine isoforms, which can be used in research, development, and manufacturing for product quality analysis, e.g., clone screening, process optimization, and purification monitoring.

Topics: Carbocyanines; Densitometry; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Erythropoietin; Europium; Fluorescent Dyes; Humans; Isoelectric Focusing; Protein Isoforms; Proteins; Recombinant Proteins; Reproducibility of Results; Staining and Labeling

Topics: Arsenicals; Carbocyanines; Fluorescent Dyes; Molecular Probe Techniques; Molecular Probes; Molecular Structure; Peptides; Protein Binding; Spectrometry, Fluorescence

Topics: Animals; Biosensing Techniques; Carbocyanines; Fluorescence; Fluorescent Dyes; Humans; Nerve Tissue Proteins; Sequence Analysis, DNA; Trinucleotide Repeats

F(1)-ATPase is a rotary molecular motor that proceeds in 120 degrees steps, each driven by ATP hydrolysis. How the chemical reactions that occur in three catalytic sites are coupled to mechanical rotation is the central question. Here, we show by high-speed imaging of rotation in single molecules of F(1) that phosphate release drives the last 40 degrees of the 120 degrees step, and that the 40 degrees rotation accompanies reduction of the affinity for phosphate. We also show, by single-molecule imaging of a fluorescent ATP analog Cy3-ATP while F(1) is forced to rotate slowly, that release of Cy3-ADP occurs at approximately 240 degrees after it is bound as Cy3-ATP at 0 degrees . This and other results suggest that the affinity for ADP also decreases with rotation, and thus ADP release contributes part of energy for rotation. Together with previous results, the coupling scheme is now basically complete.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Carbocyanines; Catalysis; Imaging, Three-Dimensional; Kinetics; Phosphates; Proton-Translocating ATPases; Rotation; Thermodynamics; Time Factors

To explore the gene which might influence endometrium receptivity during the implantation window time in normal women and patients with polycystic ovary syndrome (PCOS).. Transvaginal ultrasound were performed and serum estrogen and progestogen levels were measured in all women to monitor the exact time of ovulation. Endometrium biopsy was done in normal women and PCOS patients during implantation window time. Sixteen women were enrolled in this study, in which seven were normal women, and nine were PCOS patients. cDNA extraction was performed, matrix metalloproteinase 26 (MMP-26) primers were synthesized and real time fluorescent quantitative PCR was conducted using beta-actin gene as endogenous control.. The ratios of MMP-26 were 0.31, 0.11 and 0.05 in 3 patients with PCOS by real time fluorescent quantitative PCR, obviously decreased during implantation time compared with the normal women.. Our data suggest a lower expression of MMP-26 in implantation window time in patients with PCOS than in normal patients. This might indicate a declined capability of endometrium receptivity in implantation window time in patients with PCOS.

Topics: Adult; Carbocyanines; DNA, Complementary; Embryo Implantation; Endometrium; Female; Gene Expression Regulation, Enzymologic; Humans; Matrix Metalloproteinases, Secreted; Menstrual Cycle; Polycystic Ovary Syndrome; Reverse Transcriptase Polymerase Chain Reaction; Time Factors

Integrin alpha(IIb)beta(3), an abundant heterodimeric receptor at the surface of blood platelets, binds adhesive proteins after platelet activation and plays a primary role in haemostasis. In solution, it has been observed mainly in two conformations: the bent and the extended forms. Based on X-ray crystallography, electron microscopy and immunochemical observations of full-length integrin ectodomains and intact integrins, it has been agreed that unactivated integrins are in the bent conformation, both isolated in solution and in living cells. However, consensus is yet to emerge on the bent or extended conformation of activated integrins and on their mechanism of activation (the switchblade, the deadbolt and the S-S reduction models), which require further experimental tests at the cell level to become established facts. Here, we tested the proposed structural rearrangements undergone by integrin alpha(IIb)beta(3) after cell activation, by using Förster-type fluorescence resonance energy transfer (FRET) and attached fluorescent labels to Fab fragments of monoclonal antibodies directed to the betaA domain of the beta(3) subunit (donor, Alexa488-P97 Fab) and to the Calf-2 domain of the alpha(IIb) subunit (acceptor, Cy3-M3 Fab or Cy3-M10 Fab). The FRET efficiencies observed after ADP or TRAP platelet activation changed less than 20% from the resting values, showing that the distance between the labeled Fab fragments changes only modestly after platelet activation by physiological agonists. This observation is consistent with a conformational model of the activated integrin in the cell less extended than in the switchblade model.

Topics: Carbocyanines; Fluorescence Resonance Energy Transfer; Humans; Immunoglobulin Fab Fragments; Platelet Activation; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Conformation

The sulfoindocyanine Cy3 is one of the most commonly used fluorescent dyes in the investigation of the structure and dynamics of nucleic acids by means of fluorescence methods. In this work, we report the fluorescence and photophysical properties of Cy3 attached covalently to single-stranded and duplex DNA. Steady-state and time-resolved fluorescence techniques were used to determine fluorescence quantum yields, emission lifetimes, and fluorescence anisotropy decays. The existence of a transient photoisomer was investigated by means of transient absorption techniques. The fluorescence quantum yield of Cy3 is highest when attached to the 5' terminus of single-stranded DNA (Cy3-5' ssDNA), and decreases by a factor of 2.4 when the complementary strand is annealed to form duplex DNA (Cy3-5' dsDNA). Substantial differences were also observed between the 5'-modified strands and strands modified through an internal amino-modified deoxy uridine. The fluorescence decay of Cy3 became multiexponential upon conjugation to DNA. The longest lifetime was observed for Cy3-5' ssDNA, where about 50% of the decay is dominated by a 2.0-ns lifetime. This value is more than 10 times larger than the fluorescence lifetime of the free dye in solution. These observations are interpreted in terms of a model where the molecule undergoes a trans-cis isomerization reaction from the first excited state. We observed that the activation energy for photoisomerization depends strongly on the microenvironment in which the dye is located. The unusually high activation energy measured for Cy3-5' ssDNA is an indication of dye-ssDNA interactions. In fact, the time-resolved fluorescence anisotropy decay of this sample is dominated by a 2.5-ns rotational correlation time, which evidences the lack of rotational freedom of the dye around the linker that separates it from the terminal 5' phosphate. The remarkable variations in the photophysical properties of Cy3-DNA constructs demonstrate that caution should be used when Cy3 is used in studies employing DNA conjugates.

Topics: Anisotropy; Carbocyanines; DNA, Single-Stranded; Fluorescence; Fluorescent Dyes; Isomerism; Photolysis; Quantum Theory

A novel normalization method based on the GC content of probes is developed for two-color tiling arrays. The proposed method, together with robust estimates of the model parameters, is shown to perform superbly on published data sets. A robust algorithm for detecting peak regions is also formulated and shown to perform well compared to other approaches. The tools have been implemented as a stand-alone Java program called MA2C, which can display various plots of statistical analysis for quality control.

Topics: Algorithms; Animals; Base Sequence; Caenorhabditis elegans; Carbocyanines; Computational Biology; Fluorescent Dyes; Nucleic Acid Probes; Oligonucleotide Array Sequence Analysis; Software

Amyloid beta (Abeta) protein, a 39-43 amino acid peptide deposited in brains of individuals with Alzheimer's disease (AD), has been shown to interact directly with a number of receptor targets including neuronal nicotinic acetylcholine receptors (nAChRs) and glutamate receptors. In this study, we investigated the synaptic effects of Abeta(1-42) on glutamate-mediated neurotransmission in the diagonal band of Broca (DBB), a cholinergic basal forebrain nucleus. Glutamatergic miniature EPSCs (mEPSCs) were recorded using whole-cell patch-clamp recordings from identified cholinergic DBB neurons in rat forebrain slices. In 54% of DBB neurons, bath application of Abeta(1-42) (100 nM), but not Abeta(42-1) (inverse fragment), significantly increased the frequency of mEPSCs without affecting amplitude or kinetic parameters (rise or decay time). In 32% of DBB neurons, bath application of Abeta(1-42) significantly decreased only the frequency but not amplitude of mEPSCs. Application of dihydro-beta-erythroidine (DHbetaE) (an antagonist for the alpha4beta2 subtype of nAChRs) but not alpha-bungarotoxin (an antagonist for the alpha7 subtype of nAChRs) blocked Abeta(1-42)-mediated increases in mEPSC frequency. The Abeta(1-42)-mediated increase in glutamatergic transmission is thus presynaptic and mediated via non-alpha7 AChRs. In contrast, Abeta(1-42)-mediated decreases in mEPSC frequency could not be antagonized by either DHbetaE or alpha-bungarotoxin. However, the Abeta(1-42)-evoked depression in mEPSC frequency was antagonized by (RS)-alpha-methyl-4-carboxyphenyglycine, a nonselective group I/II metabotropic glutamate receptor antagonist. These observations provide further insight into the mechanisms whereby Abeta affects synaptic function in the brain and may be relevant in the context of synaptic failure observed in AD.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amyloid beta-Peptides; Animals; Antibodies, Monoclonal; Carbocyanines; Cholinergic Agents; Diagonal Band of Broca; Dihydro-beta-Erythroidine; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; In Vitro Techniques; N-Glycosyl Hydrolases; Neurons; Nicotinic Antagonists; Patch-Clamp Techniques; Peptide Fragments; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Receptors, Nicotinic; Ribosome Inactivating Proteins, Type 1; Saporins

Antibody-based microarray is a novel technology with great potential within high-throughput proteomics. The process of designing high-performing antibody (protein) microarrays has, however, turned out to be a challenging process. In this study, we have developed further our human recombinant single-chain variable-fragment (scFv) antibody microarray methodology by addressing two crucial technological issues, choice of sample labeling-tag and solid support. We examined the performance of a range of dyes in a one- or two-color approach on a selection of solid supports providing different surface and coupling chemistries, and surface structures. The set-ups were evaluated in terms of sensitivity, specificity, and selectivity. The results showed that a one-color approach, based on NHS-biotin (or ULS-biotin) labeling, on black polymer Maxisorb slides (or Nexterion slide H) was the superior approach for targeting low-abundant (pg/mL) analytes in nonfractionated, complex proteomes, such as human serum or crude cell supernatants. Notably, microarrays displaying adequate spot morphologies, high S/Ns, minimized nonspecific binding, and most importantly a high selectivity, specificity, and sensitivity (>or=fM range) were obtained. Taken together, we have designed the first generation of a high-performing recombinant scFv antibody microarray technology platform on black polymer Maxisorb slides for sensitive profiling of low-abundant analytes in nonfractionated biotinylated complex proteomes.

Topics: Biotinylation; Carbocyanines; Cyclic AMP; Fluorescent Dyes; Humans; Immunoglobulin Variable Region; Protein Array Analysis; Proteome; Proteomics; Recombinant Proteins; Sensitivity and Specificity

While much is known of the molecular machinery involved in protein sorting during exocytosis, less is known about the spatial regulation of exocytosis at the plasma membrane (PM). This study outlines a novel method, dual substrate display, used to formally test the hypothesis that E-cadherin-mediated adhesion directs basolateral vesicle exocytosis to specific sites at the PM. We show that vesicles containing the basolateral marker protein VSV-G preferentially target to sites of adhesion to E-cadherin rather than collagen VI or a control peptide. These results support the hypothesis that E-cadherin adhesion initiates signaling at the PM resulting in targeted sites for exocytosis.

Topics: Animals; Biological Transport; Cadherins; Carbocyanines; Cell Adhesion Molecules; Cell Line; Cell Membrane; Cytoplasmic Vesicles; Exocytosis; Fluorescent Dyes; Luminescent Proteins; Membrane Glycoproteins; Microscopy, Fluorescence; Protein Binding; Viral Envelope Proteins

The breast cancer metastasis suppressor 1 (BRMS1) gene has been shown to suppress metastasis without affecting the growth of the primary tumor in mouse models. It has also been shown to suppress the metastasis of tumors derived from breast, melanoma, and, more recently, ovarian carcinoma (see ref 1). However, how BRMS1 exerts its metastasis suppressor function remains unknown. To shed light into its metastatic mechanism of action, the sensitive 2D-DIGE analysis coupled with MS has been used to identify proteins differentially expressed by either overexpressing (Mel-BRMS1) or silencing BRMS1 (sh635) in a melanoma cell line. After comparison of the protein profiles from WT, Mel-BRMS1, and sh635 cells, 79 spots were found to be differentially expressed. Mass spectrometry analysis allowed the unambiguous identification of 55 polypeptides, corresponding to 43 different proteins. Interestingly, more than 75% of the identified proteins were down-regulated in Mel-BRMS1 cells compared to WT. In contrast, all the identified proteins in sh635 cells extracts were up-regulated compared to WT. Most of the deregulated proteins are involved in cell growth/maintenance and signal transduction among other cell processes. Six differentially expressed proteins (Hsp27, Alpha1 protease inhibitor, Cofilin1, Cathepsin D, Bone morphogenetic protein receptor2, and Annexin2) were confirmed by immunoblot and functional assays. Excellent correlation was found between DIGE analysis and immunoblot results, indicating the reliability of the analysis. Available evidence on the reported functions of the identified proteins supports the emerging role of BRMS1 as negative regulator of the metastasis development. This work opens an avenue for the molecular mechanisms' characterization of metastasis suppressor genes with the aim to understand their roles.

Topics: Bone Morphogenetic Protein Receptors, Type II; Carbocyanines; Cathepsin D; Cell Line; Cell Line, Tumor; Data Interpretation, Statistical; Electrophoresis, Gel, Two-Dimensional; Fluorescent Dyes; Humans; Melanoma; Neoplasm Metastasis; Neoplasm Proteins; Proteome; Proteomics; Repressor Proteins; RNA Interference; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Microarrays are a powerful tool for comparison and understanding of gene expression levels in healthy and diseased states. The method relies upon the assumption that signals from microarray features are a reflection of relative gene expression levels of the cell types under investigation. It has previously been reported that the classical fluorescent dyes used for microarray technology, Cy3 and Cy5, are not ideal due to the decreased stability and fluorescence intensity of the Cy5 dye relative to the Cy3, such that dye bias is an accepted phenomena necessitating dye swap experimental protocols and analysis of differential dye affects. The incentive to find new fluorophores is based on alleviating the problem of dye bias through synonymous performance between counterpart dyes. Alexa Fluor 555 and Alexa Fluor 647 are increasingly promoted as replacements for CyDye in microarray experiments. Performance relates to the molecular and steric similarities, which will vary for each new pair of dyes as well as the spectral integrity for the specific application required. Comparative analysis of the performance of these two competitive dye pairs in practical microarray applications is warranted towards this end. The findings of our study showed that both dye pairs were comparable but that conventional CyDye resulted in significantly higher signal intensities (P < 0.05) and signal minus background levels (P < 0.05) with no significant difference in background values (P > 0.05). This translated to greater levels of differential gene expression with CyDye than with the Alexa Fluor counterparts. However, CyDye fluorophores and in particular Cy5, were found to be less photostable over time and following repeated scans in microarray experiments. These results suggest that precautions against potential dye affects will continue to be necessary and that no one dye pair negates this need.

Topics: Carbocyanines; Cell Line, Tumor; Cyclic AMP; Fluorescent Dyes; Humans; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis

A high-throughput Cognate Site Identity (CSI) microarray platform interrogating all 524 800 10-base pair variable sites is correlated to quantitative DNase I footprinting data of DNA binding pyrrole-imidazole polyamides. An eight-ring hairpin polyamide programmed to target the 5 bp sequence 5'-TACGT-3' within the hypoxia response element (HRE) yielded a CSI microarray-derived sequence motif of 5'-WWACGT-3' (W = A,T). A linear beta-linked polyamide programmed to target a (GAA)3 repeat yielded a CSI microarray-derived sequence motif of 5'-AARAARWWG-3' (R = G,A). Quantitative DNase I footprinting of selected sequences from each microarray experiment enabled quantitative prediction of Ka values across the microarray intensity spectrum.

Topics: Base Pair Mismatch; Base Sequence; Carbocyanines; Deoxyribonuclease I; DNA; DNA Footprinting; Kinetics; Molecular Sequence Data; Nucleic Acid Conformation; Nylons; Oligonucleotide Array Sequence Analysis; Plasmids; Protein Structure, Secondary

Topics: Base Sequence; Carbocyanines; DNA; DNA Restriction Enzymes; Ligands; Models, Chemical; Molecular Sequence Data; Nucleic Acid Conformation; Nylons; Protein Conformation; Stereoisomerism; Temperature

Differential in-gel electrophoresis (DIGE) experiments allow three protein samples to be run per gel. The three samples are labeled with the spectrally resolvable fluorescent dyes, Cy2, Cy3, and Cy5, respectively. Here, we show that protein-specific dye effects exist, and we present a linear mixed model for analysis of DIGE data which takes dye effects into account. A Java implementation of the model, called DIGEanalyzer, is freely available at http://bioinfo.thep.lu.se/digeanalyzer.html. Three DIGE experiments from our laboratory, with 173, 64, and 24 gels, respectively, were used to quantify and verify the dye effects. DeCyder 5.0 and 6.5 were used for spot detection and matching. The fractions of proteins with a statistically significant (0.001 level) dye effect were 19, 34, and 23%, respectively. The fractions of proteins with a dye effect above 1.4-fold change were 1, 4, and 6%, respectively. The median magnitude of the dye effect was 1.07-fold change for Cy5 versus Cy3 and 1.16-fold change for Cy3 versus Cy2. The maximal dye effect was a seven-fold change. The dye effects of spots corresponding to the same protein tend to be similar within each of the three experiments, and to a smaller degree across experiments.

Topics: Algorithms; Animals; Brain Chemistry; Breast Neoplasms; Carbocyanines; Computational Biology; Electrophoresis, Gel, Two-Dimensional; Female; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Internet; Linear Models; Ovarian Neoplasms; Proteins; Proteomics; Rats; Software; Tandem Mass Spectrometry

Gene expression technology offers great potentials to generate new insights into human disease pathogenesis; however, the data quality remains a major obstacle for realizing its potentials. In the present study 60-mers oligonucleotide target immobilized on coated glass slides were utilized as a model system to investigate parameters, such as target concentration, retention, signal linearity, and fluorescence properties of fluorophores, which likely affect the quality of microarray results. An array calibration slide was used to calibrate an Axon GenePix 4000A scanner and ensure the dynamic range of the instrument. The work is a first step toward our goal of quantitative gene expression measurements.

Topics: Calibration; Carbocyanines; Humans; Oligonucleotide Array Sequence Analysis; Oligonucleotides; Spectrometry, Fluorescence

Copy-number polymorphisms at specific genomic loci have been implicated in numerous human and animal disease phenotypes. Multiplex ligation-dependent probe amplification (MLPA) is a molecular genetic technique allowing targeted quantification of genomic copy-number changes (deletions and duplications), with potential for multiplexing up to 50 loci in one assay, and resolution down to the single nucleotide level. Modification of the MLPA technique to include Cy-labeled amplification primers permits parallel product detection by capillary electrophoresis and microarray hybridization. Detection and quantification of products by sequence-specific hybridization rather than size-specific capillary electrophoresis increases the potential for probe multiplexing possible in one assay and also allows for more flexible and efficient MLPA probe design. Protocols for the printing of synthetic oligonucleotide probe-sets for the detection of MLPA products, MLPA-probe amplification using array-compatible primers, and parallel product detection by quantitative capillary electrophoresis and microarray hybridization have been optimized.

Topics: Carbocyanines; Electrophoresis, Capillary; Humans; Nucleic Acid Amplification Techniques; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes

Protein microarray is a powerful tool for identifying disease biomarkers and therapeutical targets, and for systematically studying biological pathways with high efficiency. Although the protein microarray platform has been adopted by proteomic research and discovery, what remains problematic is how to maintain the activities and structures of printed proteins on slide surface. With the recent accomplishments in the R & D laboratory, now scientists around the world are able to preserve the biological functions of spotted proteins for high throughput analysis. Full Moon BioSystems (FMB) has developed general guidelines, which helps scientists efficiently prepare forward as well as reverse-phase protein microarray using FMB's proprietary polymer-coated slides, to obtain reliable and accurate array data with FMB's unique detection and analysis technology.

Topics: Calibration; Carbocyanines; Cell Line; Humans; Protein Array Analysis; Surface Properties

We report a method for the rapid differentiation of nucleic acid sequences with 5-carboxyvinyl-2'-deoxyuridine that allows the simultaneous observation of two colors and facilitates the SNP typing of a heterozygous sample.

Topics: Base Pair Mismatch; Carbocyanines; Deoxyuridine; DNA Mutational Analysis; Fluorescent Dyes; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes; Photochemistry; Point Mutation; Polymorphism, Single Nucleotide

Topics: Candida; Carbocyanines; DNA; DNA, Bacterial; DNA, Fungal; Exodeoxyribonucleases; Fluorescent Dyes; Listeria monocytogenes; Microbiological Techniques; Neisseria meningitidis; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Polymorphism, Single Nucleotide

In this work, fluorescence anisotropy was used to study DNA binding of the DNA methyltransferase M.TaqI. For this purpose short DNA molecules labelled with three different fluorophores (Cy3, thiazole orange, and ethidium bromide) were prepared in various topologies and their suitability for detection of DNA-protein complex formation was investigated.

Topics: Benzothiazoles; Carbocyanines; DNA; Ethidium; Fluorescence Polarization; Fluorescent Dyes; Nucleic Acid Conformation; Quinolines; Site-Specific DNA-Methyltransferase (Adenine-Specific)

Different labeling methods were studied to compare various approaches to the preparation of labeled target DNA for microarray experiments. The methods under investigation included a post-PCR labeling method using the Klenow fragment and a DecaLabel DNA labeling kit, the use of a Cy3-labeled forward primer in the PCR, generating either double-stranded or single-stranded PCR products, and the incorporation of Cy3-labeled dCTPs in the PCR. A microarray that had already been designed and used for the detection of microorganisms in compost was used in the study. PCR products from the organisms Burkholderia cepacia and Staphylococcus aureus were used in the comparison study, and the signals from the probes for these organisms analyzed. The highest signals were obtained when using the post-PCR labeling method, although with this method, more non-specific hybridizations were found. Single-stranded PCR products that had been labeled by the incorporation of a Cy3-labeled forward primer in the PCR were found to give the next highest signals upon hybridization for a majority of the tested probes, with less non-specific hybridizations. Hybridization with double-stranded PCR product labeled with a Cy3-labeled forward primer, or labeled by the incorporation of Cy3-labeled dCTPs resulted in acceptable signal to noise ratios for all probes except the UNIV 1389a and Burkholderia genus probes, both located toward the 3' end of the 16S rRNA gene. The comparison of the different DNA labeling methods revealed that labeling via the Cy3-forward primer approach is the most appropriate of the studied methods for the preparation of labeled target DNA for our purposes.

Topics: Burkholderia cepacia; Carbocyanines; DNA Polymerase I; DNA, Bacterial; Fluorescent Dyes; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Staining and Labeling; Staphylococcus aureus

Differential display (DD) is one of the most commonly used approaches for identifying differentially expressed genes. Despite the great impact of the method on biomedical research, there has been a lack of automation of DD technology to increase its throughput and accuracy for a systematic gene expression analysis. Most of previous DD work has taken a "shotgun" approach of identifying one gene at a time, with limited polymerase chain reaction (PCR) reactions set up manually, giving DD a low-technology and low-throughput image. With our newly created DD mathematical model, which has been validated by computer simulations, global analysis of gene expression by DD technology is no longer a shot in the dark. After identifying the "rate-limiting" factors that contribute to the "noise" level of DD method, we have optimized the DD process with a new platform that incorporates fluorescent digital readout and automated liquid handling. The resulting streamlined fluorescent DD (FDD) technology offers an unprecedented accuracy, sensitivity, and throughput in comprehensive and quantitative analysis of gene expression. We are using this newly integrated FDD technology to conduct a systematic and comprehensive screening for p53 tumor-suppressor gene targets.

Topics: Animals; Apoptosis; Blotting, Northern; Blotting, Western; Carbocyanines; Cloning, Molecular; Deoxyribonuclease I; DNA, Complementary; Fluorescent Dyes; Gene Expression Profiling; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Genes, p53; Humans; Models, Theoretical; Neoplasms; Polymerase Chain Reaction; RNA; RNA, Messenger; Sensitivity and Specificity; Software; Time Factors; Transcription, Genetic; Tumor Suppressor Protein p53

Immobilized patterns of unmodified fibroblast growth factor-2 (FGF-2), with varying surface concentrations, were inkjet printed onto physiologically relevant fibrin substrates. Printed patterns were characterized using iodinated FGF-2 to determine FGF-2 surface concentration and retention of FGF-2 binding in vitro. MG-63 cells were uniformly seeded onto patterned substrates. Cells were exposed to defined spatial FGF-2 surface concentrations of 1-22 pg/mm(2). Cell numbers were observed to increase in register with the printed FGF-2 patterns from an initial random uniform cell distribution across the patterned and non-patterned regions. Based on time-lapse image analysis, the primary organizational response of the cells was determined to be proliferation and not migration. Cell counts on and off the FGF-2 patterns over time demonstrated an increase in cell density up to a FGF-2 surface concentration of 14 pg/mm(2). Higher surface concentrations did not result in increased cell density. In addition, the cells on the FGF-2 patterns survived longer than the cells off patterns. Our inkjet printing approach permits the systematic study of cellular responses to defined spatial surface concentrations of immobilized growth factors.

Topics: Carbocyanines; Cell Line; Cell Proliferation; Dose-Response Relationship, Drug; Fibrin; Fibroblast Growth Factor 2; Fluorescent Dyes; Humans; Printing; Surface Properties

The calcium ionophore ionomycin induces apoptosis-like events in the human embryonic kidney cell line at early times. Plasma membrane blebbing, mitochondrial depolarization, externalization of phosphatidylserine, and nuclear permeability changes can all be observed within 15 min of treatment. However, there is no activation of caspases or chromatin condensation. Expression of a fusion protein containing the enhanced green fluorescent protein (EGFP) and human cytosolic Group IVA phospholipase A(2)alpha (EGFP-cPLA(2)alpha) in these cells prevents ionomycin-induced phosphatidylserine externalization and death. Cells expressing the cPLA(2)alpha mutant D43N, which does not bind calcium, retain their susceptibility to ionomycin-induced cell death. Both nonexpressing and EGFP-D43N-cPLA(2)alpha-expressing human embryonic kidney cells can be spared from ionomycin-induced cell death by pretreating them with exogenous arachidonic acid. Moreover, during calcium overload, mitochondrial depolarization is significantly lower in the EGFP-cPLA(2)alpha-expressing cells than in cells expressing normal amounts of cPLA(2)alpha. These results suggest that early cell death events promoted by an overload of calcium can be prevented by the presence of high levels of arachidonic acid.

Topics: Animals; Annexin A5; Arachidonic Acid; Arachidonic Acids; Calcium; Carbocyanines; Cell Death; Cell Line; Enzyme Inhibitors; Fluorescent Dyes; Group IV Phospholipases A2; Humans; Ionomycin; Ionophores; Kidney; Macrophages; Mice; Organophosphonates; Phospholipases A; Phospholipases A2; Recombinant Fusion Proteins

Cell culture optimization is a labor-intensive process requiring a large number of experiments to be conducted under varying conditions. Here we describe a high-throughput bioreactor system that allows 12 mini stirred-tank bioreactors to be operated simultaneously. All bioreactors are monitored by low-cost minimally invasive optical sensors for pH and dissolved oxygen. The sensors consist of single-use patches affixed inside the bioreactors and monitored optically from the outside. Experimental results show that different sensing patches with the same composition respond consistently. The discrepancy between different pH sensors is less than 0.1 pH units over most of their responsive range. The discrepancy between different dissolved oxygen sensors is less than 10% over the whole range from 0% to 100% dissolved oxygen. The consistency of the sensing system ensures that only an initial one-time calibration is required for the sensing patches. After that, a calibration code is generated and sensing patches of the same composition can be used directly. This greatly reduces the time and cost required for monitored multi-bioreactor operations. We used SP2/0 myeloma/mouse hybridoma cell cultures to demonstrate reactor performance consistency. Transcriptional profiling, HPLC analysis, viable cell count, and viability inspection show that the presence of sensing patches and the use of optical monitoring have no apparent effect on the metabolism of the cells.

Topics: Animals; Bioreactors; Carbocyanines; Cell Culture Techniques; Cell Line, Tumor; Chromatography, Gel; Fluorescent Dyes; Hydrogen-Ion Concentration; Mice; Oligonucleotide Array Sequence Analysis; Optics and Photonics; Oxygen; Reproducibility of Results

Cellular patterning on silicon platforms is the basis for development of integrated cell-based biosensing devices, for which long-term cell selectivity and biostability remain a major challenge. We report the development of a silicon-based platform in a metal-insulator format capable of producing uniform and biostable cell patterns with long-term cell selectivity. Substrates patterned with arrays of gold electrodes were surface-engineered such that the electrodes were activated with fibronectin to mediate cell attachment and the silicon oxide background was passivated with PEG to resist protein adsorption and cell adhesion. Three types of oxide surfaces, i.e., native oxide, dry thermally grown oxide, and wet thermally grown oxide, were produced to illustrate the effect of oxide state of the surface on long-term cell selectivity. Results indicated that the cell selectivity over time differed dramatically among three patterned platforms and the best cell selectivity was found on the dry oxide surface for up to 10 days. Surface analysis results suggested that this enhancement in cell selectivity may be related to the presence of additional, more active oxide states on the dry oxide surface supporting the stability of PEG films and effectively suppressing the cell adhesion. This research offers a new strategy for development of stable and uniform cell-patterned surfaces, which is versatile for immobilization of silane-based chemicals for preparation of biostable interfaces.

Topics: Animals; Biosensing Techniques; Carbocyanines; Cell Line; Cytological Techniques; Fibronectins; Gold; Macrophages; Mice; Microscopy, Fluorescence; Oxidation-Reduction; Polyethylene Glycols; Silicon; Silicon Dioxide; Surface Properties

Inefficient trafficking of recombinant adeno-associated virus type-2 (rAAV2) to the nucleus is a major barrier for transduction. Using imaging and subcellular fractionation techniques, we evaluated the extent of rAAV2 movement through the late (Rab7) and recycling (Rab11) endosomes. Following rAAV2 infection of HeLa cells, immunoisolation of HA-Rab7- or HA-Rab11-tagged endosomes and intracellular colocalization of Cy3-labeled rAAV2 with EGFP-Rab7 or EGFP-Rab11 markers demonstrated dose-dependent trafficking of rAAV2 through the recycling and late endosomal compartments. At low multiplicities of infection (m.o.i. 100 genomes/cell), rAAV2 predominantly trafficked to the Rab7 compartment. In contrast, rAAV2 predominantly trafficked to the recycling endosome at 100-fold higher m.o.i. siRNA studies inhibiting either Rab7 or Rab11 demonstrated that reducing Rab11 protein levels more significantly inhibited rAAV2 transduction on a per genome basis compared to inhibition of Rab7. Dose-response curves, comparing the m.o.i. of AV2Luc infection to relative transduction, also supported the hypothesis that viral movement through the Rab11 compartment at high m.o.i. is more competent for transgene expression ( approximately 100-fold) than virus that moves through the Rab7 compartment at low m.o.i. These findings suggest that strategies to shunt viral movement from the late to the recycling endosome may be effective at increasing viral transduction for gene therapy.

Topics: Biomarkers; Carbocyanines; Cell Nucleus; Dependovirus; DNA, Viral; Dose-Response Relationship, Drug; Endocytosis; Endosomes; Fluorescent Dyes; Genes, Reporter; Genetic Vectors; Genome, Viral; Green Fluorescent Proteins; HeLa Cells; Humans; Luciferases; Microscopy, Fluorescence; Models, Biological; rab GTP-Binding Proteins; RNA, Small Interfering; Subcellular Fractions; Transduction, Genetic; Transgenes

Topics: Awards and Prizes; Binding Sites; Calmodulin; Carbocyanines; Fluorescent Dyes; Humans; Kinesins; Models, Biological; Molecular Motor Proteins; Movement; Myosin Heavy Chains; Myosin Type V; Nanotechnology; Rhodamines

Currently, screening for high-risk human papillomavirus (HPV) infection remains an important health concern throughout the world, because of the close association between certain types of HPV and cervical cancer. In this study, we explore the possibility of using approximately 70mer oligonucleotide microarray for detection and genotyping of HPV. The approximately 70mer type-specific oligonucleotide probes of four different types HPV were designed by using biological software Array designer 2.0, which analyzed the whole genome sequences of HPV and selected optimal probes. These probes were synthesized and printed onto the surface of glass slides in order to prepare a low-density microarray. HPV samples were labeled with fluorescence dyes Cy3 using a method of restriction display polymerase chain reaction (RD-PCR). HPV plasmid DNA was restricted with Sau3A I to produce multiple fragments that were ligated to adaptors subsequently and used as PCR template. PCR labeling was performed with the fluorescently labeled universal primer (Cy3-UP) whose sequence is designed according to the adaptor of the RD-PCR approaches. The labeled samples were hybridized with the oligonucleotide microarray. The scanning results showed that HPV DNA hybridized specifically with multiple spots correspondingly to show positive signals, whereas no signals were detected of all the negative and blank controls. These results demonstrated that approximately 70mer oligonucleotide microarray can be applied to HPV detection and genotyping. The application of RD-PCR in the sample labeling can increase significantly the sensitivity of the assay and will be especially useful for the discriminate diagnosis of multiple pathogens.

Topics: Carbocyanines; Deoxyribonucleases, Type II Site-Specific; DNA, Viral; Genotype; Humans; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes; Papillomaviridae; Plasmids; Polymerase Chain Reaction

Fluorescence resonance energy transfer (FRET) is a powerful technique to reveal interactions between membrane proteins in live cells. Fluorescence labeling for FRET is typically performed by fusion with fluorescent proteins (FP) with the drawbacks of a limited choice of fluorophores, an arduous control of donor-acceptor ratio and high background fluorescence arising from intracellular FPs. Here we show that these shortcomings can be overcome by using the acyl carrier protein labeling technique. FRET revealed interactions between cell-surface neurokinin-1 receptors simultaneously labeled with a controlled ratio of donors and acceptors. Moreover, using FRET the specific binding of fluorescent agonists could be monitored.

Topics: Acyl Carrier Protein; Carbocyanines; Cells, Cultured; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Membrane Proteins; Protein Interaction Mapping; Receptors, G-Protein-Coupled; Receptors, Neurokinin-1; Recombinant Fusion Proteins; Substance P

We have developed cell stimulative system by covalently immobilized signalling molecules on the surface of coverslips on which cells are later cultured. N-(6-maleimidocaproyloxy)sulfo-succinimide (sulfo-EMCS) cross-links the amino-terminal of epidermal growth factors (EGF) with the thiol-modified glass surface without degrading EGF's physiological activity. The glass surface was covered up to about 1.0 EGF moleculesnm(-2) with uniform density. This density can be controlled by changing concentration of the maleimide-modified EGF in the solution reacting with the thiol-modified glass coverslips. When the density of EGF was only slightly lower than that of EGF receptor dimers, cellular response was dramatically decreased. The EGF receptor molecules bound with the immobilized EGF were prevented from lateral diffusion and internalization and kept their initial position. These properties are useful for quantitative, spatial and temporal control of the input signal stimulating cells in cellular signaling system studies. In addition, the immobility of the EGF in this system makes suitable targets for stable single-molecule observation under total internal reflection fluorescence microscopy (TIR-FM) to study EGF signalling mechanism, preventing lateral diffusion and internalization of EGF receptors. Here we show results of single-molecule observations of the association and dissociation between phosphorylated EGF receptors and Cy3-labeled growth factor receptor-binding protein 2 (Grb2) proteins in A431 cells stimulated by the immobilized EGF and discuss the utility of this method for the study of intracellular signal transduction.

Topics: Biological Assay; Carbocyanines; Cell Culture Techniques; Epidermal Growth Factor; Glass; GRB2 Adaptor Protein; Humans; Microscopy, Fluorescence; Phosphorylation; Signal Transduction; Tumor Cells, Cultured

Spotted cDNA microarrays generally employ co-hybridization of fluorescently-labeled RNA targets to produce gene expression ratios for subsequent analysis. Direct comparison of two RNA samples in the same microarray provides the highest level of accuracy; however, due to the number of combinatorial pair-wise comparisons, the direct method is impractical for studies including large number of individual samples (e.g., tumor classification studies). For such studies, indirect comparisons using a common reference standard have been the preferred method. Here we evaluated the precision and accuracy of reconstructed ratios from three indirect methods relative to ratios obtained from direct hybridizations, herein considered as the gold-standard.. We performed hybridizations using a fixed amount of Cy3-labeled reference oligonucleotide (RefOligo) against distinct Cy5-labeled targets from prostate, breast and kidney tumor samples. Reconstructed ratios between all tissue pairs were derived from ratios between each tissue sample and RefOligo. Reconstructed ratios were compared to (i) ratios obtained in parallel from direct pair-wise hybridizations of tissue samples, and to (ii) reconstructed ratios derived from hybridization of each tissue against a reference RNA pool (RefPool). To evaluate the effect of the external references, reconstructed ratios were also calculated directly from intensity values of single-channel (One-Color) measurements derived from tissue sample data collected in the RefOligo experiments. We show that the average coefficient of variation of ratios between intra- and inter-slide replicates derived from RefOligo, RefPool and One-Color were similar and 2 to 4-fold higher than ratios obtained in direct hybridizations. Correlation coefficients calculated for all three tissue comparisons were also similar. In addition, the performance of all indirect methods in terms of their robustness to identify genes deemed as differentially expressed based on direct hybridizations, as well as false-positive and false-negative rates, were found to be comparable.. RefOligo produces ratios as precise and accurate as ratios reconstructed from a RNA pool, thus representing a reliable alternative in reference-based hybridization experiments. In addition, One-Color measurements alone can reconstruct expression ratios without loss in precision or accuracy. We conclude that both methods are adequate options in large-scale projects where the amount of a common reference RNA pool is usually restrictive.

Topics: Adenocarcinoma; Breast Neoplasms; Carbocyanines; Carcinoma, Renal Cell; DNA, Complementary; DNA, Neoplasm; Female; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Male; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Prostatic Neoplasms

Intravitreal administration of specific antisense oligonucleotides (ODNs) effectively downregulates gene expression in the retina but does not modulate it exclusively in retinal ganglion cells (RGCs). Expression of kynurenine aminotransferase II (KAT II) in RGCs has been well described in the literature. We describe a new method for downregulating cellular KAT II expression via transfection of RGC by retrograde transfer of ODN.. Fluorescently labeled, specific ODNs against KAT II were injected into rats either intravitreally or into the superior colliculi. Fluorescence microscopy of retinal flat-mounts and radial sections was used to compare the location, duration, and degree of transfection for both methods of delivery. The effects of both methods on KAT II expression in RGCs were studied immunohistochemically with unlabeled ODN. Retinal kynurenic acid (KYNA) contents were measured using high pressure liquid chromatography (HPLC).. After intravitreal injection, fluorescently labeled ODN reached all retinal layers, whereas injections into the superior colliculus resulted in transfection of the RGC layer alone. Immunohistochemistry showed that both methods of ODN application had a similar effect on downregulation of KAT II expression in RGC. Retinal KYNA content decreased significantly 4 days after both types of ODN administration.. This study demonstrated that retrograde transfer of specific ODN into RGC is feasible and induces downregulation of KAT II cellular expression. This may become a useful tool for modulating gene expression in the retinal ganglion cell layer in vivo without direct transfer of ODN to other retinal cell layers.

Topics: Animals; Biological Transport, Active; Carbocyanines; Chromatography, High Pressure Liquid; Down-Regulation; Fluorescent Dyes; Immunohistochemistry; Injections; Microscopy, Confocal; Microscopy, Fluorescence; Oligonucleotides, Antisense; Rats; Rats, Inbred BN; Retinal Ganglion Cells; Staining and Labeling; Stilbamidines; Superior Colliculi; Time Factors; Transaminases; Transfection; Vitreous Body

The PIN-FORMED (PIN) proteins are plasma-membrane-associated facilitators of auxin transport. They are often targeted to one side of the cell only through subcellular mechanisms that remain largely unknown. Here, we have studied the potential roles of the cytoskeleton and endomembrane system in the localisation of PIN proteins. Immunocytochemistry and image analysis on root cells from Arabidopsis thaliana and maize showed that 10-30% of the intracellular PIN proteins mapped to the Golgi network, but never to prevacuolar compartments. The remaining 70-90% were associated with yet to be identified structures. The maintenance of PIN proteins at the plasma membrane depends on a BFA-sensitive machinery, but not on microtubules and actin filaments. The polar localisation of PIN proteins at the plasmamembrane was not reflected by any asymmetric distribution of cytoplasmic organelles. In addition, PIN proteins were inserted in a symmetrical manner at both sides of the cell plate during cytokinesis. Together, the data indicate that the localisation of PIN proteins is a postmitotic event, which depends on local characteristics of the plasma membrane and its direct environment. In this context, we present evidence that microtubule arrays might define essential positional information for PIN localisation. This information seems to require the presence of an intact cell wall.

Topics: Arabidopsis; Arabidopsis Proteins; Blotting, Western; Brefeldin A; Carbocyanines; Cell Membrane; Cell Polarity; Dose-Response Relationship, Drug; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique; Fluorescent Dyes; Immunohistochemistry; Microscopy, Confocal; Models, Biological; Mutation; Plant Proteins; Plant Roots; Zea mays

We report the replication technology of DNA chip using by sequence specific localization of nucleic acids via hybridization and electric transfer of the nucleic acids onto a new substrate without losing their array information. The denatured DNA fragments are first spotted and UV-cross-linked on a nylon membrane. The membrane is then immersed and hybridized in a DNA mixture solution that contains all complementary sequences of the nucleic acids to be hybridized with the DNA fragments on the membrane. The hybridized DNA fragments are transferred to another membrane at the denatured condition. After separating two membranes, the transferred membrane contains a complementary array of DNA fragments. This method can be used for the replication of the same copy of DNA chip repeatedly and moreover could be applied for a personalized DNA chip fabrication, where specific information of each spot of DNA chip is originated from the genetic information of a personal sample.

Topics: Carbocyanines; DNA Probes; DNA, Viral; Fluorescent Dyes; Hepacivirus; Hepatitis A virus; Hepatitis B virus; Humans; Nucleic Acid Denaturation; Nucleic Acid Hybridization; Nylons; Oligonucleotide Array Sequence Analysis

T cells develop in the thymus in a highly specialized cellular and extracellular microenvironment. The basement membrane molecule, laminin-5 (LN-5), is predominantly found in the medulla of the human thymic lobules. Using high-resolution light microscopy, we show here that LN-5 is localized in a bi-membranous conduit-like structure, together with other typical basement membrane components including collagen type IV, nidogen and perlecan. Other interstitial matrix components, such as fibrillin-1 or -2, tenascin-C or fibrillar collagen types, were also associated with these structures. Three-dimensional (3D) confocal microscopy suggested a tubular structure, whereas immunoelectron and transmission electron microscopy showed that the core of these tubes contained fibrillar collagens enwrapped by the LN-5-containing membrane. These medullary conduits are surrounded by thymic epithelial cells, which in vitro were found to bind LN-5, but also fibrillin and tenascin-C. Dendritic cells were also detected in close vicinity to the conduits. Both of these stromal cell types express major histocompatibility complex (MHC) class II molecules capable of antigen presentation. The conduits are connected to blood vessels but, with an average diameter of 2 mum, they are too small to transport cells. However, evidence is provided that smaller molecules such as a 10 kDa dextran, but not large molecules (>500 kDa), can be transported in the conduits. These results clearly demonstrate that a conduit system, which is also known from secondary lymphatic organs such as lymph nodes and spleen, is present in the medulla of the human thymus, and that it might serve to transport small blood-borne molecules or chemokines to defined locations within the medulla.

Topics: Antibodies, Monoclonal; Antigen-Presenting Cells; Basement Membrane; Biological Transport; Carbocyanines; Cell Adhesion; Cell Culture Techniques; Cells, Cultured; Child; Dextrans; Epithelial Cells; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; Imaging, Three-Dimensional; Immunohistochemistry; Indoles; Laminin; Models, Biological; Ovalbumin; Precipitin Tests; Reverse Transcriptase Polymerase Chain Reaction; Thymus Gland

Maurocalcine (MCa) is a 33-amino acid residue peptide toxin initially isolated from the scorpion Scorpio maurus maurus. Its structural and functional features make it resembling many Cell Penetrating Peptides. In particular, MCa exhibits a characteristic positively charged face that may interact with membrane lipids. External application of MCa is known to produce Ca2+-release from intracellular stores within seconds. MCa binds directly to the skeletal muscle isoform of the ryanodine receptor, an intracellular channel target of the endoplasmic reticulum, and induces long-lasting channel openings in a mode of smaller conductance. The binding sites for MCa have been mapped within the cytoplasmic domain of the ryanodine receptor. In this manuscript, we further investigated how MCa proceeds to cross biological membranes in order to reach its target. A biotinylated derivative of MCa (MCab) was chemically synthesized, coupled to a fluorescent streptavidin indicator (Cy3 or Cy5) and the cell penetration of the entire complex followed by confocal microscopy and FACS analysis. The data provide evidence that MCa allows the penetration of the macro proteic complex and therefore may be used as a vector for the delivery of proteins in the cytoplasm as well as in the nucleus. Using both FACS and confocal analysis, we show that the cell penetration of the fluorescent complex is observed at concentrations as low as 10 nM, is sensitive to membrane potential and is partly inhibited by heparin. We also show that MCa interacts with the disialoganglioside GD3, the most abundant charged lipid in natural membranes. Despite its action on ryanodine receptor, MCa showed no sign of cell toxicity on HEK293 cells suggesting that it may have a wider application range. These data indicate that MCa may cross the plasma membrane directly by cell translocation and has a promising future as a carrier of various drugs and agents of therapeutic, diagnostic and technological value.

Topics: Amino Acid Sequence; Carbocyanines; Cell Membrane; Cell Nucleus; Cells, Cultured; Cytoplasm; Drug Carriers; Endocytosis; Flow Cytometry; Humans; Membrane Lipids; Membrane Potentials; Microscopy, Confocal; Molecular Sequence Data; Oncogene Protein pp60(v-src); Peptide Fragments; Peptides; Protein Conformation; Protein Transport; Ryanodine Receptor Calcium Release Channel; Scorpion Venoms

Diagnosing cancers based on serum profiling is a particularly attractive concept. However, the technical challenges to analysis of the serum proteome arise from the dynamic range of protein amounts. Cancer sera contain antibodies that react with a unique group of autologous cellular antigens, which affords a dramatic amplification of signal in the form of antibodies relative to the amount of the corresponding antigens. The serum autoantibody repertoire from cancer patients might, therefore, be exploited for antigen-antibody profiling. To date, studies of antigen-antibody reactivity using microarrays have relied on recombinant proteins or synthetic peptides as arrayed features. However, recombinant proteins and/or synthetic peptides may fail to accurately detect autoantibody binding due to the lack of proper PTMs. Here we describe the development and use of a "reverse capture" autoantibody microarray. Our "reverse capture" autoantibody microarray is based on the dual-antibody sandwich immunoassay platform of ELISA, which allows the antigens to be immobilized in their native configuration. As "proof-of-principle", we demonstrate its use for antigen-autoantibody profiling with sera from patients with prostate cancer and benign prostate hyperplasia.

Topics: Aged; Antibodies, Monoclonal; Antigens, Neoplasm; Autoantibodies; Biomarkers, Tumor; Blotting, Western; Carbocyanines; Cell Line; Enzyme-Linked Immunosorbent Assay; Fluorescent Dyes; Humans; Immunoprecipitation; Male; Middle Aged; Prostatic Hyperplasia; Prostatic Neoplasms; Protein Array Analysis; Serum

Individual prokaryotic cells from two major anoxic basins, the Cariaco Basin and the Black Sea, were enumerated throughout their water columns using fluorescence in situ hybridization (FISH) with the fluorochrome Cy3 or horseradish peroxidase-modified oligonucleotide probes. For both basins, significant differences in total prokaryotic abundance and phylogenetic composition were observed among oxic, anoxic, and transitional (redoxcline) waters. Epsilon-proteobacteria, Crenarchaeota, and Euryarchaeota were more prevalent in the redoxclines, where previous studies reported high rates of chemoautotrophic production relative to those in waters above and below the redoxclines. Relative abundances of Archaea in both systems varied between 1% and 28% of total prokaryotes, depending on depth. The prokaryotic community composition varied between the two anoxic basins, consistent with distinct geochemical and physical conditions. In the Black Sea, the relative contributions of group I Crenarchaeota (median, 5.5%) to prokaryotic communities were significantly higher (P < 0.001; n = 20) than those of group II Euryarchaeota (median, 2.9%). In contrast, their proportions were nearly equivalent in the Cariaco Basin. Beta-proteobacteria were unexpectedly common throughout the Cariaco Basin's water column, accounting for an average of 47% of 4',6'-diamidino-2-phenylindole (DAPI)-stained cells. This group was below the detection limit (<1%) in the Black Sea samples. Compositional differences between basins may reflect temporal variability in microbial populations and/or systematic differences in environmental conditions and the populations for which they select.

Topics: Anaerobiosis; Archaea; Bacteria; Carbocyanines; Epsilonproteobacteria; Horseradish Peroxidase; In Situ Hybridization, Fluorescence; Oligonucleotide Probes; Russia; Seawater; Venezuela

Microbial communities play important roles in the functioning of coral reef communities. However, extensive autofluorescence of coral tissues and endosymbionts limits the application of standard fluorescence in situ hybridization (FISH) techniques for the identification of the coral-associated bacterial communities. This study overcomes these limitations by combining FISH and spectral imaging.

Topics: Animals; Anthozoa; Bacteria; Carbocyanines; Fluorescence; Fluorescent Dyes; Image Processing, Computer-Assisted; In Situ Hybridization, Fluorescence; Microscopy, Confocal; Snails; Symbiosis

Topics: Animals; Carbocyanines; Cell Line; Dimethylpolysiloxanes; DNA, Complementary; Fluorescent Dyes; Mice; Microfluidics; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis

Combining optical tweezers with single molecule fluorescence offers a powerful technique to study the biophysical properties of single proteins and molecules. However, such integration into a combined, coincident arrangement has been severely limited by the dramatic reduction in fluorescence longevity of common dyes under simultaneous exposure to trapping and fluorescence excitation beams. We present a novel approach to overcome this problem by alternately modulating the optical trap and excitation beams to prevent simultaneous exposure of the fluorescent dye. We demonstrate the dramatic reduction of trap-induced photobleaching effects on the common single molecule fluorescence dye Cy3, which is highly susceptible to this destructive pathway. The extension in characteristic fluorophore longevity, a 20-fold improvement when compared to simultaneous exposure to both beams, prolongs the fluorescence emission to several tens of seconds in a combined, coincident arrangement. Furthermore, we show that this scheme, interlaced optical force-fluorescence, does not compromise the trap stiffness or single molecule fluorescence sensitivity at sufficiently high modulation frequencies. Such improvement permits the simultaneous measurement of the mechanical state of a system with optical tweezers and the localization of molecular changes with single molecule fluorescence, as demonstrated by mechanically unzipping a 15-basepair DNA segment labeled with Cy3.

Topics: Biophysics; Carbocyanines; DNA; Equipment Design; Fluorescent Dyes; Light; Micromanipulation; Proteins; Spectrometry, Fluorescence; Time Factors

Complexes of macromolecules that transiently self-assemble, perform a particular function, and then dissociate are a recurring theme in biology. Such systems often have a large number of possible assembly/disassembly intermediates and complex, highly branched reaction pathways. Measuring the single-step kinetic parameters in these reactions would help to identify the functionally significant pathways. We have therefore constructed a novel single-molecule fluorescence microscope capable of efficiently detecting the colocalization of multiple components in a macromolecular complex when each component is labeled using a different color fluorescent dye. In this through-objective excitation, total internal reflection instrument, the dichroic mirror conventionally used to spectrally segregate the excitation and emission pathways was replaced with small broadband mirrors. This design spatially segregates the excitation and emission pathways and thereby permits efficient collection of the spectral range of emitted fluorescence when three or more dyes are used. In a test experiment with surface-immobilized single-stranded DNA molecules, we directly monitored the time course of a hybridization reaction with three different oligonucleotides, each labeled with a different color dye. The experiment reveals which of the possible reaction intermediates were traversed by each immobilized molecule, measures the hybridization rate constants for each oligonucleotide, and characterizes kinetic interdependences of the reaction steps.

Topics: Calibration; Carbocyanines; DNA; DNA, Single-Stranded; Equipment Design; Fluorescent Dyes; Image Processing, Computer-Assisted; Kinetics; Microscopy, Fluorescence; Nucleic Acid Hybridization; Oligonucleotides; Photons; Time Factors

Human rhinoviruses (HRVs), the main etiologic agents of the common cold, transform into subviral B- or 80S particles (they sediment at 80S upon sucrose density gradient centrifugation) during infection and, in vitro, upon exposure to a temperature between 50 and 56 degrees C. With respect to the native virion they lack the genomic RNA and the viral capsid protein VP4. 80S particles are unstable and easily disintegrate into their components, VP1, VP2, and VP3 in buffers containing SDS. However, this detergent was found to be a necessary constituent of the BGE for the analysis of these viruses and their complexes with receptors and antibodies by CE. We here demonstrate that dodecylpoly(ethyleneglycol ether) (D-PEG) a nonionic detergent, is suitable for analysis of subviral particles as it preserves their integrity, in contrast to SDS. Electrophoresis of the 80S particles in borate buffer (pH 8.3, 100 mM) containing 10 mM D-PEG resulted in a well-defined electrophoretic peak. The identity of the peak was confirmed, among other means, by complexation with mAb 2G2, which recognizes a structural epitope exclusively present on subviral particles but not on native virus. Upon incubation of the 80S particles with mAb 2G2 the peak disappeared, but a new peak, attributed to the antibody complex emerged. The separation system allowed following the time course of the transformation of intact HRV serotype 2 into 80S particles upon incubation at temperatures between 40 and 65 degrees C. We also demonstrate that subviral particles derived from HRV2 labeled with the fluorescence dyes FITC or Cy3.5 were stable in the separation system containing D-PEG. Dye-modified particles were still recognized by mAb 2G2, suggesting that the exposed lysines that are derivatized by the reagent do not form part of the epitope of the antibody.

Topics: Antibodies, Monoclonal; Buffers; Carbocyanines; Cells, Cultured; Detergents; Electrophoresis, Capillary; Fluorescein-5-isothiocyanate; Humans; Hydrogen-Ion Concentration; Polyethylene Glycols; Rhinovirus; Virion

Optical inteference (OI) coated slides with unique optical properties were utilized in microarray analyses, demonstrating their enhanced detection sensitivity over traditional microarray substrates. The OI coating is comprised of a proprietary multilayered, dielectric, thin-film interference coating located beneath the functional coating (aminosilane or epoxysilane). It is designed to enhance the fluorescence in the Cy3 and Cy5 channel by increasing the light absorption of the dyes by about 6-fold and by redirecting emitted fluorescence into the detector during scanning, resulting in a theoretical limit of about 12-fold signal amplification. Two-color DNA microarray experiments conducted on the OI slides showed over 8-fold signal amplification, conservation of gene expression ratios, and increased signal-to-noise ratio when compared to control slides, indicating enhanced detection sensitivity. Protein microarray assays also exhibited over 8-fold signal amplification at three different target concentrations, demonstrating the versatility of the OI slides for different microarray applications. Further, the DNA and protein assays performed on the OI slides exhibited excellent detection sensitivity even at the low target amounts essential for diagnostic applications. The OI slides are compatible with commonly used protocols, printers, scanners and other microarray equipment. Therefore, the OI slides offer an attractive alternative to traditional microarray substrates, where enhanced detection sensitivity is desired.

Topics: Animals; Carbocyanines; DNA; Fluorescent Dyes; Gene Expression Profiling; Glass; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes; Protein Array Analysis; Proteomics; Rats; Sensitivity and Specificity; Software

Magnetic bead-based solid phases are widely used for the separation of nucleic acids from complex mixtures. The challenge to selectively separate specific DNA molecules (via complementary hybridization) in a single step is the selection of a linker between the capture probe and the solid support that can be exposed to high temperatures in the presence of a high salt media. This article presents a general platform for the fabrication of a magnetic bead-based selective solid phase that can be used for subtractive hybridization or sequence capture applications. Phosphorus dendrimers are used for the first time as linkers in a magnetic bead-based selective solid phase for capture of genomic DNA. Aside from providing a high loading capacity, they render a stable bond between the capture probe and the surface under the high temperature and salt conditions required for denaturation and capture to proceed in a single step. The thermal stability of the solid phase under these conditions is first demonstrated by hybridizing a Cy3-labeled target. The selective capture of DNA targets in a single step is then demonstrated by subtractive hybridization of fragmented human genomic DNA. The specificity and selectivity of the solid phase are demonstrated by the recovery of adenovirus serotype 4 DNA spiked into the human DNA target. The effect of steric and electrostatic constraints was also investigated by using dendrimers of different generations that vary in their size and the number of branches. The results demonstrate that this platform can be used for single-step subtractive hybridization applications with better performance over the conventional two-step method using streptavidin-coated magnetic beads.

Topics: Adenoviridae; Carbocyanines; DNA; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Genetic Techniques; Genome; Humans; Magnetics; Nucleic Acid Denaturation; Nucleic Acid Hybridization; Polymerase Chain Reaction; Salts; Sequence Analysis, DNA; Serotyping; Streptavidin; Temperature

We study the coadsorption of mercaptohexanol onto preimmobilized oligonucleotide layers on gold. Monitoring the position of the DNA relative to the surface by optical means directly shows the mercaptohexanol-induced desorption of DNA and the reorientation of surface-tethered strands in situ and in real time. By simultaneously recording the electrochemical electrode potential, we are able to demonstrate that changes in the layer conformation are predominantly of electrostatic origin and can be reversed by applying external bias to the substrate.

Topics: Adsorption; Base Sequence; Carbocyanines; Coated Materials, Biocompatible; DNA; Fluorescent Dyes; Gold; Hexanols; Materials Testing; Static Electricity; Sulfhydryl Compounds; Surface Properties

This report describes the development of a direct and rapid detection method for the pathogenic protozoan, Cryptosporidium parvum, from environmental water samples using fluorescence in situ hybridization (FISH) on a membrane filter. The hydrophilic polytetrafluoroethylene (PTFE) membrane filter with FISH-stained oocysts yielded the highest signal to noise (S/N) ratio of the different membrane filters tested. PTFE membranes retained 98.8+/-0.4% of the concentrated oocysts after washing, simultaneous permeabilization and fixation with a hot ethanol solution, and hybridization with a fluorescently labeled oligonucleotide probe. This procedure eliminates subsequent time-consuming recovery steps that often result in a loss of the actual oocysts in a given environmental water sample. Furthermore, C. parvum was successfully distinguished from Cryptosporidium muris and other species in environmental water samples with the addition of formamide into the hybridization solution. In tap water samples, the S/N ratio was heightened by washing the membrane filter prior to FISH with a 1 M HCl solution in order to reduce the large amounts of impurities and background fluorescence from the non-specific adsorption of the fluorescently labeled oligonucleotide probe.

Topics: Animals; Carbocyanines; Cryptosporidium parvum; DNA Probes; Fluorescent Dyes; Humans; In Situ Hybridization, Fluorescence; Membranes, Artificial; Microscopy, Fluorescence; Microscopy, Interference; Oocysts; Water

The synthesis of a red-shifted cy3B-GM ligand and its evaluation as a fluorescence polarization probe for Hsp90 is presented.

Topics: Benzoquinones; Carbocyanines; Color; Fluorescence Polarization; Fluorescent Dyes; HSP90 Heat-Shock Proteins; Lactams, Macrocyclic; Ligands; Molecular Structure; Rifabutin

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) still remains the most reliable and comprehensive analytical method for the evaluation of protein extracts. However, conventional SDS-PAGE is time-consuming and, thus, unpractical if several tens or hundreds of samples must be examined. We show that SDS-PAGE protein analysis can be automated using slab gel DNA sequencers and compare the instrument's performance with conventional SDS-PAGE in terms of resolution, sensitivity and sample capacity. Labeled protein bands are detected online by laser-induced fluorescence (LIF) and the acquired signals are electronically stored for further processing, avoiding gel staining and scanning. Appropriate software allows immediate display of recorded data and convenient evaluation. The method provides a higher sensitivity and dynamic range than conventional Coomassie-stained gels and the resolution of proteins with different masses is independent of the polyacrylamide concentration. Internal markers can also be used for direct quantification and assignment of the molecular masses. Additionally, we present a novel electrophoresis instrument for the simultaneous separation and online LIF detection of all samples of a microtiterplate in parallel lanes in a 3-D geometry gel cylinder. The specific gel thermostatting concept prevents irregular sample migration (smiling) and improves the reproducibility and comparability of individual separation patterns. In combination with the expected large capacity of 384 or 1,536 samples, this makes the instrument a valuable tool for high-throughput comparative screening applications.

Topics: Automation; Carbocyanines; Electrophoresis, Polyacrylamide Gel; Escherichia coli Proteins; Fluorescent Dyes; Online Systems; Proteins; Sensitivity and Specificity

Persistent cervical high-risk human papillomavirus (HPV) infection is correlated with an increased risk of developing a high-grade cervical intraepithelial lesion. A two-step method was developed for detection and genotyping of high-risk HPV. DNA was firstly amplified by asymmetrical PCR in the presence of Cy3-labelled primers and dUTP. Labelled DNA was then genotyped using DNA microarray hybridization. The current study evaluated the technical efficacy of laboratory-designed HPV DNA microarrays for high-risk HPV genotyping on 57 malignant and non-malignant cervical smears. The approach was evaluated for a broad range of cytological samples: high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL) and atypical squamous cells of high-grade (ASC-H). High-risk HPV was also detected in six atypical squamous cells of undetermined significance (ASC-US) samples; among them only one cervical specimen was found uninfected, associated with no histological lesion. The HPV oligonucleotide DNA microarray genotyping detected 36 infections with a single high-risk HPV type and 5 multiple infections with several high-risk types. Taken together, these results demonstrate the sensitivity and specificity of the HPV DNA microarray approach. This approach could improve clinical management of patients with cervical cytological abnormalities.

Topics: Base Sequence; Carbocyanines; Deoxyuracil Nucleotides; DNA Primers; DNA, Viral; Genotype; HeLa Cells; Humans; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Papillomaviridae; Papillomavirus Infections; Polymerase Chain Reaction; Sensitivity and Specificity; Sequence Alignment; Vaginal Smears

We describe fluorescent-labeled peptide (FLP) studies on living cells. The new technique is nonradioactive and it allows monitoring of the binding and internalization of Vasoactive Intestinal Peptide (VIP) in VIP receptor-expressing cells. The technique is easy to perform and the observed reaction is peptide sequence specific.

Topics: Carbocyanines; Cells, Cultured; Ligands; Microscopy, Fluorescence; Myocytes, Smooth Muscle; Protein Binding; Radioligand Assay; Receptors, Vasoactive Intestinal Peptide; Temperature; Time Factors; Vasoactive Intestinal Peptide

A microarray method was developed for simultaneous detection and identification of six species of Orthopoxvirus (OPV) including Variola, Monkeypox, Cowpox, Camelpox, Vaccinia, and Ectromelia viruses. The method allowed us to discriminate OPV species from varicella-zoster virus (VZV), Herpes Simplex 1 virus (HSV-1), and Herpes Simplex 2 virus (HSV-2) that cause infections with clinical manifestations similar to OPV infections. The nucleotide sequences of the C23L/B29R and the B19R genes identified for 86 and 72 different OPV strains, respectively, were used to design species-specific microarray oligonucleotide probes (oligoprobes). The microarray also contained several oligoprobes selected from the ORF31, US4, and US5 genes of VZV, HSV-1, and HSV-2, respectively. The samples (from HSVs or OPVs) of ssDNAs for analyses were prepared by using asymmetric PCR followed by chemical labeling of ssDNA with Cy3 dye. DNA from 52 samples of various OPV species, two isolates of VZV, two of HSV-1, and three of HSV-2 were tested using the developed microarray assay; all tested viruses were accurately identified. To ensure the robustness of the microarray assay, three additional unrelated variola virus strains with unknown sequences of the C23L/B29R and the B19R genes were tested. In each instance the microarray unambiguously identified them as Variola virus species. The results obtained in this study demonstrated that this new microarray method is a valuable tool for the rapid and accurate detection and differentiation of these important viral pathogens.

Topics: Carbocyanines; DNA, Single-Stranded; DNA, Viral; Genes, Viral; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes; Orthopoxvirus; Poxviridae Infections; Sensitivity and Specificity; Species Specificity; Staining and Labeling

A new fluorescent labeling procedure specific for the strut sequence of myosin subfragment-1's 50kDa cleft was developed using CY3 N-hydroxy succinimidyl ester as a hydrophobic tag and hydrophobic interaction chromatography to purify the major labeled species which retained actin-activated ATPase activity. Stern-Volmer analysis suggests that the CY3 is in close proximity to basic residues, consistent with inspection of the mapped labeling site in the atomic model. Fluorescence polarization indicates that the CY3 becomes more mobile upon actin binding, supporting a location near the actomyosin interface. In contrast, nucleotide binding to myosin had little impact on the CY3. Molecular mechanics and stochastic dynamics simulations suggest that this labeling site is sensitive to forced cleft opening and closure, but the upper 50kDa cleft does not move easily. In addition, there appear to be some long-range effects of forced cleft opening and closing that could impact the lever arm position.

Topics: Carbocyanines; Computer Simulation; Fluorescent Dyes; Models, Chemical; Models, Molecular; Molecular Motor Proteins; Motion; Myosins; Protein Conformation; Spectrometry, Fluorescence

Here, we describe a method for directly transferring very small amounts of reaction products from one surface to another. The approach is illustrated using a T4 DNA polymerase reaction to extend primers hybridized to a surface-confined DNA template. Following the extension reaction, the resulting oligonucleotide is transferred to a product surface. The important results are that (1) the spatial registration of the product is preserved after transfer; (2) the same reactant surface can be used to generate and transfer multiple iterations of products; and (3) the reaction products are biologically active after transfer.

Topics: Biotin; Carbocyanines; Deoxycytosine Nucleotides; Dimethylpolysiloxanes; DNA-Directed DNA Polymerase; DNA, Single-Stranded; Fluorescent Dyes; Microscopy, Fluorescence; Streptavidin; Surface Properties; Viral Proteins

The development of ECL-Plex CyDye-conjugated secondary antibodies allows the advancement of conventional Western blotting, opening up possibilities for highly sensitive and quantitative protein confirmation and identification. We report a novel proteomic method to simultaneously visualise the total protein profile as well as the specific immunodetection of an individual protein species by combining cyanine CyDye pre-labelled proteins and antibody immunoblotting. This technique proposes to revolutionise both 2-D immunoprobing and protein confirmation following MS analysis.

Topics: Amino Acid Sequence; Carbocyanines; Fluorescent Dyes; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; Humans; Immunoblotting; Immunochemistry; Molecular Chaperones; Myocardium; Neoplasm Proteins; Peptide Fragments; Protein Array Analysis; Proteins; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

We present a single particle fluorescence resonance energy transfer (spFRET) study of freely diffusing self-assembled quantum dot (QD) bioconjugate sensors, composed of CdSe-ZnS core-shell QD donors surrounded by dye-labeled protein acceptors. We first show that there is direct correlation between single particle and ensemble FRET measurements in terms of derived FRET efficiencies and donor-acceptor separation distances. We also find that, in addition to increased sensitivity, spFRET provides information about FRET efficiency distributions which can be used to resolve distinct sensor subpopulations. We use this capacity to gain information about the distribution in the valence of self-assembled QD-protein conjugates and show that this distribution follows Poisson statistics. We then apply spFRET to characterize heterogeneity in single sensor interactions with the substrate/target and show that such heterogeneity varies with the target concentration. The binding constant derived from spFRET is consistent with ensemble measurements.

Topics: Cadmium Compounds; Carbocyanines; Carrier Proteins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Maltose-Binding Proteins; Quantum Dots; Rhodamines; Selenium Compounds; Sulfides; Zinc Compounds

Filamentous fungi are a ubiquitous and diverse group of eukaryotic organisms and may contribute, along with bacteria, yeasts, protozoa and viruses, to the formation of biofilms in water distribution systems. However, fungal involvement in biofilms has not been demonstrated unambiguously. Furthermore, these fungi may be responsible for the production of tastes, odours and mycotoxins in drinking water making their early detection important. The detection of fme these problems a combination of two fluorescent techniques for direct detection was tested: (a) Fluorescence In Situ Hybridization (FISH) employing the universal rRNA probe EUK516, labelled with the red Cy3, followed by (b) staining with Calcofluor White MR2 fluorescent dye which stains fungal cell walls blue. Pure cultures of Penicillium brevicompactum were used to establish the methods followed by separate experiments with real water biofilm samples in PVC-C and cast iron coupons. FISH demonstrated eukaryotic microrganisms after approximately 5 h while the calcofluor method revealed chitinous filamentous structures in less than one hour. When the two methods were combined, additional resolution was obtained from the images of filamentous walls (blue) with intact protoplasm (red). In conclusion, FISH and Calcofluor staining provide rapid, direct and unambiguous information on the involvement of ff in biofilms which form in water.

Topics: Benzenesulfonates; Biofilms; Carbocyanines; Cell Wall; Cellulose; Chitin; Coloring Agents; Fluorescent Dyes; In Situ Hybridization, Fluorescence; Iron; Microscopy, Fluorescence; Mycology; Penicillium; Polyvinyl Chloride; RNA, Fungal; Staining and Labeling; Water Microbiology; Water Supply

We demonstrate apertureless near-field microscopy of single molecules at sub-10 nm resolution. With a novel phase filter, near-field images of single organic fluorophores were obtained with approximately sixfold improvement in the signal-to-noise ratio. The improvement allowed pairs of molecules separated by approximately 15 nm to be reliably and repeatedly resolved, thus demonstrating the first true Rayleigh resolution test for near-field images of single molecules. The potential of this technique for biological applications was demonstrated with an experiment that measured the helical rise of A-form DNA.

Topics: Algorithms; Carbocyanines; DNA, A-Form; Fluorescent Dyes; Microscopy, Atomic Force; Microscopy, Confocal; Microscopy, Fluorescence; Nucleic Acid Conformation; Silicon

As the quality of microarrays is critical to successful experiments for data consistency and validity, a reliable and convenient quality control method is needed. We describe a systematic quality control method for large-scale genome oligonucleotide arrays. This method is comprised of three steps to assess the quality of printed arrays. The first step involves assessment of the autofluorescence property of DNA. This step is convenient, quick to perform, and allowed reuse of every array. The second step involves hybridization of arrays with Cy3-labeled 9-mer oligonucleotide target to assess the quality and stability of oligonucleotides. Because this step consumed arrays, one or two arrays from each batch were used to complement the quality control data from autofluorescence. The third step involves hybridization of arrays from every batch with transcripts derived from two cell lines to assess data consistency. These hybridizations were able to distinguish two closely related tissue samples by identifying a cluster of 20 genes that were differently expressed in U87MG and T98G glioblastoma cell lines. In addition, we standardized two parameters that significantly enhanced the quality of arrays. We found that longer pin contact time and crosslinking oligonucleotides at 400 mJ/cm(2) were optimal for the highest hybridization intensity. Taken together, these results indicate that the quality of spotted oligonucleotide arrays should be assessed by at least two methods, autofluorescence and 9-mer hybridization before arrays are used for hybridization experiments.

Topics: Animals; Brain; Brain Chemistry; Carbocyanines; Cell Line, Tumor; DNA, Complementary; DNA, Neoplasm; Encephalomyelitis, Venezuelan Equine; Fluorescence; Fluorescent Dyes; Fluorometry; Gene Expression Profiling; Glioblastoma; Humans; Mice; Neoplasm Proteins; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes; Polylysine; Quality Control; Subtraction Technique; Time Factors; Transcription, Genetic; Ultraviolet Rays

Detection of specific protein-protein interactions has long been restricted to bulk biochemical methods such as immunoprecipitation and immunoblotting. Even more sensitive methods using general immunofluorescence are limited, and it is difficult to infer protein-protein interactions from the results of these tests. Fluorescence Resonance Energy Transfer (FRET) is a photophysical process that can be exploited to obtain highly sensitive information about such interactions. It can sense the presence of acceptor fluorophores in the vicinity of a donor fluorophore within a separation distance that is the size of a single protein molecule. This unit details FRET microscopy based on release of quenched donor fluorescence after acceptor photobleaching, microinjection of reagents into the nucleus or cytosol, and labeling of antibodies for these procedures.

Topics: Animals; Carbocyanines; Cells, Cultured; Fluorescence Resonance Energy Transfer; Fluorescent Antibody Technique; Green Fluorescent Proteins; Humans; Microscopy, Fluorescence; Proteins; Transfection

We assessed the ability of luminescent quantum dots (QDs) to function as energy acceptors in fluorescence resonance energy transfer (FRET) assays, with organic dyes serving as donors. Either AlexaFluor 488 or Cy3 dye was attached to maltose binding protein (MBP) and used with various QD acceptors. Steady-state and time-resolved fluorescence measurements showed no apparent FRET from dye to QD. We attribute these observations to the dominance of a fast radiative decay rate of the donor excitation relative to a slow FRET decay rate. This is due to the long exciton lifetime of the acceptor compared to that of the dye, combined with substantial QD direct excitation.

Topics: Carbocyanines; Carrier Proteins; Fluorescence Resonance Energy Transfer; Hydrazines; Luminescent Measurements; Maltose-Binding Proteins; Quantum Dots

We report the observation of a strong two-photon induced fluorescence emission of Cy5-DNA within the tunable range of a Ti:Sapphire laser. The estimated two-photon cross-section for Cy5-DNA of 400GM is about 3.5-fold higher than it was reported for rhodamine B. The fundamental anisotropies of Cy5-DNA are close to the theoretical limits of 2/5 and 4/7 for one- and two-photon excitation, respectively. We also observed an enhanced two-photon induced fluorescence (TPIF) of Cy5-DNA deposited on silver island films (SIFs). In the presence of SIFs, the TPIF is about 100-fold brighter. The brightness increase of Cy5-DNA TPIF near SIFs is mostly due to enhanced local field.

Topics: Buffers; Carbocyanines; Coated Materials, Biocompatible; DNA; Microscopy, Fluorescence, Multiphoton; Reproducibility of Results; Sensitivity and Specificity; Silver; Staining and Labeling

The sequence-specific recognitions between DNA and proteins are playing important roles in many biological functions. The double-stranded DNA microarrays (dsDNA microarrays) can be used to study the sequence-specific recognitions between DNAs and proteins in highly parallel way. In this paper, two different elongation processes in forming dsDNA from the immobilized oligonucleotides have been compared in order to optimize the fabrication of dsDNA microarrays: (1) elongation from the hairpins formed by the self-hybridized oligonucleatides spotted on a glass; (2) elongation from the complementary primers hybridized on the spotted oligonucleatides. The results suggested that the dsDNA probes density produced by the hybridized-primer extension was about four times lower than those by the self-hybridized hairpins. Meanwhile, in order to reduce the cost of dsDNA microarrays, we have replaced the Klenow DNA polymerase with Taq DNA polymerase, and optimized the reaction conditions of on-chip elongation. Our experiments showed that the elongation temperature of 50 degrees C and the Mg(2+) concentration of 2.5 mM are the optimized conditions in elongation with Taq DNA polymerase. A dsDNA microarray has been successfully constructed with the above method to detect NF-kB protein.

Topics: Carbocyanines; DNA; DNA Polymerase I; Glass; Glutaral; Magnesium Chloride; NF-kappa B; Oligonucleotide Array Sequence Analysis; Taq Polymerase; Temperature

In this paper, a simple, reliable and flexible method, which integrated in situ synthesis with the spotting technique, was reported to fabricate oligonucleotide array. Different oligonucleotide sequences are synthesized on their relative code glass slides through combinational chemistry, thus the slides are broken into smaller pieces, in which the same code pieces have the same probe sequences. An oligonucleotide array is fabricated by arbitrarily assembling these different code pieces onto another solid substrate. In principle experimentation, four different sequences of P16 gene were synthesized and a 5 x 5 array including these four sequences and the control black was fabricated. The analysis results indicated that the hybridization fluorescence intensity of the same sequences locating different sets on the array gave the approximate values, and the fluorescence intensity ratio of matched sequence to one middle location base mismatched, two base mismatched, three middle base mismatched is (1.000+/-0.080):(0.4991+/-0.0671):(0.2360+/-0.0044):(0.0493+/-0.0033). Their relative accuracies were from 6.64 to 10.2%. This result might be used to rapidly screen single-nucleotide polymorphisms (SNPs).

Topics: Carbocyanines; Combinatorial Chemistry Techniques; Molecular Probes; Oligonucleotide Array Sequence Analysis; Oligonucleotides

The behavior of single molecules of neurotrophins on growth cones was observed by the use of the fluorescent conjugate of nerve growth factor (NGF), Cy3-NGF. After the application of 0.4 nm Cy3-NGF, chick dorsal root ganglion growth cones responded within 1 min of adding the stimulus by expanding their lamellipodia. Only 40 molecules of Cy3-NGF, which occupied <5% of the estimated total binding sites on a single growth cone, were required to initiate the motile responses. After binding to the high-affinity receptor, Cy3-NGF displayed lateral diffusion on the membrane of the growth cones with a diffusion constant of 0.3 microm2 s(-1). The behavior of Cy3-NGF was shifted to a one-directional rearward movement toward the central region of the growth cone. The one-directional movement of Cy3-NGF displayed the same rate as the rearward flow of actin, approximately 4 microm/min. This movement could be stopped by the application of the potent inhibitor of actin polymerization, latrunculin B. Molecules of Cy3-NGF were suggested to be internalized in the vicinity of the central region of the growth cone during this rearward trafficking, because Cy3-NGF remained in the growth cone after the growth cones had been exposed to an acidic surrounding medium: acidic medium causes the complete dissociation of Cy3-NGF from the receptors on the surface of growth cones. These results suggested that actin-driven trafficking of the NGF receptor complex is an essential step for the accumulation and endocytosis of NGF at the growth cone and for the retrograde transport of NGF toward the cell body.

Topics: Animals; Axons; Bridged Bicyclo Compounds, Heterocyclic; Carbocyanines; Cells, Cultured; Chick Embryo; Diagnostic Imaging; Dose-Response Relationship, Drug; Drug Interactions; Endocytosis; Ganglia, Spinal; Growth Cones; Models, Biological; Nerve Growth Factor; Neurons; Protein Transport; Pseudopodia; Thiazoles; Thiazolidines; Time Factors

Adrenomedullin (AM) is a regulatory peptide widely expressed, along its receptors, in cells and tissues, of which it controls many basic and specific functions acting in an autocrine-paracrine manner. However, the unequivocal demonstration of the physiological relevance of the regulatory role of AM would require the study of cells where endogenous AM system had been suppressed. Hence, we developed a protocol to silence the human AM gene by transfection with short interfering RNAs (siRNAs). Eight possible AM-siRNA sequences were designed: six siRNAs were synthesized in our laboratory and two were provided by Ambion. As positive control the suppression of the glyceraldehyde-3-phosphate dehydrogenase (GADPH) gene was tested using the Ambion Silencer GADPH siRNA kit. Cultured human embryonal kidney cell line HEK-293 and human umbilical vein endothelial cells (HUVECs) were transfected using either the Qiagen or the Ambion transfection reagent, and transfection visualization, carried out using Cy3-labeled siRNA and examining red fluorescence within the cells, showed that the former reagent was the most efficient. AM-gene silencing was determined in HUVECs by measuring AM mRNA levels in transfected and control cells by real-time polymerase chain reaction. Only Ambion siRNAs were effective, and the best AM-gene silencing (about 80%) was observed 48 or 72 h after transfection with 3 or 6 microg of siRNAs. The conclusion is drawn that siRNA technology can be useful in the investigations on AM functions, but that the complete suppression of the AM-gene transcription is very difficult to obtain.

Topics: Adrenomedullin; Carbocyanines; Gene Silencing; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Microscopy, Fluorescence; Peptides; RNA Interference; RNA, Small Interfering

We demonstrate a novel all-optical switch consisting of two molecules: a primary fluorophore (Cy5) that can be switched between a fluorescent and a dark state by light of different wavelengths, and a secondary chromophore (Cy3) that facilitates switching. The interaction between the two molecules exhibits a distance dependence much steeper than that of conventional Fo rster resonance energy transfer. This enables the switch to act as a ruler with the capability to probe distances difficult to access by other spectroscopic methods, thus presenting a new tool for the study of biomolecules at the single-molecule level.

Topics: Bacterial Proteins; Biotin; Carbocyanines; DNA; DNA Damage; Fluorescence Resonance Energy Transfer; Models, Molecular; Optics and Photonics; RNA, Catalytic; Serum Albumin, Bovine

Expression microarrays have great potential for clinical use but variability of the results represents a challenge for reliable practical application. The amount of fluorescent dye used in microarray experiments is a significant source of variability that has not been systematically studied. Here we demonstrate that the quantity of Cy3 dye affects microarray results performed on tumor specimens. Signal-to-noise ratios and coefficients of variation are significantly improved by increasing Cy3 to 150-180 pmol, but any further increase does not improve the data. In conclusion, optimal amounts of dye reduce variability and improve reliability of expression microarray experiments.

Topics: Carbocyanines; Fluorescent Dyes; Gene Expression Profiling; Humans; Neoplasms; Oligonucleotide Array Sequence Analysis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Topics: Animals; Carbocyanines; Cell Line; Cysteine; Electrophoresis, Gel, Two-Dimensional; Epithelial Cells; Fluorescent Dyes; Gene Expression Regulation; Genes, erbB-2; Image Processing, Computer-Assisted; Lysine; Molecular Chaperones; Oxidation-Reduction; Oxidative Stress; Proteins; Sensitivity and Specificity; Sequence Analysis

Three MutS fusion systems were designed as the mutation recognition and signal elements of DNA chips for detection of DNA mutations. The expression vectors containing the encoding sequences of three recombinant proteins, Trx-His6-GFP-(Ser-Gly)6-MutS (THGLM), Trx-His6-(Ser-Gly)6-Strep tagII-(Ser-Gly)6-MutS (THLSLM) and Trx-His6-(Ser-Gly)6-MutS (THLM), were constructed by gene slicing in vitro. THGLM, THLSLM and THLM were then expressed in Escherichia coli AD494(DE3), respectively. SDS-PAGE analysis revealed that each of the expected proteins was approximately 30% of the total bacterial proteins. The recombinant proteins were purified to the purity over 90% by immobilized metal (Co2+) chelation affinity chromatography. Bioactivity assay indicated that three fusion proteins retained the mismatch-binding activity and the functions of other fusion partners. DNA chips arrayed both mismatched and unpaired DNA oligonucleotides as well as rpoB gene from Mycobacterium tuberculosis were prepared. THGLM, THLSLM and THLM that was labeled with Fluorolinktrade mark Cy3 reactive dye, were then used as both mutation recognition and labeling elements of DNA chips. The resulting DNA chips were used to detect the mismatched and unpaired mutations in the synthesized oligonucleotides and single base mutation in rpoB gene of M. tuberculosis that is resistant to rifamycin.

Topics: Carbocyanines; DNA Mutational Analysis; DNA, Bacterial; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Mutation; MutS DNA Mismatch-Binding Protein; Oligonucleotide Array Sequence Analysis; Recombinant Fusion Proteins

Microarray technology allows the analysis of whole-genome transcription within a single hybridization, and has become a standard research tool. It is extremely important to minimize variation in order to obtain high quality microarray data that can be compared among experiments and laboratories. The majority of facilities implement manual hybridization approaches for microarray studies. We developed an automated method for cDNA microarray hybridization that uses equivalent pre-hybridization, hybridization and washing conditions to the suggested manual protocol. The automated method significantly decreased variability across microarray slides compared to manual hybridization. Although normalized signal intensities for buffer-only spots across the chips were identical, significantly reduced variation and inter-quartile ranges were obtained using the automated workstation. This decreased variation led to improved correlation among technical replicates across slides in both the Cy3 and Cy5 channels.

Topics: Animals; Carbocyanines; Cell Line; Gene Expression Profiling; Mice; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Reproducibility of Results

We constructed a novel database of the proteome of DLD-1 colon cancer cells by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of fluorescence-labeled proteins followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis. The database consists of 258 functionally categorized proteins corresponding to 314 protein spots. The majority of the proteins are oxidoreductases, cytoskeletal proteins and nucleic acid binding proteins. Phosphatase treatment showed that 28% of the protein spots on the gel are phosphorylated, and mass spectrometric analysis identified 21 of them. Proteins of DLD-1 cells and of laser-microdissected colon cancer tissues showed similar distribution on 2D gels, suggesting the utility of our database for clinical proteomics.

Topics: Amino Acid Sequence; Carbocyanines; Cell Line, Tumor; Colonic Neoplasms; Databases, Protein; Electrophoresis, Gel, Two-Dimensional; Humans; Molecular Sequence Data; Phosphoproteins; Phosphoric Monoester Hydrolases; Proteins; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

We introduce the technique of FIONA, fluorescence imaging with one nanometer accuracy. This is a fluorescence technique that is able to localize the position of a single dye within approximately 1 nm in the x-y plane. It is done simply by taking the point spread function of a single fluorophore excited with wide field illumination and locating the center of the fluorescent spot by a two-dimensional Gaussian fit. We motivate the development of FIONA by unraveling the walking mechanism of the molecular motors myosin V, myosin VI, and kinesin. We find that they all walk in a hand-over-hand fashion.

Topics: Biophysical Phenomena; Biophysics; Carbocyanines; Fluorescent Dyes; Green Fluorescent Proteins; Kinesins; Microscopy, Fluorescence; Models, Molecular; Molecular Motor Proteins; Myosin Heavy Chains; Myosin Type V; Nanotechnology; Rhodamines

DIGE is a powerful tool for measuring changes in protein expression between samples. Here we assess the assumptions of normality and heterogeneity of variance that underlie the univariate statistical tests routinely used to detect proteins with expression changes. Furthermore, the technical variance experienced in a multigel experiment is assessed here and found to be reproducible within- and across-sample types. Utilising the technical variance measured, a power study is completed for several "typical" fold changes in expression commonly used as thresholds by researchers. Based on this study using DeCyder, guidance is given on the number of gel replicates that are needed for the experiment to have sufficient sensitivity to detect expression changes. A two-dye system based on utilising just Cy3 and Cy5 was found to be more reproducible than the three-dye system. A power and cost-benefit analysis performed here suggests that the traditional three-dye system would use fewer resources in studies where multiple samples are compared. Technical variance was shown to encompass both experimental and analytical noise and thus is dependent on the analytical software utilised. Data is provided as a resource to the community to assess alternative software and upgrades.

Topics: Animals; Brain; Carbocyanines; Electrophoresis, Gel, Two-Dimensional; Erwinia; Fluorescent Dyes; Gene Expression Profiling; Gene Expression Regulation; Liver; Mice; Myocardium; Proteome; Proteomics; Rats; Rats, Wistar; Reproducibility of Results; Research Design; Sensitivity and Specificity; Software

To microscopically analyze the chemotherapeutic response of tumors using in vivo staining based on an annexinV-Cy5.5 probe and independently asses their apoptotic count using quantitative histological analysis.. Lewis Lung Carcinomas cells, that are sensitive (CS-LLC) and resistant (CR-LLC) to chemotherapy were implanted in nude mice and grown to tumours. Mice were treated with cyclophosphamide and injected with a Cy5.5-annexinV fluorescent probe. In vivo imaging was performed using Fluorescence Molecular Tomography. Subsequently tumours were excised and prepared for histology. The histological tumour sections were stained for apoptosis using a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. A minimum of ten tissue sections were analyzed per tumour for apoptosis quantification by TUNEL staining and corresponding Cy5.5 distribution.. We detected higher levels of apoptosis and corresponding higher levels of Cy5.5 fluorescence in the CS-LLC vs. the CR-LLC tumours. The cell count rate on CS-LLC sections over CR-LLC was found to be approximately 2 :1 where the corresponding area observed on Cy5.5 distribution measurements revealed a approximately 1.7 :1 ratio of CS-LLC over CR-LLC. These observations are consistent with the higher apoptotic index expected from the CS-LLC cell line.. Quantitative analysis of histological slices revealed higher fluorescence and higher apoptotic count in the CS-LLC tumour images compared to the CR-LLC tumour images. These observations demonstrate that the annexinV-Cy5.5 probe sensed the chemotherapeutic effect of cyclophospamide and further confirmed in vivo FMT measurements.

Topics: Animals; Annexin A5; Annexins; Apoptosis; Carbocyanines; Carcinoma, Lewis Lung; Cell Count; Cell Line, Tumor; Cyclophosphamide; Drug Resistance, Neoplasm; Feasibility Studies; Female; Fluorescent Dyes; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Mice, Nude; Neoplasm Transplantation; Tomography, Optical

We have implemented an orthogonal 3-D intact protein analysis system (IPAS) to quantitatively profile protein differences between human serum and plasma. Reference specimens consisting of pooled Caucasian-American serum, citrate-anticoagulated plasma, and EDTA-anticoagulated plasma were each depleted of six highly abundant proteins, concentrated, and labeled with a different Cy dye (Cy5, Cy3, or Cy2). A mixture consisting of each of the labeled samples was subjected to three dimensions of separation based on charge, hydrophobicity, and molecular mass. Differences in the abundance of proteins between each of the three samples were determined. More than 5000 bands were found to have greater than two-fold difference in intensity between any pair of labeled specimens by quantitative imaging. As expected, some of the differences in band intensities between serum and plasma were attributable to proteins related to coagulation. Interestingly, many proteins were identified in multiple fractions, each exhibiting different pI, hydrophobicity, or molecular mass. This is likely reflective of the expression of different protein isoforms or specific protein cleavage products, as illustrated by complement component 3 precursor and clusterin. IPAS provides a high resolution, high sensitivity, and quantitative approach for the analysis of serum and plasma proteins, and allows assessment of PTMs as a potential source of biomarkers.

Topics: Anticoagulants; Biomarkers; Blood Proteins; Carbocyanines; Chromatography; Chromatography, Liquid; Edetic Acid; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Mass Spectrometry; Molecular Weight; Protein Isoforms; Proteome; Proteomics

Denaturing gradient gel electrophoresis (DGGE) is widely used in microbial ecology. We tested the effect of fluorophore-labeled primers on DGGE band migration, sensitivity, and normalization. The fluorophores Cy5 and Cy3 did not visibly alter DGGE fingerprints; however, 6-carboxyfluorescein retarded band migration. Fluorophore modification improved the sensitivity of DGGE fingerprint detection and facilitated normalization of samples from multiple gels by the application of intralane standards.

Topics: Bacteria; Carbocyanines; DNA Fingerprinting; DNA Primers; DNA, Bacterial; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Sensitivity and Specificity; Soil Microbiology

Typical DNA microarrays utilize diffusion of dye-labeled cDNA probes followed by sequence-specific hybridization to immobilized targets. Here we experimentally estimated the distance typical probes travel during static 16-h hybridizations. Probes labeled with Cy3 and Cy5 were individually introduced to opposite sides of a microarray with minimal convective mixing. Oppositely labeled probes diffused across the initial front separating the two solutions, generating a zone with both dyes present. Diffusion-distance estimates for Cy3- and Cy5-labeled cDNAs were 3.8 mm and 2.6 mm, respectively, despite having almost identical molecular masses. In separate 16-h hybridization experiments with oppositely labeled probes premixed, arrays that were continuously mixed had 15-20% higher signal intensities than arrays hybridized statically. However, no change was observed in the Cy3/Cy5 signal intensity ratio between continuously mixed and static hybridizations. This suggests that the observed dye bias in diffusion-distance estimates results from differences in the detection limits of Cy3 and Cy5-labeled cDNA, a potential concern for array data on low-abundance transcripts. Our conservative diffusion-distance estimates indicate that replicate targets >7.6 mm apart will not compete for scarce probes. Also, raising the microarray gap height would delay the onset of diffusion-limited hybridization by increasing the amount of available probe.

Topics: Algorithms; Biophysics; Carbocyanines; Clostridium acetobutylicum; Diffusion; DNA, Complementary; Gene Expression Regulation, Bacterial; Models, Statistical; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes; RNA; Sensitivity and Specificity; Signal Transduction; Time Factors

We examined the equilibrium binding of Escherichia coli RecBC and RecBCD helicases to duplex DNA ends possessing pre-existing single-stranded (ss) DNA ((dT)(n)) tails varying in length (n=0 to 20 nucleotides) in order to determine the contributions of both the 3' and 5' single strands to the energetics of complex formation. Protein binding was monitored by the fluorescence enhancement of a reference DNA labeled at its end with a Cy3 fluorophore. Binding to unlabeled DNA was examined by competition titrations with the Cy3-labeled reference DNA. The affinities of both RecBC and RecBCD increase as the 3'-(dT)(n) tail length increases from zero to six nucleotides, but then decrease dramatically as the 3'-(dT)(n) tail length increases from six to 20 nucleotides. Isothermal titration calorimetry experiments with RecBC show that the binding enthalpy is negative and increases in magnitude with increasing 3'-(dT)(n) tail length up to n=6 nucleotides, but remains constant for n > or =6. Hence, the decrease in binding affinity for 3'-(dT)(n) tail lengths with n > or =6 is due to an unfavorable entropic contribution. RecBC binds optimally to duplex DNA with (dT)6 tails on both the 3' and 5'-ends while RecBCD prefers duplex DNA with 3'-(dT)6 and 5'-(dT)10 tails. These data suggest that both RecBC and RecBCD helicases can destabilize or "melt out" six base-pairs upon binding to a blunt DNA duplex end in the absence of ATP. These results also provide the first evidence that a loop in the 3'-ssDNA tail can form upon binding of RecBC or RecBCD with DNA duplexes containing a pre-formed 3'-ssDNA tail with n > or =6 nucleotides. Such loops may be representative of those hypothesized to form upon interaction of a Chi site contained within the unwound 3' ss-DNA tail with the RecC subunit during DNA unwinding.

Topics: Base Sequence; Carbocyanines; DNA, Single-Stranded; Escherichia coli Proteins; Exodeoxyribonuclease V; Fluorescent Dyes; Macromolecular Substances; Molecular Sequence Data; Molecular Structure; Nucleic Acid Conformation; Protein Binding

Protein microarrays combine aspects of DNA microarrays and ELISA for the parallel interrogation of a biological sample using a multiplex of protein biomarkers. Here we report the development of a protein microarray consisting of a subset of CD antibodies and CRP. Several preparations (culture supernatant, ascites fluid and purified Ig) of each antibody were used in a forward phase protein microarray. Microarrays were fabricated using a non-contact printer delivering 300 pL (+/-30 pL) to specific locations on polyacrylamide gel-based substrates. Following production, microarrays were blocked for non-specific binding and incubated with sera conjugated directly with Cy3. Using CRP as a control biomarker, 12 clinical samples (inflammatory conditions and controls) were interrogated using the protein microarray format and results compared to CRP measured by conventional immunoassay. The data obtained from the microarray correlated with CRP assessed by immunoassay. Subsequently CRP 'positive' samples were interrogated for CD antigen expression; which revealed CD25 and CD45RO expression in all samples. Whilst this study focussed on a subset of CD antibodies, it is anticipated that this array could be expanded to include a larger number of CD antibodies and allow screening of sera from multiple conditions in order to identify disease markers.

Topics: Antibodies; Antigens, CD; C-Reactive Protein; Carbocyanines; Enzyme-Linked Immunosorbent Assay; Humans; Leukocyte Common Antigens; Protein Array Analysis; Receptors, Interleukin-2

Blue-green 488-nm laser sources are widespread in flow cytometry but suffer some drawbacks for cell analysis, including their excitation of endogenous proteins (resulting in high cellular autofluorescence) and their less-than-optimal coincidence with the excitation maxima of commonly used fluorochromes, including the phycoerythrins (PE). Longer wavelength lasers such as green helium-neons and, more recently, diode-pumped solid state (DPSS) 532-nm sources have previously been employed to overcome these difficulties and improve overall sensitivity for PE. In this study, we evaluate an even longer wavelength DPSS 561-nm for its ability to improve PE and DsRed fluorescent protein detection sensitivity.. A DPSS 561-nm laser emitting at 10 mW was mounted onto a BD LSR II. Mouse thymoma cells labeled with cell surface marker antibodies conjugated to the R- and B-forms of PE were analyzed and compared with conventional 488-nm excitation using the same bandpass filters and signal travel distances. A similar analysis was carried out with cell lines expressing the red fluorescent protein DsRed, several green-yellow excited low molecular weight fluorochromes, and a rhodamine-based caspase substrate. Additionally, cells labeled with PE and co-labeled with fluorescein or simultaneously expressing green fluorescent protein (GFP) were analyzed to determine if PE excitation at 561 nm with simultaneous fluorescein/GFP detection was feasible.. The DPSS 561-nm laser gave a several-fold improvement in the fluorochrome to autofluorescence ratios between PE-labeled cells and unlabeled controls. Analysis of cells expressing the fluorescent protein DsRed with the DPSS 561-nm source gave a 6-7-fold improvement in sensitivity over 488-nm excitation, and gave excellent excitation of yellow-green excited fluorochromes and rhodamine-based physiological probes. Yellow-green laser light also caused virtually no impingement on the spatially separated fluorescein/GFP detector, a significant problem with green laser sources, and also allowed simultaneous analysis of GFP and PE with virtually no signal overlap or requirement for color compensation.. DPSS 561-nm laser excitation gave significantly improved sensitivity for both PE-labeled and DsRed expressing cells, with little contamination of a typical fluorescein/GFP detector.

Topics: Animals; Carbocyanines; Carmine; Cell Line, Tumor; Flow Cytometry; Fluorescein; Fluorescent Dyes; Green Fluorescent Proteins; Lasers; Luminescent Proteins; Mice; Microspheres; NIH 3T3 Cells; Phycoerythrin; Rhodamines

Topics: Animals; Brain; Carbocyanines; Fluorescent Dyes; Microscopy, Fluorescence; Microtubules; Tubulin

A theoretical dynamic kinetic model was derived and a series of experiments were carried out using low-density microarrays in various concentrations of spotting probe ([P]) and labeling target ([T]). It has been shown that target and probe determined the signal intensity together. At a certain range of DNA concentration, the signal intensity was in proportion to spotting [P]. At the higher DNA concentrations, there was a decrease in hybridization signal intensity, especially in cDNA microarrays. Since the DNA microarray was constructed on a solid surface, steric hindrance, which is induced by the solid surface and the high [P], decreased the probe immobilization efficiency, leading to a decrease of the immobilized probe density. The decreased hybridization efficiency also caused the compression in signal intensity when the target increased. Nevertheless, the intensity ratio of Cy5 to Cy3 was not compressed within a microarray in the two-color system. The ratio of Cy5/Cy3 is only determined by the ratio of two targets and independent of the density and the types of probe. Therefore, the two-color fluorescent strategy is more reasonable and reliable in detection of differential gene expression. All these results indicate that the DNA microarray can be used to detect differently expressed genes, though it cannot be used to detect the absolute mRNA abundance.

Topics: Base Sequence; Carbocyanines; DNA, Complementary; Fluorescent Dyes; Humans; In Vitro Techniques; Kinetics; Models, Theoretical; Nanotechnology; Oligonucleotide Array Sequence Analysis

As the international efforts to sequence the rice genome are completed, an immediate challenge and opportunity is to comprehensively and accurately define all transcription units in the rice genome. Here we describe a strategy of using high-density oligonucleotide tiling-path microarrays to map transcription of the japonica rice genome. In a pilot experiment to test this approach, one array representing the reverse strand of the last 11.2 Mb sequence of chromosome 10 was analyzed in detail based on a mathematical model developed in this study. Analysis of the array data detected 77% of the reference gene models in a mixture of four RNA populations. Moreover, significant transcriptional activities were found in many of the previously annotated intergenic regions. These preliminary results demonstrate the utility of genome tiling microarrays in evaluating annotated rice gene models and in identifying novel transcription units that will facilitate rice genome annotation.

Topics: Carbocyanines; Chromosomes, Plant; DNA, Complementary; Genome, Plant; Oligonucleotide Array Sequence Analysis; Oryza; Pilot Projects; Reproducibility of Results; RNA, Plant; Sequence Analysis, DNA; Transcription, Genetic

In this report, we present the first case of a vulvar carcinosarcoma with squamous carcinomatous and leiomyosarcomatous differentiation. Comparative genomic hybridization was used to analyze clonality of the two tumor components. A widely identical pattern of genetic imbalances in the comparative genomic hybridization analysis in both the carcinomatous and the sarcomatous tumor component strongly supported the concept of a bidirectionally differentiated neoplasm. In both tumor components and two lymph node metastases, an amplicon was detected on chromosome 11q12-q13, homing the cyclin D1 gene locus. In contrast, exclusively in the sarcomatoid component, a characteristic amplicon on 12q13-q14 was found. The cytogenetic profile of the lymph node metastases revealed an increase in imbalances compared with the primary tumor. In summary, we found strong indications for a clonal origin of the two tumor components in a vulvar carcinosarcoma and a good correlation of the histological morphology with the pattern of genetic imbalances.

Topics: Aged; Biomarkers, Tumor; Carbocyanines; Carcinoma, Squamous Cell; Carcinosarcoma; Cell Differentiation; Chromosome Aberrations; Chromosomes, Human, Pair 12; Clone Cells; DNA, Neoplasm; Eosine Yellowish-(YS); Fatal Outcome; Female; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Hematoxylin; Humans; Immunohistochemistry; In Situ Hybridization; Indoles; Leiomyosarcoma; Lymph Node Excision; Lymphatic Metastasis; Staining and Labeling; Vulvar Neoplasms

We designed a fluorescent peptide-magnetic nanoparticle conjugate that images E-selectin expression in mouse xenograft models of Lewis lung carcinoma (LLC) by fluorescence reflectance imaging. It was synthesized by attaching the E-selectin-binding peptide (ESBP; CDSDSDITWDQLWDLMK) to a CLIO(Cy5.5) nanoparticle to yield ESBP-CLIO(Cy5.5). Internalization by activated human umbilical vein endothelial cells (HUVECs) was rapid and mediated by E-selectin, indicated by the lack of uptake of nanoparticles bearing similar numbers of a scrambled peptide (Scram). To demonstrate the specificity of E-selectin targeting to ESBP-CLIO(Cy5.5) in vivo, we coinjected ESBP-CLIO(Cy5.5) and Scram-CLIO(Cy3.5) and demonstrated a high Cy5.5/Cy3.5 fluorescence ratio using the LLC. Histology showed that ESBP-CLIO was associated with tumor cells as well as endothelial cells, but fluorescence-activated cell sorter analysis showed a far less expression of E-selectin on LLC than on HUVECs. Using immunohistochemistry, we demonstrated E-selectin expression in both endothelial cells and cancer cells in human prostate cancer specimens. We conclude that ESBP-CLIO(Cy5.5) is a useful probe for imaging E-selectin associated with the LLC tumor, and that E-selectin is expressed not only on endothelial cells but also on LLC cells and human prostate cancer specimens.

Topics: Animals; Carbocyanines; Carcinoma, Lewis Lung; Cell Line; Cell Line, Tumor; Cell Nucleus; Cell Separation; E-Selectin; Edetic Acid; Endothelial Cells; Endothelium, Vascular; Flow Cytometry; Humans; Immunohistochemistry; Interleukin-1; Male; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Nanostructures; Nanotechnology; Neoplasm Transplantation; Peptides; Platelet Endothelial Cell Adhesion Molecule-1; Prostatic Neoplasms; Sensitivity and Specificity; Substrate Specificity; Time Factors; Umbilical Veins

Biomolecule patterning by photolithographic methods has considerable advantages because a large number of different biomolecules can be assembled on a spatial area by a combinatorial method and complex biomolecule patterning can be created in situ in closed environments such as microfluidic channels. Here, a photobiotin was used as the photoactivatable reagent to create patterned arrays of biomolecules. The variability of photobiotin deposition on glass substrates modified with a variety of materials having carboxyl, lysine, aldehyde, amine groups, and BSA (bovine serum albumin) was characterized by subsequent derivatization with Cy3-labeled streptavidin. The fluorescence images of the photobiotin patterned glass surfaces showed that the BSA/aldehyde-coated glass could be considered as the most appropriate substrate to immobilize photobiotin, in view of the homogeneous immobilization of biomolecules with high density in defined regions and the reduction of nonspecific binding to the surface. In streptavidin equilibrium adsorption assays, the maximum amount of streptavidin-Cy3 bound to the BSA/aldehyde-coated glass surface continued to rise with increasing streptavidin-Cy3 concentration until 12.0 microg/mL was reached and the surface then became saturated. Also, a line array of biotin-labeled single-strand probe DNAs was created on the BSA/aldehyde-coated glass by photolysis of photobiotin through a slit-type mask and biotin/streptavidin/biotin chemistry, extended to a quantitative measurement of the concentrations of target DNA. The results of target DNA analysis showed linearity over a wide range from 0.5 ng/mL to 5 microg/mL and were reproducible.

Topics: Aldehydes; Azides; Biotin; Carbocyanines; DNA; DNA Probes; Glass; Lysine; Microfluidic Analytical Techniques; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Photolysis; Serum Albumin, Bovine; Streptavidin

Topics: Bacillus Phages; Carbocyanines; DNA Replication; DNA-Directed DNA Polymerase; DNA, Circular; Glass; Nucleotides; Reagent Kits, Diagnostic; Streptavidin

A critical function of trabecular meshwork cells is to degrade cellular debris, including DNA. We hypothesize that low transfection efficiencies of primary human trabecular meshwork (HTM) cell cultures with plasmid DNA are a function of retained capacity to efficiently degrade exogenous DNA in vitro.. To determine mechanisms responsible for low transfection efficiencies of cultured HTM cells, steps of DNA entry into cytoplasm and nucleus were characterized. Following synchronization with sequential serum starvation and serum reintroduction, the HTM cell cycle was characterized using 5-bromo-2-deoxyuridine incorporation into replicating DNA. HTM cells were transfected during S-phase with plasmid DNA encoding green fluorescence protein (GFP) or plasmid DNA conjugated with Cy3. In some experiments, cells were treated with a DNase I inhibitor, 100 nM aurintricarboxylic acid. Uptake of plasmid DNA was measured by intracellular fluorescence of Cy3 and productive transfection efficiency was measured by intracellular fluorescence of GFP.. HTM cells enter S-phase between 18 and 20 h after synchronization. Plasmid DNA reached the cytosolic compartment in 95% of transfected cells, regardless of synchronization. Synchronization dramatically increased productive transfection efficiency in HTM cells, from 3.0 to 9.0%. DNase I inhibition increased productive transfection efficiency of HTM cells two fold.. Cultured HTM cells have a lower transfection efficiency than other primary ocular cell cultures, likely due partially to cytoplasmic digestion of DNA. We suggest that the difficulties in transfecting cultured HTM cells may be related to the filter function of the cells in vivo where the cells must degrade exogenous DNA.

Topics: Adult; Aged; Aurintricarboxylic Acid; Bromodeoxyuridine; Carbocyanines; Cell Cycle; Cell Division; Cells, Cultured; Deoxyribonuclease I; DNA; DNA Replication; Green Fluorescent Proteins; Humans; Infant; Pigment Epithelium of Eye; Plasmids; Trabecular Meshwork; Transfection

Specialized lytic transglycosylases are muramidases capable of locally degrading the peptidoglycan meshwork of Gram-negative bacteria. Specialized lytic transglycosylase genes are present in clusters encoding diverse macromolecular transport systems. This paper reports the analysis of selected members of the specialized lytic transglycosylase family from type III and type IV secretion systems. These proteins were analysed in vivo by assaying their ability to complement the DNA transfer defect of the conjugative F-like plasmid R1-16 lacking a functional P19 protein, the specialized lytic transglycosylase of this type IV secretion system. Heterologous complementation was accomplished using IpgF from the plasmid-encoded type III secretion system of Shigella sonnei and TrbN from the type IV secretion system of the conjugative plasmid RP4. In contrast, neither VirB1 proteins (Agrobacterium tumefaciens, Brucella suis) nor IagB (Salmonella enterica) could functionally replace P19. In vitro, IpgF, IagB, both VirB1 proteins, HP0523 (Helicobacter pylori) and P19 displayed peptidoglycanase activity in zymogram analyses. Using an established test system and a newly developed assay it was shown that IpgF degraded peptidoglycan in solution. IpgF was active only after removal of the chaperonin GroEL, which co-purified with IpgF and inhibited its enzymic activity. A mutant IpgF protein in which the predicted catalytic amino acid, Glu42, was replaced by Gln, was completely inactive. IpgF-catalysed peptidoglycan degradation was optimal at pH 6 and was inhibited by the lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A.

Topics: Bacterial Proteins; Carbocyanines; Conjugation, Genetic; Fluorescent Dyes; Genetic Complementation Test; Glycosyltransferases; Gram-Negative Bacteria; Muramidase; Mutation; Peptidoglycan; Protein Transport

Brain microglia are phagocytic cells that are the major inflammatory response cells of the central nervous system and widely held to play important pathophysiologic roles in Alzheimer's disease (AD) in both potentially neurotoxic responses and potentially beneficial phagocytic responses. In the study, we examined whether ginsonoside Rg3, a by-product of red ginseng, enhances the microglial phagocytosis of Abeta. We found that Rg3 promoted Abeta uptake, internalization, and digestion. Increased maximal Abeta uptake was observed at 4 and 8 h after Rg3 pre-treatment (25 microg/mL), and the internalized Abeta was almost completely digested from cells within 36 h when pretreated with Rg3 comparing with single non-Rg3-treated groups. The expression of MSRA (type A MSR) was also up-regulated by Rg3 treatment in a dose- and time-dependent manner which was coincidently identified in western blots for MSRA proteins in cytosol. These results indicate that microglial phagocytosis of Abeta may be enhanced by Rg3 and the effect of Rg3 on promoting clearance of Abeta may be related to the MSRA-associated action of Rg3. Thus, stimulation of the MSRA might contribute to the therapeutic potentials of Rg3 in microglial phagocytosis and digestion in the treatment of AD.

Topics: Amyloid beta-Peptides; Animals; Animals, Newborn; Blotting, Western; Carbocyanines; Cells, Cultured; Dose-Response Relationship, Drug; Fluorescent Dyes; Gene Expression; Ginsenosides; Lipoproteins, LDL; Mice; Microglia; Microscopy, Fluorescence; Peptide Fragments; Phagocytosis; Rats; Rats, Sprague-Dawley; Receptors, Scavenger; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors

A unique array-based pathogen chip has been developed for the detection of viral RNA or DNA relevant to pathologies of the central nervous system. A total of 715 unique oligonucleotides (60-mer) representing approximately 100 pathogens were designed based on open reading frames (ORFs) from highly conserved and heterogenic regions within viral families. In addition, viral genes reflecting different stages of pathogen infection were also included to potentially define the stage of the viral infection. Viruses (double-stranded DNA, double- or single-stranded RNA, delta, retroid), parasites, and bacteria were included. Test samples labeled with Cy5 were examined by cohybridization with a reference RNA, labeled with Cy3, to the pathogen microarray chip. Good reproducibility of experiments was observed, based on data generated from duplicate hybridizations and duplicate spots on the microarray platform. A viral transcript detection sensitivity of 1 x 10(3) plaque-forming units (pfus) was achieved using selected cell lines and viruses. These findings suggest that the array-based platform described here is capable of detecting a broad spectrum of viruses in a single assay with relatively high sensitivity, specificity, and reproducibility. This method may be used to provide evidence of viral infection in postmortem tissue from psychiatric patients as well as a wide range of other diagnostic categories.

Topics: Autopsy; Brain; Carbocyanines; Cell Line; Cell Line, Tumor; Cluster Analysis; Databases, Genetic; DNA, Viral; Genome; Herpesvirus 1, Human; Humans; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Oligonucleotides; Open Reading Frames; RNA, Antisense; RNA, Messenger; RNA, Viral; Sensitivity and Specificity

To detect the differentially expressed genes in human polycystic kidney by cDNA microarray.. The PCR products of 8398 genes were spotted onto a chip in array. Both mRNAs isolated from polycystic kidney tissue and normal kidney tissue were reversely transcribed to cDNAs with the incorporation of fluorescent dUTP (Cy5-dUTP and Cy3-dUTP) for preparing the hybridization probes. The mixed probes were hybridized to the cDNA microarray. Then the cDNA microarray was scanned for the fluorescent signals and the display of differences between the 2 tissues. IGF1 mRNA, one of the up regulated genes was detected by in situ hybridization technique in the two tissues to validate the result from cDNA microarray.. The result indicated that the expressions of 263 genes were up regulated while the expressions of 94 genes were down regulated in the polycystic kidney tissue among the 8398 target genes. Bioinformatical analysis of those genes had been performed. The up-regulated genes were mainly the ones of oncogene, cellular skeleton and movement, apoptosis related protein, cell signal transduction protein, and cytokine. The down regulated genes were mainly the ones of anti-oncogene, DNA binding and transcription factors, cell signal transduction protein, and metabolism protein. The IGF1 mRNA expression detected by in situ hybridization was consequently consistent with the cDNA microarray.. cDNA microarray is an effective and quick method for studying differential expressed genes. Three hundred and fifty-seven differentially expressed genes with different functions were revealed in the polycystic kidney tissue, which may play some roles in the progression of polycystic kidney.

Topics: Carbocyanines; Computational Biology; DNA, Complementary; Fluorescent Dyes; Gene Expression Profiling; Humans; In Situ Hybridization; Insulin-Like Growth Factor I; Oligonucleotide Array Sequence Analysis; Polycystic Kidney, Autosomal Dominant; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction

The highly processive motor, myosin V, has an extremely long neck containing six calmodulin-binding IQ motifs that allows it to take multiple 36 nm steps corresponding to the pseudo-repeat of actin. To further investigate how myosin V moves processively on actin filaments, we altered the length of the neck by adding or deleting IQ motifs in myosin constructs lacking the globular tail domain. These myosin V IQ mutants were fluorescently labeled by exchange of a single Cy3-labeled calmodulin into the neck region of one head. We measured the step-size of these individual IQ mutants with nanometer precision and subsecond resolution using FIONA. The step-size was proportional to neck length for constructs containing 2, 4, 6, and 8 IQ motifs, providing strong support for the swinging lever-arm model of myosin motility. In addition, the kinetics of stepping provided additional support for the hand-over-hand model whereby the two heads alternately assume the leading position. Interestingly, the 8IQ myosin V mutant gave a broad distribution of step-sizes with multiple peaks, suggesting that this mutant has many choices of binding sites on an actin filament. These data demonstrate that the step-size of myosin V is affected by the length of its neck and is not solely determined by the pseudo-repeat of the actin filament.

Topics: Actin Cytoskeleton; Amino Acid Sequence; Animals; Calmodulin; Carbocyanines; Fluorescent Dyes; Models, Molecular; Muscle, Skeletal; Mutation; Myosin Type V; Protein Structure, Tertiary; Rabbits

Human microarrays are readily available, and it would be advantageous if they could be used to study gene expression in other species, such as pigs. The objectives of this research were to validate the use of human microarrays in the analysis of porcine gene expression, to assess the variability of the data generated, and to compare gene expression in boars with different levels of steroidogenesis. Cytochrome b5 (CYB5) expression was used to assess array detection sensitivity. Samples having high or low CYB5 RNA levels were hybridized to microarrays to determine if the known expression difference could be detected. Six hybridizations were conducted using human microarrays containing 3840 total spots representing 1718 characterized human ESTs. To analyze gene expression in boars with different levels of steroidogenesis, testis RNA from four boars with high levels of plasma estrone sulphate was hybridized to testis RNA from four boars with lower levels. Eight microarray hybridizations were conducted including fluor-flips. Self-self hybridizations were also conducted to assess the variability of array experiments. The Cy5 and Cy3 intensity values for each array were normalized using a locally weighted linear regression (LOESS). Statistical significance was assessed using a Student's t-test followed by the Benjamini and Hochberg multiple testing correction procedure. Quantitative real-time PCR (Q-RT-PCR) was used to verify select gene expression differences. The results show that CYB5 was significantly overexpressed in the high CYB5 sample by 1.8 fold (P < 0.05), verifying the known expression difference. The average log2 ratio of the majority of genes (1643) falls within one standard deviation of the mean, indicating the data were reproducible. In the high versus low steroidogenesis experiment, seven genes were significantly overexpressed in the high group (P < 0.05). Quantitative real-time PCR was used to validate five genes with the highest fold change, and the results corroborated those found by the microarray experiments. The results of the self-self hybridizations showed that no genes were significantly differentially expressed following the application of the Benjamini and Hochberg multiple testing correction procedure. The results presented in this report show that human arrays can be used for gene expression analysis in pigs.

Topics: Animals; Carbocyanines; Cytochromes b5; Estrone; Fluorescent Dyes; Gene Expression Regulation, Enzymologic; Humans; Male; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA; Swine; Testis

To elucidate the significance of beta-1,4-galactosyltransferase IV (beta-1,4-GT-IV) in the clinical presentation and prognostication of colorectal cancer.. Tissue lysates from paired tumor and nontumor tissues of a colon cancer patient were labeled separately with fluorescent dyes Cy5 and Cy3 for two-dimensional difference in-gel electrophoresis. Subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and immunoblot analyses identified a down-regulated level of beta-1,4-GT-IV in the tumor tissue. In the follow-up study, paired tissue lysates were obtained from 100 colorectal cancer patients with immunoblot analyses done to compare the levels of beta-1,4-GT-IV expression in these patients.. Of 100 colorectal patients studied, 48% had down-regulated expression of beta-1,4-GT-IV in the tumor tissue but 28% of patients exhibited elevated beta-1,4-GT-IV levels. Increased beta-1,4-GT-IV in the tumor tissue was significantly coexistent with raised serum level of CA-199 and the presence of tumor metastasis (P=0.006 and P<0.001, respectively) but was independent of age and gender of patient, tumor site, tumor size, serum level of carcinoembryonic antigen, grade of tumor cell differentiation, and depth of tumor invasion. The results of logistic regression analyses suggested that tumor beta-1,4-GT-IV overexpression and tumor invasion, but not other patient variables such as tumor size and serum levels of carcinoembryonic antigen and CA19-9, were significantly correlated with the occurrence of metastases (P<0.05). In a multivariate regression analysis, the patient group with tumor beta-1,4-GT-IV overexpression strongly predicted for tumor metastasis (odds ratio, 10.009; 95% confidence interval, 2.992-33.484; P<0.001). Likewise, tumor beta-1,4-GT-IV overexpression was significantly associated with poor overall survival (P<0.01). By Cox regression analysis, this association remained significant even after adjustment for tumor metastasis (P=0.048).. Increased beta-1,4-GT-IV expression in tumor tissue was strongly associated with tumor metastases and poor prognosis in colorectal cancer.

Topics: Aged; Carbocyanines; Colorectal Neoplasms; Electrophoresis, Gel, Two-Dimensional; Female; Galactosyltransferases; Humans; Male; Middle Aged; Neoplasm Metastasis; Prognosis; Risk Factors; Up-Regulation

RecG is a DNA helicase involved in the repair of damage at a replication fork and catalyzes the reversal of the fork to a point beyond the damage in the template strand. It unwinds duplex DNA in reactions that are coupled to ATP hydrolysis. The kinetic mechanism of duplex DNA unwinding by RecG was analyzed using a quantitative fluorescence assay based on the process of contact quenching between Cy3 and Dabcyl groups attached to synthetic three-way DNA junctions. The data show that the protein moves at a rate of 26 bp s(-1) along the duplex DNA during the unwinding process. RecG ATPase activity during translocation indicates a constant rate of 7.6 s(-1), measured using a fluorescent phosphate sensor, MDCC-PBP. These two rates imply a movement of approximately 3 bp per ATP hydrolyzed. We demonstrate in several trapping experiments that RecG remains attached to DNA after translocation to the end of the arm of the synthetic DNA junction. ATPase activity continues after translocation is complete. Dissociation of RecG from the product DNA occurs only very slowly, suggesting strong interactions between them. The data support the idea that interactions of the duplex template arm with the protein are the major sites of binding and production of translocation.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Base Sequence; Carbocyanines; Coumarins; DNA; DNA Helicases; Kinetics; Molecular Sequence Data; Nucleic Acid Conformation; Oligonucleotides; p-Dimethylaminoazobenzene; Phosphate-Binding Proteins; Phosphates; Protein Binding; Spectrometry, Fluorescence; Thermotoga maritima

To determine the site of insulin exocytosis in the pancreatic beta cell plasma membrane, we analyzed the interaction between the docking/fusion of green fluorescent protein-tagged insulin granules and syntaxin 1 labeled by TAT-conjugated Cy3-labeled antibody (Ab) using total internal reflection fluorescence microscopy (TIRFM). Monoclonal Ab against syntaxin 1 was labeled with Cy3 then conjugated with the protein transduction domain of HIV-1 TAT. TAT-conjugated Cy3-labeled anti-syntaxin 1 Ab was transduced rapidly into the subplasmalemmal region in live MIN6 beta cells, which enabled us to observe the spatial organization and distribution of endogenous syntaxin 1. TIRFM imaging revealed that syntaxin 1 is distributed in numerous separate clusters in the intact plasma membrane, where insulin secretory granules were docked preferentially to the sites of syntaxin 1 clusters, colocalizing with synaptosomal-associated protein of 25 kDa (SNAP-25) clusters. TIRFM imaging analysis of the motion of single insulin granules demonstrated that the fusion of insulin secretory granules stimulated by 50 mm KCl occurred exclusively at the sites of the syntaxin 1 clusters. Cholesterol depletion by methyl-beta-cyclodextrin treatment, in which the syntaxin 1 clusters were disintegrated, decreased the number of docked insulin granules, and, eventually the number of fusion events was significantly reduced. Our results indicate that 1) insulin exocytosis occurs at the site of syntaxin 1 clusters; 2) syntaxin 1 clusters are essential for the docking and fusion of insulin granules in MIN6 beta cells; and 3) the sites of syntaxin 1 clusters are distinct from flotillin-1 lipid rafts.

Topics: Animals; Antibodies, Monoclonal; Antigens, Surface; Carbocyanines; Cell Line; Cell Membrane; Exocytosis; Fluorescein; Fluorescent Dyes; Gene Products, tat; Green Fluorescent Proteins; HIV-1; Insulin; Insulin Secretion; Intracellular Membranes; Islets of Langerhans; Luminescent Proteins; Membrane Fusion; Membrane Proteins; Mice; Microscopy, Fluorescence; Nerve Tissue Proteins; Recombinant Fusion Proteins; Secretory Vesicles; Synaptosomal-Associated Protein 25; Syntaxin 1; tat Gene Products, Human Immunodeficiency Virus; Transfection

Graphical representation of fluorescence lifetime imaging microscopy data demonstrates that a mixture of two components with single exponential decays can be resolved by single frequency measurements. We derive a method based on linear fitting that allows the calculation of the fluorescence lifetimes of the two components. We show that introduction of proper error-weighting results in a non-linear method that is mathematically identical to a global analysis algorithm that was recently derived. The graphical approach was applied to cellular data obtained from a lifetime-based phosphorylation assay for the epidermal growth factor receptor and yielded results similar to those obtained by a global analysis algorithm.

Topics: Carbocyanines; Cell Line; ErbB Receptors; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Green Fluorescent Proteins; Humans; Image Processing, Computer-Assisted; Luminescent Proteins; Microscopy, Fluorescence; Phosphorylation; Phosphotyrosine; Recombinant Fusion Proteins; Tumor Cells, Cultured

Kinesin is a processive motor that takes 8.3-nm center-of-mass steps along microtubules for each adenosine triphosphate hydrolyzed. Whether kinesin moves by a "hand-over-hand" or an "inchworm" model has been controversial. We have labeled a single head of the kinesin dimer with a Cy3 fluorophore and localized the position of the dye to within 2 nm before and after a step. We observed that single kinesin heads take steps of 17.3 +/- 3.3 nm. A kinetic analysis of the dwell times between steps shows that the 17-nm steps alternate with 0-nm steps. These results strongly support a hand-over-hand mechanism, and not an inchworm mechanism. In addition, our results suggest that kinesin is bound by both heads to the microtubule while it waits for adenosine triphosphate in between steps.

Topics: Adenosine Triphosphate; Carbocyanines; Dimerization; Fluorescence; Fluorescent Dyes; Humans; Kinesins; Kinetics; Microtubules; Models, Biological; Models, Molecular; Molecular Motor Proteins; Mutation; Protein Conformation

Neuroblastomas show remarkable biological heterogeneity, resulting in favorable or unfavorable outcomes. To survey the differences in gene expression profiles between favorable and unfavorable neuroblastomas, we analyzed ten favorable neuroblastoma samples from patients whose tumors consequently regressed or matured and ten unfavorable tumor samples from patients who consequently died of disease using the microarray technique. In each sample, total RNA was labeled with Cy3 or Cy5 in reverse-trancriptase reaction and hybridized with our original microarray prepared with a cDNA library of human fetal brain. Microarray analysis revealed that 43 genes, including MYCN, hTERT, NME1 and cell cycle regulatory protein-coding genes, were highly expressed in unfavorable neuroblastomas, while another 80 genes were detected as highly expressed in favorable tumors, including neuronal differentiating genes and apoptotic inducing genes. Among favorable neuroblastoma samples, highly expressing genes in regressing tumors were different from those in maturing tumors. Expression profiling data revealed the existence of up-regulated and down-regulated gene clusters in favorable and unfavorable tumors. This cluster analysis is a powerful procedure to distinguish unfavorable tumors from favorable tumors as well as regressing tumors from maturing tumors among favorable tumors. The information obtained from expression profiling would clarify the key genes for cell growth, regression or maturation of neuroblastoma cells, and these genes will become diagnostic and therapeutic targets in human neuroblastoma in the future.

Topics: Apoptosis; Apoptosis Inducing Factor; Carbocyanines; Cause of Death; Cell Cycle Proteins; Down-Regulation; Flavoproteins; Fluorescent Dyes; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histocompatibility Antigens Class I; Humans; Infant; Membrane Proteins; N-Myc Proto-Oncogene Protein; Neoplasm Regression, Spontaneous; Nerve Tissue Proteins; Neuroblastoma; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Oncogene Proteins; Up-Regulation

Established procedures use different and seemingly incompatible experimental protocols for fluorescent in situ hybridization (FISH) with Gram-negative and Gram-positive bacteria. The aim of this study was to develop a procedure, based on FISH and confocal laser scanning microscopy (CLSM), for the analysis of the spatial organization of in vitro biofilms containing both Gram-negative and Gram-positive oral bacteria. Biofilms composed of the six oral species Actinomyces naeslundii, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus sobrinus, and Veillonella dispar were grown anaerobically for 64.5 h at 37 degrees C on hydroxyapatite disks preconditioned with saliva. Conditions for the simultaneous in situ hybridization of both Gram-negative and Gram-positive bacteria were sought by systematic variation of fixation and exposure to lysozyme. After fixation and permeabilization biofilms were labeled by FISH with 16S rRNA-targeted oligonucleotide probes ANA103 (for the detection of A. naeslundii), EUK116 (C. albicans), FUS664 (F. nucleatum), MIT447 and MIT588 (S. oralis), SOB174 (S. sobrinus), and VEI217 (V. dispar). Probes were used as 6-FAM, Cy3 or Cy5 conjugates, resulting in green, orange-red or deep-red fluorescence of target cells, respectively. Thus, with two independent triple-hybridizations with three probes carrying different fluorescence-tags, all six species could be visualized. Results show that the simultaneous investigation by FISH of complex biofilms composed of multiple bacterial species with differential Gram-staining properties is possible. In combination with the optical sectioning properties of CLSM the technique holds great promise for the analysis of spatial alterations in biofilm composition in response to environmental challenges.

Topics: Biofilms; Carbocyanines; Dental Plaque; DNA Probes; DNA, Bacterial; Fluoresceins; Fluorescent Dyes; Gram-Negative Bacteria; Gram-Positive Bacteria; In Situ Hybridization, Fluorescence; Microscopy, Confocal; Specimen Handling

Investigation of a terminally modified oligodeoxynucleotide (ODN) directed against p53 mRNA (p53-3' polyethylene glycol-5' tocopherol ODN as a novel drug for pancreatic ductal carcinoma therapy in vitro and in vivo.. The impact of lipophilic modifications at the 5' end of p53-directed ODNs on cellular uptake was analyzed in vitro using proliferation assays, fluorescence-activated cell sorting analysis, and confocal laser scanning microscopy. The in vivo effects of p53-PT-ODN on the growth of orthotopically xenografted human pancreatic ductal carcinoma cells (PancTuI) were studied in SCID beige mice. Distribution was examined in vitro and in vivo using Cy3-labeled ODNs.. Terminally modified p53-PT-ODN showed excellent cellular uptake without using transfection reagents. Microscopically detectable levels of p53-PT-ODN were reached in vivo within 3 hours after intraperitoneal injection, even in extraperitoneal organs. At this time, Cy3-labeled p53-PT-ODN was found in solid tumor formations. We observed a significant inhibition of tumor growth (50%) in vivo at low doses of p53-PT-ODN, whereas at high doses, 2 of 9 animals had no detectable tumors at necropsy. When p53-PT-ODN was injected on the day of tumor cell inoculation, the growth inhibition of solid tumors was significantly stronger compared with that with delayed treatment.. p53-Directed modified ODNs might be of therapeutic value in pancreatic ductal carcinoma, particularly as adjuvant therapy after pancreatic tumor resection.

Topics: Animals; Carbocyanines; Carcinoma, Pancreatic Ductal; Cell Division; Cell Line, Tumor; Female; Humans; Mice; Mice, SCID; Neoplasm Transplantation; Oligonucleotides, Antisense; Pancreatic Neoplasms; Tissue Distribution; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

We used luminescent CdSe-ZnS core-shell quantum dots (QDs) as energy donors in fluorescent resonance energy transfer (FRET) assays. Engineered maltose binding protein (MBP) appended with an oligohistidine tail and labeled with an acceptor dye (Cy3) was immobilized on the nanocrystals via a noncovalent self-assembly scheme. This configuration allowed accurate control of the donor-acceptor separation distance to a range smaller than 100 A and provided a good model system to explore FRET phenomena in QD-protein-dye conjugates. This QD-MBP conjugate presents two advantages: (1) it permits one to tune the degree of spectral overlap between donor and acceptor and (2) provides a unique configuration where a single donor can interact with several acceptors simultaneously. The FRET signal was measured for these complexes as a function of both degree of spectral overlap and fraction of dye-labeled proteins in the QD conjugate. Data showed that substantial acceptor signals were measured upon conjugate formation, indicating efficient nonradiative exciton transfer between QD donors and dye-labeled protein acceptors. FRET efficiency can be controlled either by tuning the QD photoemission or by adjusting the number of dye-labeled proteins immobilized on the QD center. Results showed a clear dependence of the efficiency on the spectral overlap between the QD donor and dye acceptor. Apparent donor-acceptor distances were determined from efficiency measurements and corresponding Förster distances, and these results agreed with QD bioconjugate dimensions extracted from structural data and core size variations among QD populations.

Topics: Cadmium Compounds; Carbocyanines; Carrier Proteins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Histidine; Luminescent Measurements; Maltose-Binding Proteins; Quantum Dots; Selenium Compounds; Spectrometry, Fluorescence; Sulfides; Titrimetry; Zinc Compounds

Cystic fibrosis (CF) is a common indication for preimplantation genetic diagnosis (PGD). A 3-bp deletion (DeltaF508) in the cftr gene, which accounts for approximately 80% of all CF mutations in the Caucasian population, is normally diagnosed in IVF embryos using fluorescent PCR (FL-PCR) and allelic sizing. In PGD, the possibility of using microarrays for genetic diagnosis is largely unexplored. Therefore, the aim of this study was to prove the diagnostic capability of microarrays for PGD, using DeltaF508 as a model mutation. To this end, oligonucleotide probes representing both the normal and DeltaF508 disease alleles were used to construct a single microarray platform. Target DNA, which was generated by PCR and labelled with the fluorescent dye Cy3, was hybridized to the array and the DeltaF508 genotypes assigned from the fluorescence bound to each allelic probe. The performance of the array was evaluated by its ability to detect DeltaF508 mutations in target DNA. Strong binding of the target to the probes was observed, allowing the expected DeltaF508 genotypes to be assigned. The reliability and accuracy of the microarray diagnosis for DeltaF508 was blindly assessed on 10 samples with either a homozygous normal, homozygous affected or heterozygous genotype. All samples were correctly genotyped. In addition, PCR products from a previous PGD case involving DeltaF508 were re-evaluated on the array, with results in complete concordance with allelic sizing methods used to make the original diagnosis. Together, these findings prove the concept that the DeltaF508 mutation of CF can be reliably and accurately diagnosed at the single cell level using microarray analysis. The availability of more cost-effective array platforms comprising mutation probes for common single-gene disorders and a reliable method of whole genome amplification (WGA) would allow PGD to be offered to the majority of PGD patients with minimal or no change to methodology.

Topics: Adenine; Alleles; Carbocyanines; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytosine; Female; Fluorescent Dyes; Gene Deletion; Guanine; Heterozygote; Homozygote; Humans; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Point Mutation; Polymerase Chain Reaction; Pregnancy; Preimplantation Diagnosis

We have designed a reversible fluorescent DNA probe that can be used to determine the concentration of single-stranded DNA in solution by a ratiometric fluorescence measurement. The probe consists of a single-stranded dual fluorescently labeled DNA molecule that adopts a stem-loop conformation in its nonhybridized state. The stem length and the length of the loop region complementary to the target were chosen to allow for reversible binding. The excitation and emission wavelengths of the two labels Cy3 and Cy5 allow for fluorescence resonance energy transfer in the closed state. Upon hybridization, the probe opens up resulting in a fluorescence intensity increase of the donor and a fluorescence intensity decrease of the acceptor. The ratio of the acceptor-to-donor fluorescence intensities is independent of the amount of probe and provides a quantitative measure of the free target concentration.

Topics: Carbocyanines; DNA; DNA Probes; Fluorescence Resonance Energy Transfer; Nucleic Acid Conformation; Nucleic Acid Hybridization

In two-colour microarrays, the ratio of signal intensities of two co-hybridized samples is used as a relative measure of gene expression. Ratio-based analysis becomes complicated and inefficient in multi-class comparisons. We therefore investigated the validity of an intensity-based analysis procedure. To this end, two different cRNA targets were hybridized together, separately, with a common reference and in a self-self fashion on spotted 65mer oligonucleotide microarrays. We found that the signal intensity of the cRNA targets was not influenced by the presence of a target labelled in the opposite colour. This indicates that targets do not compete for binding sites on the array, which is essential for intensity-based analysis. It is demonstrated that, for good-quality arrays, the correlation of signal intensity measurements between the different hybridization designs is high (R > 0.9). Furthermore, ratio calculations from ratio- and intensity-based analyses correlated well (R > 0.8). Based on these results, we advocate the use of separate intensities rather than ratios in the analysis of two-colour long-oligonucleotide microarrays. Intensity-based analysis makes microarray experiments more efficient and more flexible: It allows for direct comparisons between all hybridized samples, while circumventing the need for a reference sample that occupies half of the hybridization capacity.

Topics: Animals; Carbocyanines; Color; Mice; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; Sensitivity and Specificity; Templates, Genetic

RNA interference is rapidly becoming a powerful tool for gene silencing in mammalian cells. Introduction of siRNA into primary cells, however, remains one of the major difficulties of this novel technique. Using cationic lipid-based transfection reagents satisfactory transfection results are observed in cell lines, but low transfection efficiency and cytotoxicity limit applications in primary cells, especially primary neurons. The application of "naked" siRNA has been previously used successfully in nematodes and mammals in vivo. We therefore evaluated the effects of non-cationic-lipid-based siRNA application to primary hippocampal neuron cultures. "Naked" siRNA was bound to the cell surface and was taken up into endosomes. No significant silencing effect of endogenous or reporter genes was observed, rather application of "naked" siRNA was accompanied by a moderate downregulation of metabolic activity in culture. We postulate that endosomal degradation of "naked" siRNA in neurons prevents the induction of significant RNAi-mediated mRNA-downregulation and is accompanied by a global impairment of the cell metabolism. Transfection methods circumventing the endosomal pathway therefore might prove useful for siRNA transduction of primary neurons.

Topics: Animals; Carbocyanines; Cells, Cultured; Endosomes; Female; Genes, Reporter; Green Fluorescent Proteins; Hippocampus; Indicators and Reagents; Liposomes; Luminescent Proteins; Lysosomes; Microscopy, Fluorescence; Microtubule-Associated Proteins; Neurons; Pregnancy; Rats; Rats, Wistar; RNA, Small Interfering; Transduction, Genetic; Transfection

To evaluate the role of fluorescent antibodies as a means of enhancing the detection of retinal breaks during vitrectomy for rhegmatogenous retinal detachment.. In ex vivo studies, unfixed, porcine retinal flatmounts were incubated with Cy3 anti-GFAP. Experiments were repeated in the presence of excess soluble GFAP and after surface excimer laser ablation through the internal limiting membrane, into the Müller cell foot processes. Tissue was also incubated with trypan blue, and cross-species immunoreactivity was determined in bovine, rabbit, and human retina. In vivo studies were conducted in a porcine model of rhegmatogenous retinal detachment. Cy3 anti-GFAP was injected into the vitreous cavity of eyes with retinal breaks and then rinsed from the eye. Barrier filters were fitted to the operating microscope to allow intraoperative visualization of tissue stained with Cy3. Excitation endoillumination was provided by a 532-nm diode-pumped laser.. In ex vivo studies, retinal flatmounts exposed to Cy3 anti-GFAP showed minimal surface fluorescence, but exposed glial elements at the cut edge of the flatmount stained brightly, as did those exposed by excimer ablation of the Müller cell membrane. Blocking studies confirmed that binding was antigen specific. Trypan blue colocalized to the cut edge of retinal flatmounts. All species showed high levels of immunoreactivity except rabbit. In vivo studies demonstrated selective intraoperative staining of retinal breaks with a high level of specificity.. Intraoperative vital staining of retinal breaks is possible in an animal model of retinal detachment. Ex vivo studies indicate that this occurs because the Cy3 anti-GFAP selectively binds the intermediate filaments of glial cells with damaged or destroyed cell membranes.

Topics: Aged; Aged, 80 and over; Animals; Antibodies, Monoclonal; Carbocyanines; Cattle; Fluorescent Dyes; Glial Fibrillary Acidic Protein; Humans; Microscopy, Fluorescence; Monitoring, Intraoperative; Neuroglia; Rabbits; Retinal Detachment; Retinal Perforations; Species Specificity; Staining and Labeling; Swine; Vitrectomy

Expression microarrays have evolved into a powerful tool with great potential for clinical application and therefore reliability of data is essential. RNA amplification is used when the amount of starting material is scarce, as is frequently the case with clinical samples. Purification steps are critical in RNA amplification and labelling protocols, and there is a lack of sufficient data to validate and optimise the process.. Here the purification steps involved in the protocol for indirect labelling of amplified RNA are evaluated and the experimentally determined best method for each step with respect to yield, purity, size distribution of the transcripts, and dye coupling is used to generate targets tested in replicate hybridisations. DNase treatment of diluted total RNA samples followed by phenol extraction is the optimal way to remove genomic DNA contamination. Purification of double-stranded cDNA is best achieved by phenol extraction followed by isopropanol precipitation at room temperature. Extraction with guanidinium-phenol and Lithium Chloride precipitation are the optimal methods for purification of amplified RNA and labelled aRNA respectively.. This protocol provides targets that generate highly reproducible microarray data with good representation of transcripts across the size spectrum and a coefficient of repeatability significantly better than that reported previously.

Topics: Carbocyanines; Cell Line, Tumor; DNA, Complementary; Gene Expression Profiling; Guanidine; Humans; Lithium Chloride; Oligonucleotide Array Sequence Analysis; Phenol; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm

Apoptosis, a mechanism of programmed cell death that removes superfluous and harmful cells, is important both during development and in tissue homeostasis. Although Zn2+ is believed to be critical in apoptosis, the precise details of its role have yet to be elucidated. The macrocyclic Zn2+ ligand dansylamidoethylcyclen [L1*(HCl)4*(H2O)2], which is found primarily in a diprotonated form (H2L1), is cell-permeable and forms a strongly fluorescent 1:1 Zn2+ complex when Zn2+ entry into cells is facilitated by the Zn2+ ionophore pyrithione. H2L1 can be used to readily identify HeLa cells undergoing the early stages of etoposide-induced apoptosis because of the increased level of free Zn2+ that occurs at this time. The selectivity of H2L1 for the detection of apoptotic cells was verified by a conventional probe for apoptosis, annexin V-Cy3. Here, we describe methods for detecting apoptotic cells with H2L1 and for comparing detection of apoptosis with H2L1 to detection with annexin V-Cy3 and Zinquin.

Topics: Annexin A5; Apoptosis; Carbocyanines; Cell Line, Tumor; Dansyl Compounds; DNA; DNA Fragmentation; Fluorescent Dyes; HeLa Cells; Heterocyclic Compounds, 1-Ring; Humans; Indicators and Reagents; Intercalating Agents; Ionophores; Necrosis; Propidium; Quinolones; Staining and Labeling; Tosyl Compounds; Zinc

Microarray technology is a powerful tool to speed up genomics study, yet many technical aspects need to be improved. The hybridization reaction of microarray experiments is carried out for 16h or overnight in order to obtain reasonably strong signals for analysis in the presence of high salt buffer, like SSC. However, the quantitative aspect of microarray hybridization has seldom been investigated. In this study, we showed that higher overall signals from hybridization were achieved in a buffer system containing dextran sulfate, which can accelerate the kinetics of reaction by increasing the local concentration of the reactants. The dextran sulfate containing hybridization solution increases the reaction 4-fold (median) for cDNA microarray and 29-fold for oligonucleotide microarray. More importantly, the solution also provides a quantitative hybridization reaction, where the hybridization signals are proportional to the abundance of transcript added. The enhancement in the kinetics of hybridization is due to both dextran sulfate and formamide present in the solution, but the effect is not due to the higher temperature used during the reaction. With a slightly longer reaction time the hybridization reaction with the solution allows the detection of hybridization signals from rare transcripts that is not possible with regular hybridization buffers. With appropriate washing, the enhancement of kinetics by the solution does not increase the background signals at all, allowing higher signal-to-noise ratios to be achieved.

Topics: Arabidopsis; Buffers; Carbocyanines; Cell Line; Dextran Sulfate; DNA, Complementary; Formamides; Humans; Kinetics; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; RNA, Messenger; Sensitivity and Specificity

High-throughput, parallel gene expression analysis by means of microarray technology has become a widely used technique in recent years. There are currently two main dye-labelling strategies for microarray studies based on custom-spotted cDNA or oligonucleotides arrays: (I) Dye-labelling of a single target sample with a particular dye, followed by subsequent hybridisation to a single microarray slide, (II) Dye-labelling of two different target samples with two different dyes, followed by subsequent co-hybridisation to a single microarray slide. The two dyes most frequently used for either method are Cy3 and Cy5. We propose and evaluate a novel experiment set-up utilising three differently labelled targets co-hybridised to one microarray slide. In addition to Cy3 and Cy5, this incorporates Alexa 594 as a third dye-label. We evaluate this approach in line with current data processing and analysis techniques for microarrays, and run separate analyses on Alexa 594 used in single-target, dual-target and the intended triple-target experiment set-ups (a total of 18 microarray slides). We follow this by pointing out practical applications and suitable analysis methods, and conclude that triple-target microarray experiments can add value to microarray research by reducing material costs for arrays and related processes, and by increasing the number of options for pragmatic experiment design.. The addition of Alexa 594 as a dye-label for an additional--third--target sample works within the framework of more commonplace Cy5/Cy3 labelled target sample combinations. Standard normalisation methods are still applicable, and the resulting data can be expected to allow identification of expression differences in a biological experiment, given sufficient levels of biological replication (as is necessary for most microarray experiments).. The use of three dye-labelled target samples can be a valuable addition to the standard repertoire of microarray experiment designs. The method enables direct comparison between two experimental populations as well as measuring these two populations in relation to a third reference sample, allowing comparisons within the slide and across slides. These benefits are only offset by the added level of consideration required in the experimental design and data processing of a triple-target study design. Common methods for data processing and analysis are still applicable, but there is scope for the development of custom models for triple-target data. In summary, we do not consider the triple-target approach to be a new standard, but a valuable addition to the existing microarray study toolkit.

Topics: Animals; Carbocyanines; DNA, Complementary; Fluorescent Dyes; Gene Expression Profiling; Male; Mice; Mice, Inbred C57BL; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Organic Chemicals; Reproducibility of Results; Testis

Spotted 70-mer oligonucleotide arrays offer potentially greater specificity and an alternative to expensive cDNA library maintenance and amplification. Since microarray fabrication is a considerable source of data variance, we previously directly tagged cDNA probes with a third fluorophore for prehybridization quality control. Fluorescently modifying oligonucleotide sets is cost prohibitive, therefore, a co-spotted Staphylococcus aureus-specific fluorescein-labeled "tracking" oligonucleotide is described to monitor fabrication variables of a Mycobacterium tuberculosis oligonucleotide microarray.. Significantly (p < 0.01) improved DNA retention was achieved printing in 15% DMSO/1.5 M betaine compared to the vendor recommended buffers. Introduction of tracking oligonucleotide did not effect hybridization efficiency or introduce ratio measurement bias in hybridizations between M. tuberculosis H37Rv and M. tuberculosis mprA. Linearity between the mean log Cy3/Cy5 ratios of genes differentially expressed from arrays either possessing or lacking the tracking oligonucleotide was observed (R2 = 0.90, p < 0.05) and there were no significant differences in Pearson's correlation coefficients of ratio data between replicates possessing (0.72 +/- 0.07), replicates lacking (0.74 +/- 0.10), or replicates with and without (0.70 +/- 0.04) the tracking oligonucleotide. ANOVA analysis confirmed the tracking oligonucleotide introduced no bias. Titrating target-specific oligonucleotide (40 microM to 0.78 microM) in the presence of 0.5 microM tracking oligonucleotide, revealed a fluorescein fluorescence inversely related to target-specific oligonucleotide molarity, making tracking oligonucleotide signal useful for quality control measurements and differentiating false negatives (synthesis failures and mechanical misses) from true negatives (no gene expression).. This novel approach enables prehybridization array visualization for spotted oligonucleotide arrays and sets the stage for more sophisticated slide qualification and data filtering applications.

Topics: Analysis of Variance; Carbocyanines; DNA, Bacterial; Gene Expression Profiling; Mycobacterium tuberculosis; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes; Quality Control; Reference Standards; Reproducibility of Results; RNA, Bacterial; Staphylococcus aureus

High throughput gene expression data from spotted cDNA microarrays are collected by scanning the signal intensities of the corresponding spots by dedicated fluorescence scanners. The major scanner settings for increasing the spot intensities are the laser power and the voltage of the photomultiplier tube (PMT). It is required that the expression ratios are independent of these settings. We have investigated the relationships between PMT voltage, spot intensities, and expression ratios for different scanners, in order to define an optimal scanning procedure.. All scanners showed a limited intensity range from 200 to 50 000 (mean spot intensity), for which the expression ratios were independent of PMT voltage. This usable intensity range was considerably less than the maximum detection range of the PMTs. The use of spot and background intensities outside this range led to errors in the ratios. The errors at high intensities were caused by saturation of pixel intensities within the spots. An algorithm was developed to correct the intensities of these spots, and, hence, extend the upper limit of the usable intensity range.. It is suggested that the PMT voltage should be increased to avoid intensities of the weakest spots below the usable range, allowing the brightest spots to reach the level of saturation. Subsequently, a second set of images should be acquired with a lower PMT setting such that no pixels are in saturation. Reliable data for spots with saturation in the first set of images can easily be extracted from the second set of images by the use of our algorithm. This procedure would lead to an increase in the accuracy of the data and in the number of data points achieved in each experiment compared to traditional procedures.

Topics: Algorithms; Carbocyanines; DNA, Complementary; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; Uterine Cervical Neoplasms

Bone marrow (BM)-derived cells differentiate into a wide variety of cell types. BM contains a heterogeneous population of stem and progenitor cells including hematopoietic stem cells, marrow stromal cells, and perhaps other progenitor cells. To establish unequivocally the transdifferentiation capability of a hematopoietic cell to a nonhematopoietic cell (endothelial cells, neurons, and glial cells), it is imperative to demonstrate that a single cell or clone of that single cell (clonal analysis) differentiates into cells comprising vessels or other cells in the brain.. We generated mice that exhibited a high level of hematopoietic reconstitution from a single enhanced green fluorescent protein (EGFP) stem cell. To achieve this, we combined FACS sorting and cell culture to generate a population of cells derived from a single hematopoietic stem cell (Lin-, CD34-, c-kit+, and Sca-1+). Clonal populations of cells were then transplanted into lethally irradiated recipient mice. After 3-4 months of engraftment, some mice underwent middle cerebral artery (MCA) suture occlusion. EGFP immunocytochemistry and dual labeling was performed with cell-specific markers on tissue from various time points.. In all transplanted mice, EGFP+ highly ramified cells were seen in the brain parenchyma. These cells stained with RCA120 lectin and had the characteristics of parenchymal microglial cells. In brains without infarction and in uninfarcted brain regions of mice that underwent MCA occlusion, there were many EGFP+ cells in a perivascular distribution, associated with both small and larger blood vessels. The cells were tightly apposed to the vessel wall and some had long processes that enveloped the endothelial cells. After MCA occlusion, there was an influx of EGFP expressing cells in the ischemic tissue that colocalized with the "neovascularization." These EGFP+ cells were wrapped around endothelial cells in an albuminal location and did not coexpress von Willebrand Factor or CD31. We detected rare dual-labeled EGFP and NeuN-expressing cells. We detected two staining patterns. The more frequent pattern was phagocytosis of NeuN cells by EGFP expressing cells. However, we also detected rarer cells where the EGFP and NeuN appeared to be colocalized by confocal microscopy.. HSC differentiate into parenchymal microglial cells and perivascular cells in the brain. The numbers of these cells increase after cerebral ischemia. The HSC is therefore one source of parenchymal microglial cells and a source for perivascular cells. After a cerebral infarction, there are rare HSC-derived cells that stain with the neuronal marker, NeuN. However, the more common pattern appears to represent phagocytosis of damaged neurons by EGFP+ microglial cells.

Topics: Animals; Antigens; Benzimidazoles; Brain; Carbocyanines; Cell Differentiation; Cells, Cultured; Endothelial Cells; Endothelium, Vascular; Female; Flow Cytometry; Functional Laterality; Green Fluorescent Proteins; Hematopoiesis; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Immunohistochemistry; Infarction, Middle Cerebral Artery; Luminescent Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Microscopy, Confocal; Neurons; Phosphopyruvate Hydratase; Plant Lectins; Platelet Endothelial Cell Adhesion Molecule-1; Radiation Chimera; Time Factors; von Willebrand Factor

Macrolide-resistant Helicobacter (H.) pylori represent an increasing therapeutic problem. Macrolide resistance is usually determined phenotypically in vitro with methods such as E-test or agar dilution test. A prerequisite for those tests, however, is bacterial culture that is not routinely set up in the course of gastroscopy. In contrast, formalin-fixed, paraffin-embedded biopsies are regularly available from patients who have undergone gastroscopy. In such biopsies macrolide-resistant H. pylori can be detected by the genotype-based technique of fluorescence in situ hybridization (FISH). Experience gained by this new method, however, is still extremely limited, especially in formalin-fixed tissue. Therefore, we retrospectively investigated formalin-fixed, paraffin-embedded biopsy specimens by FISH in 104 patients suffering from therapy-resistant H. pylori gastritis. To test the accuracy of FISH, we initially examined specimens from 53 patients for whom results of the E-test were available. Next we analyzed biopsies from another 51 patients that had been selected since phenotypical resistance testing had failed despite documented culturing attempts. In all 104 patients, H. pylori was detected by FISH and could thus be investigated for macrolide resistance. Overall, macrolide-resistant bacteria were found in 71 patients (68.3%). In 49 of 53 patients (92.4%), FISH and E-test returned identical results (no significant discordance according to McNemar's chi(2)-test). Taken together, our study demonstrates that FISH is a highly sensitive and reliable method for detecting macrolide-resistant H. pylori in formalin-fixed, paraffin-embedded biopsy specimens, which represents the routine method of processing tissue obtained upon gastroscopy.

Topics: Anti-Bacterial Agents; Carbocyanines; Clarithromycin; Drug Resistance, Bacterial; Fluorescein; Formaldehyde; Gastritis; Helicobacter Infections; Helicobacter pylori; Humans; In Situ Hybridization, Fluorescence; Macrolides; Reproducibility of Results; Stomach; Tissue Fixation

We present an improved FISH strategy for differentiating the sex chromosomes of the dioecious model plant, Silene latifolia. Fixed mitotic protoplasts were dropped on a polyethylene naphthalate membrane, the X or Y chromosomes were isolated using nitrogen laser beam microdissection, catapulted by laser pressure, and amplified by DOP-PCR. A modified FAST-FISH protocol based on a short hybridization time combined with a low concentration of probe was used. The success of this approach is demonstrated by the differential labeling of the X and Y chromosomes and it could represent a quick method for comparing organization of plant genomes.

Topics: Carbocyanines; Chromosomes, Plant; DNA Probes; Fluorescent Dyes; In Situ Hybridization, Fluorescence; Lasers; Metaphase; Microdissection; Polymerase Chain Reaction; Primed In Situ Labeling; Protoplasts; Sex Chromosomes; Silene

On the background of the neurotoxicity of mercury compounds, the interaction of mercury(II) with the cytoskeleton was investigated in vitro using IMR-32 neuroblastoma cells. Conditions for culture of these cells on microscopic slides and procedures for immunofluorescence staining of the microtubule network were optimised. Both morphology and viability of IMR-32 cells were affected by mercury(II) at concentrations higher than 15 microM. Pronounced disintegration of the microtubule cytoskeleton was detected at 30 microM mercury(II). Compared to previous studies with fibroblasts, the no-observed-effect concentration was markedly lower, pointing to a particular sensitivity of nerve cells to mercury. This could be due to disturbed information transfer processes depending on an intact microtubule system.

Topics: Carbocyanines; Cell Line, Tumor; Cytoskeleton; Humans; Mercuric Chloride; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Microtubules; Neurons

Two-dimensional difference gel electrophoresis (DIGE) is a tool for measuring changes in protein expression between samples involving pre-electrophoretic labeling with cyanine dyes. Here we assess a common method to analyze DIGE data using the DeCyder software system. Experimental error was studied by a series of same sample comparisons. Aliquots of sample were labeled with N-hydroxyl succinimidyl ester-derivatives of Cy2, Cy3, and Cy5 dyes and run together on one gel. This allowed assessment of how experimental error influenced differential expression analysis. Bias in the log volume ratios was observed, which could be explained by differences in dye background. Further complications are caused by significant gel-to-gel variation in the spot volume ratio distributions. Using DeCyder alone results in an inability to define ratio thresholds for 90 or 95% confidence. An alternative normalization method was thus applied which resulted in improved data distribution and allowed greater sensitivity in analysis. When combined with a standardizing function, this allowed gel-independent thresholds for 90% confidence. The new approach, detailed here, represents a method to greatly improve the success of DIGE data analysis.

Topics: Bacterial Proteins; Bias; Carbocyanines; Confidence Intervals; Electrophoresis, Gel, Two-Dimensional; Erwinia; Fluorescent Dyes; Proteins; Reproducibility of Results; Sensitivity and Specificity; Software; Staining and Labeling

The basis for differences in activity-dependent trafficking of AMPA receptors (AMPARs) and NMDA receptors (NMDARs) remains unclear. Using single-molecule tracking, we found different lateral mobilities for AMPARs and NMDARs: changes in neuronal activity modified AMPAR but not NMDAR mobility, whereas protein kinase C activation modified both. Differences in mobility were mainly detected for extrasynaptic AMPARs, suggesting that receptor diffusion between synaptic and extrasynaptic domains is involved in plasticity processes.

Topics: Animals; Animals, Newborn; Antibodies; Carbocyanines; Cells, Cultured; Hippocampus; Neurons; Potassium Chloride; Protein Kinase C; Protein Transport; Rats; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Statistics, Nonparametric; Synapses; Tetradecanoylphorbol Acetate; Tetrodotoxin; Time Factors

Dystrophin is a cytoskeletal protein normally expressed underneath the sarcolemma of muscle fibers. The lack of dystrophin in Duchenne muscular Dystrophy (DMD) muscles results in fiber necrosis, which was proposed to be mediated by chronic calcium mishandling. The extensive comparison of dystrophic cells from human or mdx mice with normal muscles have suggested that the lack of dystrophin may alter the resting calcium permeability and steady-state levels of calcium, but this latter observation remains controversial. It is also not clear, whether calcium mishandling is resulting from the dystrophic process or if dystrophin can directly regulate calcium handling in muscle cells. This prompted us to determine if transfection of full-length dystrophin or Becker Muscular Dystrophy (BMD) minidystrophin, a candidate for viral-mediated gene therapy, could change calcium handling properties. We took advantage of specific properties of Sol8 cell line showing the absence of dystrophin expression together with a drastic calcium mishandling. Here, we show that full-length dystrophin allowed the recovery of a low resting intracellular-free calcium concentration together with lower calcium transients. We also show for the first time that stable expression of minidystrophin was able to restore normal calcium handling in Sol8 myotubes through a better control of steady-state levels, calcium transients, and subcellular calcium events. It suggests that dystrophin could play a regulatory role on calcium homeostasis apparatus and that functional links exist between calcium signaling and cytoskeleton.

Topics: Animals; Calcium; Carbocyanines; Cells, Cultured; Dystrophin; Fluorescent Dyes; Green Fluorescent Proteins; Homeostasis; Immunohistochemistry; Luminescent Proteins; Mice; Mice, Inbred C3H; Microinjections; Microscopy, Confocal; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Plasmids; Retroviridae

The detailed characterization of virus DNA is a challenge, and the genotyping that has been achieved to date has only been possible because researchers have sent a great deal of time and effort to do so. Instead of the simultaneous detection of hundreds of viruses on a single high-density DNA-chip at very high costs per chip, we present here an alternative approach using a well-designed and tailored microarray which can establish whether or not a handful of viral genes are present in a clinical sample.. In this study we applied a new concept of microarray-based, optimized and robust biochemistry for molecular diagnostics of the herpesviruses. For comparison, all samples were genotyped using standard procedures.. The biochemical procedure of a knowledge-based, low-density microarray was established based on the molecular diagnostics of human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The study attempted to optimize parameters of microarray design, surface chemistry, oligonucleotide probe spotting, sample labeling and DNA hybridization to the developed DNA microarray. The results of 12 900 hybridization reactions on about 150 configured herpes virus microarrays showed that the established microarray-based typing procedure was reproducible, virus-specific and sufficiently sensitive with a lower limit of 100 viral copies per mL sample.. The developed method utilizes low-fluorescence background coverslips, epoxy surface chemistry, standardized oligonucleotide probe spotting, PCR-labeling with Cy3 of isolated DNA, array hybridization, and detecting of specific spot fluorescence by an automatic microarray reader. We expect the configured microarray approach to be the method for high-throughput associated studies on human herpes viruses.

Topics: Carbocyanines; DNA, Viral; Fluorescent Dyes; Herpesviridae; Herpesviridae Infections; Humans; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes

Herein we report that crotamine, a small lysine- and cysteine-rich protein from the venom of the South American rattlesnake, can rapidly penetrate into different cell types and mouse blastocysts in vitro. In vivo crotamine strongly labels cells from mouse bone marrow and spleen and from peritoneal liquid, as shown by fluorescent confocal laser-scanning microscopy. Nuclear localization of crotamine was observed in both fixed and unfixed cells. In the cytoplasm, crotamine specifically associates with centrosomes and thus allows us to follow the process of centriole duplication and separation. In the nucleus, it binds to the chromosomes at S/G2 phase, when centrioles start dividing. Moreover, crotamine appears as a marker of actively proliferating cells, as shown by 5-BrdU cell-proliferation assay. Crotamine in the micromolar range proved nontoxic to any of the cell cultures tested and did not affect the pluripotency of ES cells or the development of mouse embryos.

Topics: Animals; Biomarkers; Carbocyanines; Cell Cycle; Cell Line; Cell Membrane Permeability; Cell Nucleus; Cell Proliferation; Centrioles; Centrosome; Chromosomes, Mammalian; Crotalid Venoms; Crotalus; Embryo, Mammalian; Embryo, Nonmammalian; Humans; Mice; Stem Cells

Neurogenesis occurs throughout adult life in dentate gyrus of mammal hippocampus. Therefore, neurons at different stages of electrophysiological and morphological maturation and showing various, if any, synaptic inputs co-exist in the adult granule cell layer, as occurs during dentate gyrus development. The knowledge of functional properties of new neurons throughout their maturation can contribute to understanding their role in the hippocampal function. In this study electrophysiological and morphological features of granule layer cells, characterized as immature or mature neurons, without and with synaptic input, were comparatively described in adult rats. The patch-clamp technique was used to perform electrophysiological recordings, the occurrence of synaptic input evoked by medial perforant pathway stimulation was investigated and synaptic input was characterized. Cells were then identified and morphologically described via detection of biocytin injected through the patch pipette. The neuronal phenotype of recorded cells was assessed by immunohistochemistry and single-cell RT-PCR. Cells with very low capacitance, high input resistance, depolarized resting membrane potential and without synaptic activity were found exclusively at the border of the GCL facing hilus; this type of cell expressed the class III beta-tubulin neuronal marker (mRNA and protein) and did not express a glial marker. Immature neuronal cells with progressively increasing capacitance, decreasing input resistance and resting membrane potential getting more hyperpolarized showed only depolarizing GABAergic synaptic input at first and then also glutamatergic synaptic input. Finally, cells showing electrophysiological, synaptic, and morphological features of mature granule, expressing the mature neuron marker NeuN, were identified.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bicuculline; Calcium Channel Blockers; Carbocyanines; Cellular Senescence; Dentate Gyrus; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; GABA Antagonists; Glial Fibrillary Acidic Protein; Immunohistochemistry; In Vitro Techniques; Male; Membrane Potentials; Microscopy, Confocal; Neural Cell Adhesion Molecule L1; Neurons; Patch-Clamp Techniques; Perforant Pathway; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sialic Acids; Synapses; Synaptic Transmission; Tetrodotoxin; Tubulin; Valine; Verapamil

DNA oligo-nucleotides, localized at Au metal electrodes in aqueous solution, are found to be released when applying a negative bias voltage to the electrode. The release was confirmed by monitoring the intensity of the fluorescence of cyanine dyes (Cy3) linked to the 5' end of the DNA. The threshold voltage of the release changes depending on the kind of linker added to the DNA 3'-terminal. The amount of released DNA depends on the duration of the voltage pulse. Using this technique, we can retain DNA at Au electrodes or Au needles, and release the desired amount of DNA at a precise location in a target. The results suggest that DNA injection into living cells is possible with this method.

Topics: Biosensing Techniques; Carbocyanines; DNA; Electrochemistry; Electrodes; Fluorescent Dyes; Gold; Reproducibility of Results; Sensitivity and Specificity; Static Electricity; Time Factors

Response to photodynamic therapy depends on adequate tumor oxygenation as well as sufficient accumulation of photosensitizer in the tumor. The goal of this study was to investigate the presence of hypoxia and retention of the photosensitizer Photofrin in the tumors of patients with intra-abdominal carcinomatosis or sarcomatosis.. Tumor nodules from 10 patients were studied. In nine of these patients, hypoxia was identified in histological sections of biopsied tumor after administration of the hypoxia marker 2-(2-nitroimidazol-1[H]-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5). In separate tumor nodules from 10 patients, Photofrin uptake was measured by fluorescence after tissue solubilization.. Hypoxia existed in the tumors of five patients, with three of these patients demonstrating at least one severely hypoxic nodule. Physiological levels of oxygen were present in the tumors of four patients. An association between tumor size and hypoxia was not evident because some tumor nodules as small as approximately 2 mm in diameter were severely hypoxic. However, even these tumor nodules contained vascular networks. Three patients with severely hypoxic tumor nodules exhibited moderate levels of Photofrin uptake of 3.9 +/- 0.4 to 3.9 +/- 0.5 ng/mg (mean +/- SE). The four patients with tumors of physiological oxygenation did not consistently exhibit high tumor concentrations of Photofrin: mean +/- SE drug uptake among these patients ranged from 0.6 +/- 0.8 to 5.8 +/- 0.5 ng/mg.. Carcinomatosis or sarcomatosis of the i.p. cavity may exhibit severe tumor hypoxia. Photofrin accumulation in tumors varied by a factor of approximately 10x among all patients, and, on average, those with severe hypoxia in at least one nodule did not demonstrate poor Photofrin uptake in separate tumor samples. These data emphasize the need for reconsideration of the generally accepted paradigm of small tumor size, good oxygenation, and good drug delivery because this may vary on an individual tumor basis.

Topics: Appendiceal Neoplasms; Benzimidazoles; Binding, Competitive; Carbocyanines; Colonic Neoplasms; Dihematoporphyrin Ether; Etanidazole; Female; Gastrointestinal Neoplasms; Gastrointestinal Stromal Tumors; Humans; Hydrocarbons, Fluorinated; In Vitro Techniques; Intestine, Small; Male; Microscopy, Fluorescence; Ovarian Neoplasms; Oxygen; Photochemotherapy; Sarcoma

Double stranded targets on the cDNA microarray contain representatives of both the coding and noncoding strands, which will introduce hybridization competition with probes. Here, the effect of double and single strands of targets on the signal intensity and the ratios of Cy5/Cy3 within the same slide were compared. The results show that single stranded targets can increase the hybridization efficiency without changing the Cy5/Cy3 ratio. Based on these results, a new strategy was established by generating cDNA targets with asymmetric PCR, instead of conventional PCR, to increase the sensitivity of the cDNA microarray. Furthermore, the feasibility of this approach was validated. The results indicate that the cDNA microarray system based on asymmetric PCR is more sensitive, with no decrease in the reliability and reproducibility as compared with that based on conventional symmetric PCR.

Topics: Carbocyanines; DNA; DNA, Complementary; DNA, Single-Stranded; Fluorescent Dyes; Hybridization, Genetic; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Reproducibility of Results; RNA

A reproducible, transcriptionally diverse common reference RNA is required for accurate comparisons of data generated from most spotted microarray experiments in different experiments. Several methods have been proposed to make such a reference RNA, such as pooling RNAs isolated from multiple cell lines or tissues, amplifying pooled RNAs, or synthesizing RNAs or DNAs complementary to microarray features. We report an approach to prepare a large amount of mouse reference RNA from whole neonatal mice. This approach is simple, quick, reliable, reproducible, and inexpensive. The whole mouse reference RNA is highly representative when compared to two commercially available universal mouse reference RNAs isolated and pooled from multiple cell lines or organs.

Topics: Animals; Animals, Newborn; Carbocyanines; DNA, Complementary; Fluorescent Dyes; Gene Expression Profiling; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; RNA

We evaluated a lymphoid RNA standard prepared in our laboratory for spotted microarrays against the Universal Human Reference standard from Stratagene. Our goal was to determine if the Stratagene standard, which contains only two lymphoid cell lines out of a pool of 10 human cancer cell lines, had acceptable gene coverage to serve as a comprehensive standard for gene expression profiling of lymphoid tissues. Our lymphoid standard was prepared from thymus, spleen, tonsil, and cell lines representing immature B cells, plasma cells, and natural killer (NK) cells, thus covering the entire spectrum of lymphoid cells and most stromal elements present in specialized lymphoid tissues. The two standards were co-hybridized on oligonucleotide microarrays containing 17,260 genes, and both had fluorescence intensities above background for approximately 85% of the genes. Despite the limited representation of lymphoid cells in the Stratagene standard, only 4.2% genes exhibited expression differences greater than 2-fold including only 0.35% with differences greater than 4-fold. Although the lymphoid standard reflected a more comprehensive representation of immune system-associated genes, the Stratagene standard has the advantage of being commercially available, enabling easier comparison across laboratories and allowing comparative studies across a long period of time.

Topics: Carbocyanines; Cell Line, Tumor; Fluorescent Dyes; Gene Expression Profiling; Humans; Lymphoid Tissue; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes; Reference Standards; Reproducibility of Results; RNA

The structure and dynamics of the polyadenylation inhibition element (PIE) RNA, free and bound to the U1A protein, have been examined using time-resolved FRET and 2-aminopurine (2AP) fluorescence. This regulatory RNA, located at the 3' end of the U1A pre-mRNA, adopts a U-shaped structure, with binding sites for a single U1A protein at each bend (box 1 and box 2). The distance between the termini of the arms of the RNA is sensitive to its three-dimensional structure. Using Cy3/Cy5 FRET efficiency to monitor binding of Mg(2+), we show that the PIE RNA binds two Mg(2+) ions, which results in a restriction of its distance distribution of conformations. Local RNA structure probing using 2AP fluorescence shows that the structure of box 2 changes in response to Mg(2+) binding, thus tentatively locating the ion binding sites. Steady-state FRET data show that the distance R between the termini of the PIE RNA stems decreases from 66 A in the free RNA, to 58 A when N-terminal RNA binding domains (RBD1) of U1A are bound, and to 53 A when U1A proteins bind. However, anisotropy measurements indicate that both Cy3 and Cy5 stack on the ends of the RNA. To examine the consequences of the restricted motion of the fluorophores, FRET data are analyzed using two different models of motion and then compared to analogous data from the Cy3/fluorescein FRET pair. We conclude that the error introduced into distance calculations by stacking of the dyes is within the error of our measurements. Distance distributions of the RNA structures show that the intramolecular distance between the arms of the PIE RNA varies on the time scale of the fluorescence measurements; the mean distance is dependent on protein binding, but the breadth of the distributions indicates that the RNA retains structural heterogeneity.

Topics: 2-Aminopurine; Amino Acid Sequence; Base Sequence; Binding Sites; Carbocyanines; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Magnesium; Molecular Sequence Data; Nucleic Acid Conformation; Poly A; Polyadenylation; Protein Conformation; Ribonucleoprotein, U1 Small Nuclear; RNA Precursors; RNA-Binding Proteins; RNA, Messenger; Solutions; Thermodynamics

Secreted signaling molecules released from a restricted source are of great importance during embryonic development because they elicit induction, proliferation, differentiation, and patterning events in target cells . Fgf8 is a member of the fibroblast growth factor family with key inductive functions during vertebrate development of, for example, the forebrain , midbrain , cerebellum , heart , inner ear , and mesoderm . However, the mechanism by which the signaling range of Fgf8 is controlled in a field of target cells is unknown. We studied Fgf8 as a potential morphogen in the nascent neuroectoderm of living zebrafish embryos. We find that spreading of epitope-tagged Fgf8 through target tissue is carefully controlled by endocytosis and subsequent degradation in lysosomes, or "restrictive clearance," from extracellular spaces. If internalization is inhibited, Fgf8 protein accumulates extracellularly, spreads further, and activates target gene expression over a greater distance. Conversely, enhanced internalization increases Fgf8 uptake and shortens its effective signaling range. Our results suggest that Fgf8 spreads extracellularly by a diffusion-based mechanism and demonstrate that target cells can actively influence, through endocytosis and subsequent degradation, the availability of Fgf8 ligand to other target cells.

Topics: Animals; Carbocyanines; DNA-Binding Proteins; Ectoderm; Embryo, Nonmammalian; Endocytosis; Fibroblast Growth Factor 8; Fibroblast Growth Factors; Gene Expression Regulation, Developmental; Microscopy, Electron; Models, Biological; Nerve Tissue Proteins; Protein Transport; Signal Transduction; Transcription Factors; Zebrafish; Zebrafish Proteins

Non-linearities in observed log-ratios of gene expressions, also known as intensity dependent log-ratios, can often be accounted for by global biases in the two channels being compared. Any step in a microarray process may introduce such offsets and in this article we study the biases introduced by the microarray scanner and the image analysis software.. By scanning the same spotted oligonucleotide microarray at different photomultiplier tube (PMT) gains, we have identified a channel-specific bias present in two-channel microarray data. For the scanners analyzed it was in the range of 15-25 (out of 65,535). The observed bias was very stable between subsequent scans of the same array although the PMT gain was greatly adjusted. This indicates that the bias does not originate from a step preceding the scanner detector parts. The bias varies slightly between arrays. When comparing estimates based on data from the same array, but from different scanners, we have found that different scanners introduce different amounts of bias. So do various image analysis methods. We propose a scanning protocol and a constrained affine model that allows us to identify and estimate the bias in each channel. Backward transformation removes the bias and brings the channels to the same scale. The result is that systematic effects such as intensity dependent log-ratios are removed, but also that signal densities become much more similar. The average scan, which has a larger dynamical range and greater signal-to-noise ratio than individual scans, can then be obtained.. The study shows that microarray scanners may introduce a significant bias in each channel. Such biases have to be calibrated for, otherwise systematic effects such as intensity dependent log-ratios will be observed. The proposed scanning protocol and calibration method is simple to use and is useful for evaluating scanner biases or for obtaining calibrated measurements with extended dynamical range and better precision. The cross-platform R package aroma, which implements all described methods, is available for free from http://www.maths.lth.se/bioinformatics/.

Topics: Calibration; Carbocyanines; DNA; Image Interpretation, Computer-Assisted; Models, Statistical; Nucleic Acid Hybridization; Observer Variation; Oligonucleotide Array Sequence Analysis; RNA

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that eliminates mRNAs containing premature termination codons (PTCs). The proteins UPF1, SMG5, SMG6, and SMG7 are essential NMD factors in metazoa. SMG5 and SMG7 form a complex with UPF1 and interact with each other via their N-terminal domains. Here we show that SMG5 and SMG7 colocalize in cytoplasmic mRNA decay bodies, while SMG6 forms separate cytoplasmic foci. When SMG7 is tethered to a reporter transcript, it elicits its degradation, bypassing the requirement for a PTC, UPF1, SMG5, or SMG6. This activity is mediated by the C-terminal domain of SMG7. In contrast, SMG5 requires SMG7 to trigger mRNA decay and to localize to decay bodies. Our findings indicate that SMG7 provides a link between the NMD and the mRNA degradation machinery by interacting with SMG5 and UPF1 via its N-terminal domain and targeting bound transcripts for decay via its C-terminal domain.

Topics: Blotting, Western; Carbocyanines; Carrier Proteins; Cytoplasm; Dactinomycin; Frameshift Mutation; Genes, Reporter; HeLa Cells; Humans; Luciferases; Microscopy, Fluorescence; Mutation; Nucleic Acid Synthesis Inhibitors; Plasmids; Protein Structure, Tertiary; Recombinant Fusion Proteins; Ribonucleoproteins, Small Nuclear; RNA Helicases; RNA Interference; RNA, Messenger; RNA, Small Interfering; Templates, Genetic; Time Factors; Trans-Activators; Transfection

Escherichia coli UvrD protein is a 3' to 5' SF1 DNA helicase involved in methyl-directed mismatch repair and nucleotide excision repair of DNA. Using stopped-flow methods we have examined the kinetic mechanism of translocation of UvrD monomers along single-stranded DNA (ssDNA) in vitro by monitoring the transient kinetics of arrival of protein at the 5'-end of the ssDNA. Arrival at the 5'-end was monitored by the effect of protein on the fluorescence intensity of fluorophores (Cy3 or fluorescein) attached to the 5'-end of a series of oligodeoxythymidylates varying in length from 16 to 124 nt. We find that UvrD monomers are capable of ATP-dependent translocation along ssDNA with a biased 3' to 5' directionality. Global non-linear least-squares analysis of the full kinetic time-courses in the presence of a protein trap to prevent rebinding of free protein to the DNA using the methods described in the accompanying paper enabled us to obtain quantitative estimates of the kinetic parameters for translocation. We find that UvrD monomers translocate in discrete steps with an average kinetic step-size, m=3.68(+/-0.03) nt step(-1), a translocation rate constant, kt=51.3(+/-0.6) steps s(-1), (macroscopic translocation rate, mkt=189.0(+/-0.7) nt s(-1)), with a processivity corresponding to an average translocation distance of 2400(+/-600) nt before dissociation (10 mM Tris-HCl (pH 8.3), 20 mM NaCl, 20% (v/v) glycerol, 25 degrees C). However, in spite of its ability to translocate rapidly and efficiently along ssDNA, a UvrD monomer is unable to unwind even an 18 bp duplex in vitro. DNA helicase activity in vitro requires a UvrD dimer that unwinds DNA with a similar kinetic step-size of 4-5 bp step(-1), but an approximately threefold slower unwinding rate of 68(+/-9) bp s(-1) under the same solution conditions, indicating that DNA unwinding activity requires more than the ability to simply translocate directionally along ss-DNA.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Carbocyanines; Catalysis; DNA Helicases; DNA, Single-Stranded; Escherichia coli; Escherichia coli Proteins; Fluorescein; Heparin; Kinetics; Molecular Structure; Movement; Nucleic Acid Conformation; Spectrometry, Fluorescence; Time Factors

The spectral properties of a rigidified trimethine cyanine dye, Cy3B have been characterised. This probe has excellent fluorescent properties, good water solubility and can be bioconjugated. The emission properties of this fluorophore have also been investigated upon conjugation to an antibody. This study compared the conjugated emission properties of Cy3B with other commercially available fluorophores emitting at similar wavelengths.

Topics: Benzenesulfonates; Carbocyanines; Fluorescence; Fluorescent Dyes; Immunoglobulin G

Topics: Carbocyanines; Cell Nucleus; Cells, Cultured; Humans; JC Virus; Microscopy, Confocal

DNA microarrays are becoming increasingly popular in toxicogenomic studies. It is common knowledge that multiple repeats of microarray experiments have to be performed with multiple samples to get reliable data, due to experimental variations (both technical and biological) contained in microarray experiments. However, the use of multiple samples and replicate experiments is not practical in the field of environmental toxicology, since environmental samples are limited and microarray experiments are expensive. Thus it is desirable to obtain reliable data from a minimum number of repeats of microarray experiments. To further establish and extend gene expression profiling for toxicogenomics using microarrays, it is necessary to establish an analytical method of microarrays for toxicogenomics. In this study, we attempted to construct a "Data Dependent Analysis (DDA)" yeast cDNA microarray experiment from 100 microarray datasets in order to get reliable data from one microarray experiment.

Topics: Carbocyanines; Data Interpretation, Statistical; DNA, Fungal; Fluorescent Dyes; Oligonucleotide Array Sequence Analysis; Saccharomyces cerevisiae; Toxicogenetics

We report here an approach for simultaneous fluorescence imaging and electrical recording of single ion channels in planar bilayer membranes. As a test case, fluorescently labeled (Cy3 and Cy5) gramicidin derivatives were imaged at the single-molecule level using far-field illumination and cooled CCD camera detection. Gramicidin monomers were observed to diffuse in the plane of the membrane with a diffusion coefficient of 3.3 x 10(-8) cm(2)s(-1). Simultaneous electrical recording detected gramicidin homodimer (Cy3/Cy3, Cy5/Cy5) and heterodimer (Cy3/Cy5) channels. Heterodimer formation was observed optically by the appearance of a fluorescence resonance energy transfer (FRET) signal (irradiation of Cy3, detection of Cy5). The number of FRET signals was significantly smaller than the number of Cy3 signals (Cy3 monomers plus Cy3 homodimers) as expected. The number of FRET signals increased with increasing channel activity. In numerous cases the appearance of a FRET signal was observed to correlate with a channel opening event detected electrically. The heterodimers also diffused in the plane of the membrane with a diffusion coefficient of 3.0 x 10(-8) cm(2)s(-1). These experiments demonstrate the feasibility of simultaneous optical and electrical detection of structural changes in single ion channels as well as suggesting strategies for improving the reliability of such measurements.

Topics: Carbocyanines; Electrochemistry; Feasibility Studies; Fluorescence Resonance Energy Transfer; Gramicidin; Ion Channels; Lipid Bilayers; Liposomes; Membrane Potentials; Membranes, Artificial; Microscopy, Video; Motion; Patch-Clamp Techniques; Protein Conformation

Here we describe a new opportunity in methodology for increasing the detectability of fluorescently labeled DNA on solid substrates. We show that the use of glass substrates coated with metallic silver particles results in an approximate 5-fold increase in the intensity of Cy3- or Cy5-labeled DNA oligomers. Proximity to these silver particles also increases the photostability of Cy3- and Cy5-labeled oligomers. These results suggest the use of DNA array substrates with silver particles for increased sensitivity in genetic analysis.

Topics: Carbocyanines; DNA; Fluorescent Dyes; Oligonucleotides; Quality Control; Sensitivity and Specificity; Silver; Spectrometry, Fluorescence; Staining and Labeling; Surface Plasmon Resonance

Microarray based gene expression studies allow simultaneous analysis of relative amounts of messenger RNA (mRNA) for thousands of genes using fluorescently labeled nucleic acid targets. Most common methods use enzymatic techniques, such as oligo-dT primed reverse transcription to produce labeled cDNA. These labeling methods have a number of shortcomings, including enzyme- introduced labeling and sequence bias, laborious protocols, high experiment-to-experiment variability and an inability to detect small changes in expression levels. Here, we describe a novel labeling methodology that uses platinum-linked cyanine dyes to directly chemically label mRNA from as little as 2 micro g of total RNA. We show that the gene expression data produced using the labeled mRNA method has very high precision, low error, no labeling bias and a dynamic range over several orders of magnitude. This allows a greater accuracy in the identification of differentially expressed genes and cuts down on the need for running too many replicate assays. Small changes in gene expression can now be detected in large-scale gene expression profiling assays using this simple, easy and quick procedure.

Topics: Carbocyanines; DNA, Complementary; Fluorescent Dyes; Gene Expression Profiling; HL-60 Cells; Humans; Jurkat Cells; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; RNA, Messenger; Sensitivity and Specificity

Gene expression studies using microarrays have great potential to generate new insights into human disease pathogenesis, but data quality remains a major obstacle. In particular, there does not exist a method to determine prior to hybridization whether an array will yield high quality data, given good study design and target preparation. We have solved this problem through development of a three-color cDNA microarray platform where printed probes are fluorescein labeled, but are spectrally compatible with Cy3 and Cy5 dye-labeled targets when using confocal laser scanners possessing narrow bandwidths. This approach enables prehybridization evaluation of array/spot morphology, DNA deposition and retention and background levels. By using these measurements and the intra-slide coefficient of variation for fluorescence intensity we show that slides in the same batch are not equivalent and measurable prehybridization parameters can be predictive of hybridization performance as determined by replicate consistency. When hybridizing target derived from two cell lines to high and low quality replicate pairs (n = 50 pairs), a direct and significant relationship between prehybridization signal-to-background noise and post-hybridization reproducibility (R2 = 0.80, P < 0.001) was observed. We therefore conclude that slide selection based upon prehybridization quality scores will greatly benefit the ability to generate reliable gene expression data.

Topics: Carbocyanines; DNA Probes; DNA, Complementary; Fluorescein; Humans; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; Sensitivity and Specificity

Microarray analysis is a critically important technology for genome-enabled biology, therefore it is essential that the data obtained be reliable. Current software and normalization techniques for microarray analysis rely on the assumption that fluorescent background within spots is essentially the same throughout the glass slide and can be measured by fluorescence surrounding the spots. This assumption is not valid if background fluorescence is spot-localized. Inaccurate estimates of background fluorescence under the spot create a source of error, especially for low expressed genes. We have identified spot-localized, contaminating fluorescence in the Cy3 channel on several commercial and in-house printed microarray slides. We determined through mock hybridizations (without labeled target) that pre-hybridization scans could not be used to predict the contribution of this contaminating fluorescence after hybridization because the change in spot-to-spot fluorescence after hybridization was too variable. Two solutions to this problem were identified. First, allowing 4 h of exposure to air prior to printing on to Corning UltraGAPS slides significantly reduced contaminating fluorescence intensities to approximately the value of the surrounding glass. Alternatively, application of a novel, hyperspectral imaging scanner and multivariate curve resolution algorithms, allowed the spectral contributions of Cy3 signal, glass, and contaminating fluorescence to be distinguished and quantified after hybridization.

Topics: Artifacts; Calibration; Carbocyanines; DNA, Complementary; Fluorescence; Fluorescent Dyes; Genome, Fungal; Oligonucleotide Array Sequence Analysis; Reference Standards; Saccharomyces cerevisiae

Recent studies have demonstrated that silver metallic particles can increase the quantum yield and decrease the lifetimes of nearby fluorophores. These studies are extended to double stranded DNA oligomers labeled with N,N'-(dipropyl)-tetramethylindocarbocyanine (Cy3) or N,N-(dipropyl)-tetramethylindodicarbocyanine (Cy5). The proximity to silver particles increases the apparent quantum yields and decreases the lifetimes of the double helical DNA 23-mer labeled individually with Cy3 or Cy5. The decreased lifetimes are accompanied by apparently increased photostability of the labeled oligomers near silver particles. Because of spatial averaging across the sample these results are likely to significantly underestimate the effects of silver particles on labeled DNA localized at an optimal distance from the metallic surface. These results suggest that DNA arrays fabricated on substrates with silver particles can display increased sensitivity and photostability in the analysis of gene expression.

Topics: Base Sequence; Carbocyanines; DNA; Fluorescence; Molecular Sequence Data; Nucleic Acid Conformation; Oligonucleotide Array Sequence Analysis; Silver; Spectrometry, Fluorescence

Microarrays of oligonucleotide expression libraries can be hybridised with either cDNA, generated from mRNA during reverse transcription, or cRNA, generated in an Eberwine mRNA amplification procedure. While methods for fluorescent labelling of cDNA have been thoroughly investigated, methods for cRNA labelling have not. To this purpose, we developed an aminoallyl-UTP (aa-UTP) driven cRNA labelling protocol and compared it in expression profiling studies using spotted 7.5 K 65mer murine oligonucleotide arrays with labelling via direct incorporation of Cy-UTPs. The presence of dimethylsulfoxide during coupling of aa-modified cRNA with N-hydroxysuccinimide-modified, fluorescent Cy dyes greatly enhanced the labelling efficiency, as analysed by spectrophotometry and fluorescent hybridisation signals. Indirect labelling using aa-UTP resulted in 2- to 3-fold higher degrees of labelling and fluorescent signals than labelling by direct incorporation of Cy-UTP. By variation of the aa-UTP:UTP ratio, a clear optimal degree of labelling was found (1 dye per 20-25 nt). Incorporation of more label increased Cy3 signal but lowered Cy5 fluorescence. This effect is probably due to quenching, which is more prominent for Cy5 than for Cy3. In conclusion, the currently developed method is an efficient, robust and inexpensive technique for fluorescent labelling of cRNA and allows sensitive detection of gene expression profiles on oligonucleotide microarrays.

Topics: Carbocyanines; Fluorescent Dyes; Oligonucleotide Array Sequence Analysis; RNA, Complementary; Spectrophotometry; Uridine Triphosphate

Low accessibility of the rRNA is together with cell wall impermeability and low cellular ribosome content a frequent reason for failure of whole-cell fluorescence hybridization with fluorescently labeled oligonucleotide probes. In this study we compare accessibility data for the 16S rRNA of Escherichia coli (gamma Proteobacteria, Bacteria) with the phylogenetically distantly related organisms Pirellula sp. strain 1 (Planctomycetes, Bacteria) and Metallosphaera sedula (Crenarchaeota, Archaea) and the 18S rRNA accessibility of Saccharomyces cerevisiae (Eucarya). For a total of 537 Cy3-labeled probes, the signal intensities of hybridized cells were quantified under standardized conditions by flow cytometry. The relative probe-conferred fluorescence intensities are shown on color-coded small-subunit rRNA secondary-structure models. For Pirellula sp., most of the probes belong to class II and III (72% of the whole data set), whereas most of the probes targeting sites on M. sedula were grouped into class V and VI (46% of the whole data set). For E. coli, 45% of all probes of the data set belong to class III and IV. A consensus model for the accessibility of the small-subunit rRNA to oligonucleotide probes is proposed which uses 60 homolog target sites of the three prokaryotic 16S rRNA molecules. In general, open regions were localized around helices 13 and 14 including target positions 285 to 338, whereas helix 22 (positions 585 to 656) and the 3' half of helix 47 (positions 1320 to 1345) were generally inaccessible. Finally, the 16S rRNA consensus model was compared to data on the in situ accessibility of the 18S rRNA of S. cerevisiae.

Topics: Bacteria; Carbocyanines; Crenarchaeota; Escherichia coli; Flow Cytometry; Fluorescent Dyes; In Situ Hybridization, Fluorescence; Oligonucleotide Probes; RNA, Ribosomal, 16S; RNA, Ribosomal, 18S; Saccharomyces cerevisiae

The impact of a peptide that contains a nuclear localisation sequence (NLS) on intracellular DNA trafficking was studied. We used the adenoviral core peptide mu and an SV40 NLS peptide to condense plasmid DNA (pDNA) prior to formulation with 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol/dioleoyl-L-alpha-phosphatidyl ethanolamine (DC-Chol/DOPE) liposomes to give LMD and LND vectors, respectively. Fluorescent-labelled lipid and peptides plus dye-labelled pDNA components were used to investigate gene delivery in dividing and S-phase growth-arrested cells. Confocal microscopic analyses reveal little difference in intracellular trafficking events. Strikingly, mu peptide associates with nuclei and nucleoli of cells within less than 15 mins incubation of LMD with cells, which suggests that mu peptide has an NLS function. These NLS properties were confirmed by cloning of a mu-beta-galactosidase fusion protein that localises in the nuclei of cells after cytosolic translation. In dividing cells both LMD and LND deliver pDNA(Cy3) to nuclei within 30-45 min incubation with cells. By contrast, pDNA is detected only in the cytoplasm in growth-arrested cells over the period of time investigated, and not in the nuclei. LD systems prepared from DC-Chol/DOPE cationic liposomes and pDNA(Cy3) behave similarly to LMD systems, which suggests that mu peptide is unable to influence trafficking events in this current LMD formulation, in spite of its strong NLS capacity. We further describe the effect of polyethyleneglycol (PEG) on cellular uptake. "Stealth" systems obtained by post-coating LMD particles with fluorescent-labelled PEG molecules (0.5, 5 and 10 mol % fluorescein-PEG(5000)-N-hydroxysuccinimide) were prepared and shown to be internalised rapidly (mins) by cells, without detectable transgene expression. This result indicates that PEG blocks intracellular trafficking of pDNA.

Topics: Adenoviridae; Carbocyanines; Cell Line; Cholesterol; DNA; Electrophoretic Mobility Shift Assay; Fluorescent Dyes; Gene Transfer Techniques; Genetic Vectors; Glycoside Hydrolases; Humans; Intracellular Space; Liposomes; Microscopy, Confocal; Nuclear Localization Signals; Oligopeptides; Peptides; Phosphatidylethanolamines; Plasmids; Polyethylene Glycols; Recombinant Fusion Proteins; Transfection

We examined the fluorescence spectral properties of Cy3- and Cy5-labeled oligonucleotides at various distances from the surface of silver island films. The distance to the surface was controlled by alternating layers of biotinylated bovine serum albumin (BSA) and avidin, followed by binding of a biotinylated oligonucleotide. The maximum enhancement of fluorescence near a factor of 12 was observed for the first BSA-avidin layer, with the enhancement decreasing to 2-fold for six layers. The minimum lifetimes were observed for the first BSA-avidin layer, and were about 25-fold shorter than on quartz slides without silver, with the lifetimes being about 2-fold shorter for six BSA-avidin layers. These results suggest that maximum fluorescence enhancements occur about 90A from the silver surface, a distance readily obtained by one or two layers of proteins.

Topics: Animals; Avidin; Base Sequence; Biotin; Carbocyanines; Cattle; Colloids; DNA; Fluorescent Dyes; Half-Life; Microscopy, Atomic Force; Oligonucleotides; Quartz; Serum Albumin, Bovine; Silver; Spectrometry, Fluorescence; Surface Plasmon Resonance; Surface Properties; Time Factors

In cDNA microarray experiments all samples are labelled with either Cy3 dye or Cy5 dye. Certain genes exhibit dye bias-a tendency to bind more efficiently to one of the dyes. The common reference design avoids the problem of dye bias by running all arrays 'forward', so that the samples being compared are always labelled with the same dye. But comparison of samples labelled with different dyes is sometimes of interest. In these situations, it is necessary to run some arrays 'reverse'-with the dye labelling reversed-in order to correct for the dye bias. The design of these experiments will impact one's ability to identify genes that are differentially expressed in different tissues or conditions. We address the design issue of how many specimens are needed, how many forward and reverse labelled arrays to perform, and how to optimally assign Cy3 and Cy5 labels to the specimens.. We consider three types of experiments for which some reverse labelling is needed: paired samples, samples from two predefined groups, and reference design data when comparison with the reference is of interest. We present simple probability models for the data, derive optimal estimators for relative gene expression, and compare the efficiency of the estimators for a range of designs. In each case, we present the optimal design and sample size formulas. We show that reverse labelling of individual arrays is generally not required.

Topics: Algorithms; Artifacts; Carbocyanines; Cluster Analysis; Computer-Aided Design; Equipment Design; Equipment Failure Analysis; Fluorescent Dyes; Models, Statistical; Oligonucleotide Array Sequence Analysis; Quality Control; Reproducibility of Results; Sample Size; Sensitivity and Specificity; Sequence Analysis, DNA; Staining and Labeling

Data from one-channel cDNA microarray studies may exhibit poor reproducibility due to spatial heterogeneity, non-linear array-to-array variation and problems in correcting for background. Uncorrected, these phenomena can give rise to misleading conclusions.. Spatial heterogeneity may be corrected using two-dimensional loess smoothing (Colantuoni et al., 2002). Non-linear between-array variation may be corrected using an iterative application of one-dimensional loess smoothing. A method for background correction using a smoothing function rather than simple subtraction is described. These techniques promote within-array spatial uniformity and between-array reproducibility. Their application is illustrated using data from a study of the effects of an insulin sensitizer, rosiglitazone, on gene expression in white adipose tissue in diabetic db/db mice. They may also be useful with data from two-channel cDNA microarrays and from oligonucleotide arrays.. R functions for the methods described are available on request from the author.

Topics: Adipose Tissue; Animals; Artifacts; Carbocyanines; Gene Expression Profiling; Male; Mice; Models, Genetic; Models, Statistical; Nonlinear Dynamics; Oligonucleotide Array Sequence Analysis; Quality Control; Reproducibility of Results; RNA; Sensitivity and Specificity; Sequence Analysis, DNA; Stochastic Processes

Fluorescence in situ hybridization (FISH) has proven to be most useful for the identification of microorganisms. However, species-specific oligonucleotide probes often fail to give satisfactory results. Among the causes leading to low hybridization signals is the reduced accessibility of the targeted rRNA site to the oligonucleotide, mainly for structural reasons. In this study we used flow cytometry to determine whole-cell fluorescence intensities with a set of 32 Cy3-labeled oligonucleotide probes covering the full length of the D1 and D2 domains in the 26S rRNA of Saccharomyces cerevisiae PYCC 4455(T). The brightest signal was obtained with a probe complementary to positions 223 to 240. Almost half of the probes conferred a fluorescence intensity above 60% of the maximum, whereas only one probe could hardly detect the cells. The accessibility map based on the results obtained can be extrapolated to other yeasts, as shown experimentally with 27 additional species (14 ascomycetes and 13 basidiomycetes). This work contributes to a more rational design of species-specific probes for yeast identification and monitoring.

Topics: Base Sequence; Carbocyanines; Flow Cytometry; Fluorescent Dyes; In Situ Hybridization, Fluorescence; Nucleic Acid Conformation; Oligonucleotide Probes; RNA, Fungal; RNA, Ribosomal; Saccharomyces cerevisiae; Species Specificity

One approach to the accurate determination of the pathogenic potential (pathotype) of isolated Escherichia coli strains would be through a complete assessment of each strain for the presence of all known E. coli virulence factors. To accomplish this, an E. coli virulence factor DNA microarray composed of 105 DNA PCR amplicons printed on glass slides and arranged in eight subarrays corresponding to different E. coli pathotypes was developed. Fluorescently labeled genomic DNAs from E. coli strains representing known pathotypes were initially hybridized to the virulence gene microarrays for both chip optimization and validation. Hybridization pattern analysis with clinical isolates permitted a rapid assessment of their virulence attributes and determination of the pathogenic group to which they belonged. Virulence factors belonging to two different pathotypes were detected in one human E. coli isolate (strain H87-5406). The microarray was also tested for its ability to distinguish among phylogenetic groups of genes by using gene probes derived from the attaching-and-effacing locus (espA, espB, tir). After hybridization with these probes, we were able to distinguish E. coli strains harboring espA, espB, and tir sequences closely related to the gene sequences of an enterohemorrhagic strain (EDL933), a human enteropathogenic strain (E2348/69), or an animal enteropathogenic strain (RDEC-1). Our results show that the virulence factor microarray is a powerful tool for diagnosis-based studies and that the concept is useful for both gene quantitation and subtyping. Additionally, the multitude of virulence genes present on the microarray should greatly facilitate the detection of virulence genes acquired by horizontal transfer and the identification of emerging pathotypes.

Topics: Animals; Base Sequence; Carbocyanines; DNA, Bacterial; Escherichia coli; Fluorescent Dyes; Genes, Bacterial; Genetic Variation; Humans; Multigene Family; Oligonucleotide Array Sequence Analysis; Phylogeny; Virulence

We examined the fluorescence spectral properties of DNA oligomers, labeled with Cy3 or Cy5, when bound to quartz surfaces coated with metallic silver particles. Prior to binding of labeled DNA the surfaces were treated with polylysine or 3-aminopropyl triethoxysilane or were coated with avidin for binding of biotinylated oligomers. The fluorescence intensities were increased an average of 8-fold on these surfaces. Despite the increased emission intensity, the photostability of the labeled DNA was the same or higher on the silver-coated surfaces than on the uncoated slides. The time-integrated intensities, that is the area under the intensity plots with continuous illumination, increased an average of 6-fold. In all cases the lifetimes were dramatically shortened on the silver particles, indicating an over 100-fold increase in the radiative decay rates. These results suggest the use of substrates containing silver particles for increased sensitivity of DNA detection on DNA arrays.

Topics: Avidin; Base Sequence; Biotin; Carbocyanines; DNA; Oligonucleotides; Polylysine; Silver; Spectrometry, Fluorescence; Surface Properties; Time Factors

Topics: Base Pair Mismatch; Base Sequence; Binding Sites; Carbocyanines; Deoxyribonucleases, Type II Site-Specific; Heteroduplex Analysis; Nucleic Acid Heteroduplexes; Oligonucleotides

For visualization of proteins or nucleic acids, direct and indirect in situ fluorescence and absorption methods (immunohistochemistry and cytochemistry) have existed for many years. The authors describe a new experimental approach using microarray as a model to quantitatively compare both visualization methods. The spots obtained with the microarray robot had a progressive twofold decrease in concentrations and are used as objects with known amounts of DNA. Subsequent hybridization resulted in a direct fluorescence (DF) label or in hapten for indirect fluorescence (IF) and absorption. The results show that the image of the object in the IF method is larger than that in the DF method because of an edge effect, with stronger staining at the circumference. This leads to a higher plateau level and an 8- to 10-fold reduction in the detection threshold for IF compared with DF. These features are especially useful for one-color DNA-related microarray analysis, such as single nucleotide polymorphism, loss of heterozygosity, and mutation analysis, provided that the spots are not designed directly adjacent to each other, so that the edge effect is taken into account. The slope of the linear range for the IF method is much steeper than for the DF method, pointing to a narrow dynamic range in immunohistochemistry. It is noteworthy that the detection limit for absorption images after indirect immunoenzyme visualization is lower than for the DF images. The indirect immunohistochemistry semiquantitative absorption signal was at least similar compared with the DF fluorescence. In conclusion, an explanation for the difficulties experienced in quantitative immunohistochemistry is provided, and the data emphasize that in general, for daily pathology, semiquantitative patterns should suffice. Indirect labeling of DNA has useful characteristics for application in microarray analyses because of the large signal enhancement.

Topics: Carbocyanines; Diagnostic Imaging; Fluorescent Dyes; Immunoenzyme Techniques; In Situ Hybridization, Fluorescence; Oligonucleotide Array Sequence Analysis; Oligonucleotides; Robotics

Myosin V is a dimeric molecular motor that moves processively on actin, with the center of mass moving approximately 37 nanometers for each adenosine triphosphate hydrolyzed. We have labeled myosin V with a single fluorophore at different positions in the light-chain domain and measured the step size with a standard deviation of <1.5 nanometers, with 0.5-second temporal resolution, and observation times of minutes. The step size alternates between 37 + 2x nm and 37 - 2x, where x is the distance along the direction of motion between the dye and the midpoint between the two heads. These results strongly support a hand-over-hand model of motility, not an inchworm model.

Topics: Actin Cytoskeleton; Actins; Adenosine Triphosphate; Binding Sites; Calmodulin; Carbocyanines; Catalytic Domain; DNA; Fluorescence; Fluorescent Dyes; Kinetics; Mathematics; Microscopy, Fluorescence; Models, Biological; Molecular Motor Proteins; Myosin Light Chains; Myosin Type V; Protein Structure, Tertiary; Rhodamines

Ischemia-reperfusion injury is a major complication occurring in acute myocardial infarction, cardiopulmonary bypass surgery, and heart transplantation. The aim of this study was to identify proteins that were involved in ischemia-reperfusion injury using fluorescence two-dimensional difference gel electrophoresis. We compared the 100,000 x g precipitate fractions of normal, ischemic and ischemia-reperfused rat hearts and detected six spots which changed more than two-fold in expression level and two additional spots related to these spots. Using peptide mass fingerprinting by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, we identified five of these spots as protein disulfide isomerase A3 (PDA3), one as 60 kDa heat shock protein (HSP60) and two as elongation factor Tu (EF-Tu). HSP60 was increased during ischemia and decreased to normal expression level after reperfusion. EF-Tu was increased in ischemia but not decreased by reperfusion. We also found that several protein spots of PDA3 shifted towards a higher isoelectric point in ischemia and ischemia-reperfusion. Our data strongly suggested that PDA3 underwent dephosphorylation during ischemia and reperfusion and serine 343 of PDA3 was one of the phosphorylation sites.

Topics: Animals; Binding Sites; Carbocyanines; Chaperonin 60; Databases as Topic; Electrophoresis, Gel, Two-Dimensional; Fluorescent Dyes; Image Processing, Computer-Assisted; Ischemia; Male; Microscopy, Fluorescence; Myocardium; Peptide Elongation Factor Tu; Phosphorylation; Protein Disulfide-Isomerases; Proteome; Rats; Rats, Wistar; Reperfusion Injury; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Subcellular Fractions

One- and two-photon fluorescence resonance energy transfer (FRET) microscopy, using different bandwidth emission filters and a novel spectral spillover correction algorithm (PFRET algorithm), provides the basis for a quantitative approach to measure receptor clustering in endocytic membranes. Emission filters with wider bandwidth allow for an increased FRET signal and corresponding spillover. Treatment with the PFRET correction algorithm results in increasing correction levels and comparable energy transfer efficiency (E%) values, thus validating our algorithm-based approach. The relationship between E% and acceptor and donor levels and donor:acceptor (D:A) ratio is used to characterize the distribution of receptor-ligand complexes in endocytic membranes. In addition to the standard test for clustering (E%'s independence from acceptor levels), we describe a second parameter: the negative dependence of E% on increasing donor levels and D:A ratio. A donor geometric exclusion hypothesis is proposed to explain this phenomenon. One- and two-photon FRET microscopy assays show that polymeric IgA-receptor-ligand complexes are organized in clusters within apical endocytic membranes of polarized Madin-Darby canine kidney cells.

Topics: Algorithms; Animals; Artifacts; Carbocyanines; Cell Membrane; Cluster Analysis; Dogs; Endocytosis; Endosomes; Energy Transfer; Fluorescence Resonance Energy Transfer; Hydrazines; Image Enhancement; Kidney; Microscopy, Confocal; Microscopy, Fluorescence, Multiphoton; Rabbits; Receptors, Fc; Recombinant Proteins; Tissue Distribution

Observations of cells or tissues with fluorescence microscopy can provide unique insights into cellular physiology and structure. Such information may reveal the pathological state of a tissue to the physician or information on cytoskeletal dynamics to the research scientist. However, problems of overlapping spectra, low signal, and light scatter impose serious limitations on what can be achieved in practice with fluorescence microscopy. These problems can be addressed in part by the development of new imaging modalities that make maximum use of the information present in the fluorescence signal. We describe the application of a new technology to the study of standard histological pathology specimens: a multiphoton excitation fluorescence microscope that incorporates a novel, photon-counting detector that measures the excited-state lifetimes of fluorescent probes. In initial investigations, we have applied this system to the observation of C. elegans embryos and primate histology specimens, with the objective of identifying potentially diagnostic signatures. Our findings demonstrate that lifetime multiphoton microscopy has considerable potential as a diagnostic tool for pathological investigations.

Topics: Animals; Caenorhabditis elegans; Carbocyanines; Cell Nucleus; Cytoplasm; Cytoskeletal Proteins; DNA; Feasibility Studies; Indoles; Microscopy, Confocal; Microscopy, Fluorescence, Multiphoton

Fluorescence resonance energy transfer (FRET) was used to monitor interactions between Cy3-labeled plasmid DNA and NBD-labeled cationic liposomes. FRET data show that binding of cationic liposomes to DNA occurs immediately upon mixing (within 1 min), but FRET efficiencies do not stabilize for 1-5 h. The time allowed for complex formation has effects on in vitro luciferase transfection efficiencies of DOPE-based lipoplexes; i.e., lipoplexes prepared with a 1-h incubation have much higher transfection efficiencies than samples with 1-min or 5-h incubations. The molar charge ratio of DOTAP to negatively charged phosphates in the DNA (DOTAP+/DNA-) also affected the interaction between liposomes and plasmid DNA, and interactions stabilized more rapidly at higher charge ratios. Lipoplexes formulated with DOPE were more resistant to high ionic strength than complexes formulated with cholesterol. Taken together, our data demonstrate that lipid-DNA interactions and in vitro transfection efficiencies are strongly affected by the time allowed for complex formation. This effect is especially evident in DOPE-based lipoplexes, and suggests that the time allowed for lipoplex formation is a parameter that should be carefully controlled in future studies.

Topics: Carbocyanines; DNA; Fatty Acids, Monounsaturated; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Lipids; Liposomes; Osmolar Concentration; Particle Size; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Sodium Chloride; Transfection

The ability to extract meaningful information from transcriptome technologies such as cDNA microarrays relies on the precision, sensitivity and reproducibility of the measured values for a given gene across multiple samples. Given the lack of a 'gold standard' for the production of microarrays using current technologies, there is a high degree of variation in the quality of data derived from microarray experiments. Poor reproducibility not only adds to the cost of a given study but also leads to data sets that are difficult to interpret. For glass slide DNA microarrays, much of this variation is introduced systematically, during the spotting, or deposition, of the DNA onto the slide surface. In order to reduce this type of systematic variation we tested spotting solutions containing different detergent additives in the presence of one of two different denaturants and determined their effect on spot quality. We show that spotting cDNA in a solution consisting of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in the presence of formamide or dimethyl sulfoxide yields spots of superior quality in terms of morphology, size homogeneity and signal reproducibility, as well as overall intensity, when used with popular, commercially available slides.

Topics: Carbocyanines; Cholic Acids; Dimethyl Sulfoxide; DNA, Complementary; DNA, Fungal; Formamides; Gene Expression Regulation, Fungal; Oligonucleotide Array Sequence Analysis; Open Reading Frames; Reproducibility of Results; Saccharomyces cerevisiae; Sensitivity and Specificity; Solutions

Fluorescent and haptenized, monofunctionally binding platinum compounds are increasingly used for chemically labeling nucleic acids for hybridization detection purposes. Here we analyze in detail the effect of labeling density of the cyanin-3 and -5 platinum DNA adducts on fluorescence and thermal stability. We also analyzed the kinetics of the reaction of the cyanin platinum compounds with DNA. The data provided are important for the design of optimal platinum DNA labeling and hybridization conditions for fluorescence hybridization applications.

Topics: Animals; Carbocyanines; Diethylamines; Dinitrophenols; DNA; Fluorescence; Kinetics; Mice; Molecular Structure; Nucleic Acid Denaturation; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Platinum; Spectrometry, Fluorescence; Temperature; Time Factors

Fluorescence polarization (FP) is an established technique for the study of biological interactions and is frequently used in the high-throughput screening (HTS) of potential new drug targets. This work describes the miniaturization of FP receptor assays to 1536-well formats for use in HTS. The FP assays were initially developed in 384-well microplates using CyDye-labeled nonpeptide and peptide ligands. Receptor expression levels varied from approximately 1 to 10 pmols receptor per mg protein, and ligand concentrations were in the 0.5- to 1.0-nM range. The FP assays were successfully miniaturized to 1536-well formats using Cy3B-labeled ligands, significantly reducing reagent consumption, particularly the receptor source, without compromising assay reliability. Z' factor values determined for the FP receptor assays in both 384- and 1536-well formats were found to be > 0.5, indicating the assays to be robust, reliable, and suitable for HTS purposes.

Topics: Animals; Atropine; Binding, Competitive; Carbocyanines; CHO Cells; Cricetinae; Fluorescence Polarization; Fluorescent Dyes; Humans; Kinetics; Miniaturization; Neurokinin A; Peptides; Pirenzepine; Receptors, Cell Surface; Receptors, Muscarinic; Receptors, Neurokinin-1; Sensitivity and Specificity; Substance P

The restriction display PCR is a useful technique for studying the diversity of gene expression. This method involves ligating the digested genes with adapters and amplifying the gene fragments by PCR using universal and selective primers. In this study, we improved this restriction display PCR method by using Cy3-UP, a fluorescently labelled universal primer, in place of Cy3-dCTP in sample-labelling for DNA microarray. The results show that this new method increases significantly the sensitivity of the assay, and will have a wide application in the DNA microarray field.

Topics: Carbocyanines; DNA Primers; DNA, Viral; Fluorescent Dyes; HIV-1; Humans; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Specimen Handling

A data anomaly was observed that affected the uniformity and reproducibility of fluorescent signal across DNA microarrays. Results from experimental sets designed to identify potential causes (from microarray production to array scanning) indicated that the anomaly was linked to a batch process; further work allowed us to localize the effect to the posthybridization array stringency washes. Ozone levels were monitored and highly correlated with the batch effect. Controlled exposures of microarrays to ozone confirmed this factor as the root cause, and we present data that show susceptibility of a class of cyanine dyes (e.g., Cy5, Alexa 647) to ozone levels as low as 5-10 ppb for periods as short as 10-30 s. Other cyanine dyes (e.g., Cy3, Alexa 555) were not significantly affected until higher ozone levels (> 100 ppb). To address this environmental effect, laboratory ozone levels should be kept below 2 ppb (e.g., with filters in HVAC) to achieve high quality microarray data.

Topics: Artifacts; Atmosphere; Carbocyanines; Desiccation; Fluorescence; Oligonucleotide Array Sequence Analysis; Ozone; Quality Control; Reproducibility of Results

Resonance energy transfer (RET) is typically limited to distances below 60 A, which can be too short for some biomedical assays. We examined a new method for increasing the RET distances by placing donor- and acceptor-labeled DNA oligomers between two slides coated with metallic silver particles. A N,N'-(dipropyl)-tetramethylindocarbocyanine donor and a N,N'-(dipropyl)-tetramethylindodicarbocyanine acceptor were covalently bound to opposite 5' ends of complementary 23 base pair DNA oligomers. The transfer efficiency was 25% in the absence of silver particles or if only one slide was silvered, and it increased to an average value near 64% between two silvered slides. The average value of the Forster distance increased from 58 to 77 A. The energy transfer data were analyzed with a model assuming two populations of donor-acceptor pairs: unaffected and affected by silver island films. In an affected fraction of about 28%, the apparent energy transfer efficiency is near 87% and the Forster distance increases to 119 A. These results suggest the use of metallic silver particles to increase the distances over which RET occurs in biomedical and biotechnology assays.

Topics: Carbocyanines; DNA; Fluorescence Resonance Energy Transfer; Quartz; Silver; Staining and Labeling

Connexin 36 (Cx36) is a channel-forming protein found in the membranes of apposed cells, forming the hexameric hemichannels of intercellular gap junction channels. It localizes to certain neurons in various regions of the brain including the retina. We characterized the expression pattern of neuronal Cx36 in the guinea pig retina by immunocytochemistry using specific antisera against Cx36 and green/red cone opsin or recoverin. Strong Cx36 immunoreactivity was visible in the ON sublamina of the inner plexiform layer and in the outer plexiform layer, as punctate labelling patterns. Double-labelling experiments with antibody directed against Cx36 and green/red cone opsin or recoverin showed that strong clustered Cx36 immunoreactivity localized to the axon terminals of cone or close to rod photoreceptors. By electron microscopy, Cx36 immunoreactivity was visible in the gap junctions as well as in the cytoplasmic matrices of both sides of cone photoreceptors. In the gap junctions between cone and rod photoreceptors, Cx36 immunoreactivity was only visible in the cytoplasmic matrices of cone photoreceptors. These results clearly indicate that Cx36 forms homologous gap junctions between neighbouring cone photoreceptors, and forms heterologous gap junctions between cone and rod photoreceptors in guinea pig retina. This focal location of Cx36 at the terminals of the photoreceptor suggests that rod photoreceptors can transmit rod signals to the pedicle of a neighbouring cone photoreceptor via Cx36, and that the cone in turn signals to corresponding ganglion cells via ON and OFF cone bipolar cells.

Topics: Animals; Blotting, Western; Calbindins; Calcium-Binding Proteins; Carbocyanines; Cells, Cultured; Connexins; Eye Proteins; Female; Gap Junction delta-2 Protein; Gap Junctions; Guinea Pigs; Hippocalcin; Immunohistochemistry; Lipoproteins; Male; Microscopy, Confocal; Microscopy, Immunoelectron; Nerve Tissue Proteins; Recoverin; Retina; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; S100 Calcium Binding Protein G

Maternal care during the first week of postnatal life influences hippocampal development and function (Liu et al., 2000; Nature Neurosci., 3, 799-806). Offspring reared by mothers who exhibit increased levels of pup licking/grooming (LG) show increased hippocampal synaptic density and enhanced spatial learning and memory. Using 5-bromo-2'-deoxyuridine (BrdU), a thymidine analogue incorporated into cells during DNA synthesis, we examined the effects of early maternal care on hippocampal cell proliferation and neuronal survival in the rat. Twenty-four hours following injection on day 7 of life (P7) there were no differences in BrdU labelling in the offspring of high- compared with low-LG mothers, suggesting no maternal effect on the rate of proliferation at this age. However, 14 and 83 days following injection (P21 and P90), the offspring of high-LG mothers had significantly more surviving BrdU-labelled cells and BrdU-NeuN+-colabelled neurons in the dentate gyrus subgranular zone and granule cell layer. At P21, the offspring of high-LG mothers showed increased protein expression of basic fibroblast growth factor and significantly decreased levels of pyknosis. These findings suggest an influence of maternal care on neuronal survival in the hippocampus. Conversely, at the same time point there was a significantly higher level of hippocampal glial fibrillary acidic protein expression in the offspring of low-LG mothers. These findings emphasize the importance of early maternal care for hippocampal development.

Topics: Animals; Animals, Newborn; Behavior, Animal; Blotting, Western; Bromodeoxyuridine; Carbocyanines; Cell Count; Cell Survival; Female; Fibroblast Growth Factors; Glial Fibrillary Acidic Protein; Hippocampus; Immunohistochemistry; Male; Maternal Behavior; Neurons; Oxazines; Phosphopyruvate Hydratase; Rats; Rats, Long-Evans; Time Factors

A highly efficient and specific small interfering (siRNA) (PsiR4) for the serine/threonine kinase Pim-1 has been generated that silences the expression of a Pim1-green fluorescent protein (GFP) fusion gene at low nanomolar concentrations (approximately 5 nM). Only one of four siRNAs tested against Pim-1 had high potency, whereas the three other siRNAs were completely inefficient up to a concentration of 100 nM. PsiR4 was labeled with Cy3 at the 5' -end of the sense strand to investigate cellular uptake and localization in living COS-7 and F-11 cells. This modification has only minor effects on the potency of PsiR4 to inhibit Pim1-GFP. Cellular uptake of the Cy3-labeled siRNA by lipofection was observed in more than 90% of the cells and reaches a plateau 4-6 hours after transfection. Cotransfection studies with low PsiR4-Cy3 concentrations demonstrated that most cells that still expressed Pim1-GFP did not show siRNA uptake. Localization studies with PsiR4-Cy3 in the neuronal hybridoma cell line F-11 displayed a dotted, perinuclear accumulation of siRNAs. Moreover, cells with neuritelike structures contain PsiR4 in this cellular compartment.

Topics: Animals; Base Sequence; Carbocyanines; Chlorocebus aethiops; COS Cells; Fluorescent Dyes; Gene Silencing; Green Fluorescent Proteins; Luminescent Proteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-pim-1; Recombinant Fusion Proteins; RNA, Small Interfering; Transfection

A variety of electronic DNA array devices and techniques have been developed that allow electric field enhanced hybridization to be carried out under special low-conductance conditions. These devices include both planar microelectronic DNA array/chip devices as well as electronic microtiter plate-like devices. Such "active" electronic devices are able to provide controlled electric (electrophoretic) fields that serve as a driving force to move and concentrate nucleic acid molecules (DNA/RNA) to selected microlocation test-sites on the device. In addition to ionic strength, pH, temperature and other agents, the electric field provides another controllable parameter that can affect and enhance DNA hybridization. With regard to the planar microelectronic array devices, special low-conductance buffers were developed in order to maintain rapid transport of DNA molecules and to facilitate hybridization within the constrained low current and voltage ranges for this type of device. With regard to electronic microtiter plate type devices (which do not have the low current/voltage constraints), the use of mixed buffers (low conductance upper chamber/high conductance lower chamber) can be used in a unique fashion to create favorable hybridization conditions in a microzone within the test site location. Both types of devices allow DNA molecules to be rapidly and selectively hybridized at the array test sites under conditions where the DNA in the bulk solution can remain substantially denatured.

Topics: Buffers; Carbocyanines; DNA; DNA Probes; Electrons; Electrophoresis, Capillary; Fluorescent Dyes; Hydrogel, Polyethylene Glycol Dimethacrylate; Indicators and Reagents; Nucleic Acid Hybridization; Oligodeoxyribonucleotides; Oligonucleotide Array Sequence Analysis; Streptavidin

Cholinergic basal forebrain neurons (CBFN) expressing the low-affinity neurotrophin receptor p75 (p75(NTR)) were previously selectively labeled in vivo with carbocyanine 3 (Cy3)-tagged anti-p75(NTR), but the applied 192IgG-conjugates recognized p75(NTR) only in rat. The antibody ME 20.4 raised against human p75(NTR) had been shown to cross-react with the receptor in monkey, raccoon, sheep, cat, dog, pig and rabbit. Hence, for in vivo labeling of rabbit CBFN in the present study, ME 20.4 was fluorochromated with Cy3-N-hydroxysuccinimide ester and purified Cy3-ME 20.4 was injected intracerebroventricularly. Two days post-injection, clusters of Cy3-ME 20.4 were found in CBFN displaying choline acetyltrans-ferase-immunoreactivity. Following photoconversion, electron microscopy revealed fluorochromated antibodies in secondary lysosomes. In conclusion, Cy3-ME 20.4 might become an appropriate marker for CBFN in live and fixed tissues of various mammalian species.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Carbocyanines; Cholinergic Fibers; Female; Fluorescent Dyes; Immunotoxins; Injections, Intraventricular; Male; Microscopy, Confocal; N-Glycosyl Hydrolases; Neurons; Prosencephalon; Rabbits; Ribosome Inactivating Proteins, Type 1; Saporins

We report high time-resolution measurements of photon statistics from pairs of dye molecules coupled by fluorescence resonance energy transfer (FRET). In addition to quantum-optical photon antibunching, we observe photon bunching on a time scale of several nanoseconds. We show by numerical simulation that configuration fluctuations in the coupled fluorophore system could account for minor deviations of our data from predictions of basic Förster theory. With further characterization we believe that FRET photon statistics could provide a unique tool for studying DNA mechanics on time scales from 10(-9)-10(-3) s.

Topics: Carbocyanines; DNA; Energy Transfer; Fluorescent Dyes; Models, Chemical; Monte Carlo Method; Proteins; Spectrometry, Fluorescence

Normalization of the data of cDNA microarray is an obligatory step during microarray experiments due to the relatively frequent non-specific errors. Generally, normalization of microarray data is based on the null hypothesis and variance model. In the Yang's model (Yang et al., 2001), at least two types of noises are included. The one is additive noise and the other is multiplicative noise. Usually, background is considered as one of additive noise to the signal and the variation between the signal pixels is the representative multiplicative noise. In this study, the relation between the signal (spot intensity minus background intensity) and background was observed and the influence of background on normalization as a representative additive factor was investigated. Although the relation has not been considered as a factor affecting the normalization, it could improve the accuracy of microarray data when the normalization was carried out considering signal/background ratio. The background dependent normalization decreased the number of genes whose expression levels were changed significantly and it could make their distribution more consistent through the whole range of signal intensities. In this study, printing pin dependent normalization was also carried out regarding the printing pin as a representative multiplicative noise. It improved the distribution of spots in the Cy3-Cy5 scatter plot, but its effect was slight. These studies suggest that there are some influences of the signals on the local backgrounds and they must be considered for the normalization of cDNA microarray data.

Topics: Carbocyanines; DNA, Complementary; Gene Expression Profiling; Linear Models; Oligonucleotide Array Sequence Analysis; Reference Standards; Reproducibility of Results; Sensitivity and Specificity

The kinetic mechanism of transfer of the homotetrameric Escherichia coli SSB protein between ssDNA molecules was studied using stopped-flow experiments. Dissociation of SSB from the donor ssDNA was monitored after addition of a large excess of unlabeled acceptor ssDNA by using either SSB tryptophan fluorescence or the fluorescence of a ssDNA labeled with an extrinsic fluorophore [fluorescein (F) or Cy3]. The dominant pathway for SSB dissociation occurs by a "direct transfer" mechanism in which an intermediate composed of two DNA molecules bound to one SSB tetramer forms transiently prior to the release of the acceptor DNA. When an initial 1:1 SSB-ssDNA complex is formed with (dT)(70) in the fully wrapped (SSB)(65) mode so that all four SSB subunits are bound to (dT)(70), the formation of the ternary intermediate complex occurs slowly with an apparent bimolecular rate constant, k(2,app), ranging from 1.2 x 10(3) M(-1) s(-1) (0.2 M NaCl) to approximately 5.1 x 10(3) M(-1) s(-1) (0.4 M NaBr), and this rate limits the overall rate of the transfer reaction (pH 8.1, 25 degrees C). These rate constants are approximately 7 x 10(5)- and approximately 7 x 10(4)-fold lower, respectively, than those measured for binding of the same ssDNA to an unligated SSB tetramer to form a singly ligated complex. However, when an initial SSB-ssDNA complex is formed with (dT)(35) so that only two SSB subunits interact with the DNA in an (SSB)(35) complex, the formation of the ternary intermediate occurs much faster with a k(2,app) ranging from >6.3 x 10(7) M(-1) s(-1) (0.2 M NaCl) to 2.6 x 10(7) M(-1) s(-1) (0.4 M NaBr). For these experiments, the rate of dissociation of the donor ssDNA determines the overall rate of the transfer reaction. Hence, an SSB tetramer can be transferred from one ssDNA molecule to another without proceeding through a free protein intermediate, and the rate of transfer is determined by the availability of free DNA binding sites within the initial SSB-ssDNA donor complex. Such a mechanism may be used to recycle SSB tetramers between old and newly formed ssDNA regions during lagging strand DNA replication.

Topics: Binding Sites; Carbocyanines; DNA-Binding Proteins; DNA, Single-Stranded; Escherichia coli Proteins; Fluorescent Dyes; Kinetics; Models, Chemical; Protein Binding; Protein Subunits; Protein Transport; Spectrometry, Fluorescence; Thymidine

With the advent of protein and antibody microarray technology several different coatings and protocols have been published, which may be broadly divided into two types: gel-coated surfaces and plain non-gel-coated glass or plastic surfaces, some with chemical groups attached. We have screened 11 different array surfaces of both types and compared them with respect to their detection limit, inter- and intrachip variation, and storage characteristics. Five different antibodies were immobilized onto each type of microarray support, with total protein concentrations ranging from 40 fmol to 25 amol per spot. From these results, it was seen that some antibodies were more suited for use on antibody arrays. All measurements were performed in quadruplicate, and the results revealed high signal uniformity and reproducibility of most plain glass and plastic slides. Lower detection limits were obtained with polyacrylamide-coated slides, making them more suitable for the detection of very low concentrations of antigen. All microarray coatings could be stored for a period of 8 weeks; however, improved results were seen after 2 weeks of storage. In conclusion, the results indicate the need to test each antibody to be used on an antibody array and to select the microarray coating based on experimental requirements.

Topics: Acrylic Resins; Antibodies; Carbocyanines; Drug Stability; Drug Storage; Fibrinogen; Glass; Humans; Hydrogel, Polyethylene Glycol Dimethacrylate; Polylysine; Polymers; Protein Array Analysis; Reproducibility of Results; Sensitivity and Specificity; Surface Properties; Time Factors

We have developed a method for the parallel analysis of multiple CpG sites in genomic DNA for their state of methylation. Hypermethylation of CpG islands within the promoters and 5' exons of genes has been found to be a mechanism of transcriptional inactivation associated with a variety of tumors. The method that we developed relies on the differential reactivity of methylated and unmethylated cytosines with sodium bisulfite, which exclusively converts unmethylated cytosines to deoxyuracils. The resulting sequence changes are determined with single-nucleotide resolution by hybridization to an oligonucleotide array. Cohybridization with a reference sample containing a different label provides an internal standard for assessment of methylation state. This method provides advantages in parallelism over existing methods of methylation analysis. We have demonstrated this technique with a region from the promoter of the tumor suppressor gene p16, which is hypermethylated in many cancers.

Topics: Base Sequence; Carbocyanines; Cloning, Molecular; CpG Islands; Cytosine; DNA Methylation; DNA Primers; DNA, Neoplasm; Genes, Tumor Suppressor; Humans; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Reference Standards; Sulfites; Tumor Cells, Cultured

We have developed a method for simultaneous recording of high-resolution topography and cell surface fluorescence in a single scan which we call scanning surface confocal microscopy. The resolution of the system allows imaging of individual fluorescent particles in the nanometer range on fixed or live cells. We used this technique to record the interaction of single virus-like particles with the cell surface and demonstrated that single particles sink into the membrane in invaginations reminiscent of caveolae or pinocytic vesicles. This method provides a technique for elucidating the interaction of individual viruses and other nanoparticles, such as gene therapy vectors, with target cells. Furthermore, this technique should find widespread application for studying the relationship of fluorescently tagged molecules with components of the cell plasma membrane.

Topics: Animals; Capsid; Carbocyanines; Cell Membrane; Chlorocebus aethiops; COS Cells; Equipment Design; Fluorescent Dyes; Genes, Synthetic; Microscopy, Confocal; Microscopy, Fluorescence; Nanotechnology; Particle Size; Surface Properties

An immunohistochemical technique was developed to visualize nitric oxide synthase (NOS)-immunopositive neurons in fresh-frozen tissue sections of rat brain for laser capture microdissection (LCM) and mRNA analysis. The effect of tissue fixation and the choice of fluorophore were investigated. Here we describe a rapid immunofluorescence protocol that allows the processing of fresh-frozen tissue sections within eight minutes and subsequent mRNA extraction and real-time PCR from pools of 20 NOS-immunopositive LCM neurons. The cellular complement of a subset of ionotropic glutamate receptors, specifically N-methyl-D-aspartate receptor subunit mRNAs, was examined because these receptor complexes are thought to mediate the effects of fast and slow glutamate excitotoxicity. Real-time PCR data revealed that striatal NOS interneurons express the mRNAs encoding NR1, NR2A, NR2B, and NR2D but not NR2C. These LCM mRNA data are consistent with previous in situ hybridization studies and demonstrate the utility of rapid immuno-LCM with real-time quantitative PCR for the study of mRNA abundance in discrete populations of neurons within the mammalian brain.

Topics: Animals; Carbocyanines; Cell Separation; Computer Systems; Corpus Striatum; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Frozen Sections; Hydrazines; Interneurons; Isoenzymes; Lasers; Nerve Tissue Proteins; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Rhodamines; RNA, Messenger; Specimen Handling; Time Factors

An image analysis procedure was developed for bacterial cells after staining with the DNA-intercalating dye 4'-6-diamidino-2-phenylindole (DAPI), and after in situ hybridization with Cy3-labeled, rRNA-targeted oligonucleotide probes. DAPI- and Cy3-images were captured separately from the same scenery with a cooled digital video camera with three CCD chips for the basic colors red (R), green (G) and blue (B). Using the appropriate filter sets, images of DAPI-stained cells were captured with the red channel shut down, while Cy3-stained cells were captured with the green and blue channels shut down. DAPI images and Cy3 images were subsequently merged to produce virtual color (RGB)-images. Processing of all color channels allowed to specifically enumerate DAPI-stained and hybridized bacteria, to measure their cell sizes, to subsequently calculate their biovolumes and to estimate their biomass. Using this procedure, significant differences were detected in bacterial populations in food resources, digestive tract and cast of the earthworm L. terrestris L. In leaves, bacteria were on average ten times more abundant and two times larger than in soil. In the digestive tract of L. terrestris, however, numbers and volumes of bacteria were comparable to those in soil indicating the disruption of cells originating from leaves before arriving in the foregut. Passage through the digestive tract of L. terrestris significantly reduced bacterial populations belonging to the alpha-, beta- and gamma-subdivisions of Proteobacteria. While these populations did not recover during incubation of cast, populations of the delta-subdivision of Proteobacteria and the Cytophaga-Flavobacterium cluster of the CFB phylum increased in cast. These results suggest a large impact of passage through the digestive tract of L. terrestris on bacterial community structure and demonstrate the usefulness of our image analysis procedure for the determination of cell sizes and biovolumes and thus biomass of specific bacterial populations in different terrestrial habitats.

Topics: Animals; Bacteria; Carbocyanines; Ecosystem; Feces; Fluorescent Dyes; Image Processing, Computer-Assisted; In Situ Hybridization; Indoles; Microscopy, Atomic Force; Microscopy, Fluorescence; Oligochaeta; Soil; Soil Microbiology; Stomach

We compared the detection of bacteria and archaea in the coastal North Sea and at Monterey Bay, Calif., after fluorescence in situ hybridization (FISH) either with rRNA-targeted oligonucleotide probes monolabeled with the cyanin dye Cy3 (oligoFISH) or with fluorescein-labeled polyribonucleotide probes (polyFISH). During an annual cycle in German Bight surface waters, the percentages of bacteria visualized by polyFISH (annual mean, 77% of total counts) were significantly higher than those detected by oligoFISH (53%). The fraction of total bacteria visualized by oligoFISH declined during winter, whereas cell numbers determined by polyFISH remained constant throughout the year. Depth profiles from Monterey Bay showed large differences in the fraction of bacterial cells visualized by polyFISH and oligoFISH in the deeper water layers irrespective of the season. Image-analyzed microscopy indicated that the superior detection of cells by polyFISH with fluorescein-labeled probes in bacterioplankton samples was less a consequence of higher absolute fluorescence intensities but was rather related to quasi-linear bleaching dynamics and to a higher signal-to-background ratio. The relative abundances of archaea in North Sea and Monterey Bay spring samples as determined by oligoFISH were on average higher than those determined by polyFISH. However, simultaneous hybridizations with oligonucleotide probes for bacteria and archaea suggested that the oligoFISH probe ARCH915 unspecifically stained a population of bacteria. Using either FISH technique, blooms of archaea were observed in North Sea surface waters during the spring and summer months. Marine group II archaea (Euryarchaeota) reached >30% of total picoplankton abundances, as determined by polyFISH. We suggest that studies of pelagic microbial community structure using oligoFISH with monolabeled probes should focus on environments that yield detections > or =70% of total cell counts, e.g., coastal surface waters during spring and summer.

Topics: Archaea; Bacteria; Carbocyanines; Fluorescein; Fluorescent Dyes; Image Processing, Computer-Assisted; In Situ Hybridization, Fluorescence; Oligonucleotide Probes; Polyribonucleotides; RNA, Ribosomal; Seawater

We examined whether CO2 affects colonic Na+ absorption by endosome recycling of the Na+/H+ exchanger NHE3.. Rat distal colon segments exposed to various acid-base conditions were examined by transmission electron microscopy at 27,500x magnification and subapical vesicles quantified. Immunocytochemistry was used to identify vesicular NHE3. Endocytosis was tested for by observing internalization of apical membrane labeled with fluorescein isothiocyanate-phytohemagglutinin and Cy-3-NHE3 antibody using confocal microscopy. The effects of mucosal 5-(N,N-dimethyl)-amiloride (DMA), which inhibits NHE2 and/or NHE3, and wortmannin, which inhibits phosphatidylinositol 3-kinase, on CO2-stimulated Na+ absorption were measured in the Ussing chamber.. The number of (coated and uncoated) subapical vesicles in epithelial cells was specifically and inversely related to net colonic Na+ absorption and PCO2. Immunoperoxidase labeling localized NHE3 on microvilli and vesicle membranes. Under the confocal microscope, a fluorescent band along apical membranes at PCO2 70 mm Hg became a subapical haze at PCO2 21 mm Hg. This pattern was not affected by carbonic anhydrase inhibition or when pH or [HCO3-] was changed, but PCO2 was held constant. DMA inhibition indicated that NHE3 mediates CO2-stimulated Na+ absorption. Wortmannin inhibited CO2-stimulated vesicle movement (exocytosis) and Na+ absorption.. CO2 affects Na+ absorption in rat distal colon epithelium in part by modulating the movement of NHE3-containing vesicles to and from the apical membrane.

Topics: Acid-Base Equilibrium; Androstadienes; Animals; Carbocyanines; Carbon Dioxide; Colon; Cytoplasmic Vesicles; Endocytosis; Enzyme Inhibitors; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Intestinal Absorption; Male; Phytohemagglutinins; Rats; Rats, Sprague-Dawley; Sodium; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers; Wortmannin

Analysis of complex biochemical processes at the level of the proteome requires methods that quantitatively solubilize cytosolic and membrane bound proteins yet are compatible with isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, it is often necessary to employ several highly sensitive detection methods to identify key proteins that are modified or exhibit a change in expression levels in response to a given experimental stimulus or condition. Methods were developed that efficiently extract tissues or lyse cultured cells and quantitatively solubilize proteins in a single step without the need to shear nucleic acids. These approaches utilize urea, thiourea, a mixture of detergents, low levels of an ampholyte blend, reductant and a combination of alcohols. To aid in the detection of low abundance proteins and the accurate identification of specific proteins of interest in these samples, two approaches were pursued. In one, proteins are transferred from two-dimensional (2-D) gels to blot membranes. Proteins are then detected by staining with SYPRO Ruby and the resulting 2-D protein pattern is captured using a charge-coupled device (CCD) camera. The blots are then probed with antibodies directed against the protein(s) or functionalities of interest. The resulting chemiluminescent blot image is also generated with the CCD camera and the fluorescent SYPRO Ruby image is recaptured again without moving the membrane. It is thereby possible to generate a direct image overlay of the blot pattern on that of the stained protein pattern. This approach significantly aids in the accurate identification of the dye-stained protein that is detected by the specific antibody. In addition to detecting protein post-gel transfer, a second approach utilizes protein samples labeled with fluorescent dyes prior to 2-D electrophoresis in an effort to increase the sensitivity of protein detection and to facilitate protein quantitation. It is also possible to stain the blots with different dyes and overlay these images as well. Using these approaches, it is possible to perform more rapid and accurate comparative analyses and proteomic, post-gel characterization of proteins of interest than using comparative image analysis of multiple gels.

Topics: Blotting, Western; Carbocyanines; Electrophoresis, Gel, Two-Dimensional; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Proteome; Solubility; U937 Cells

Excitotoxic lesions in the magnocellular nucleus basalis (MBN) lead to a significant damage of cholinergic neurons concomitant with increased amyloid precursor protein (APP) expression in the cerebral cortex. However, the sensitivity of non-cholinergic neurons to excitotoxicity, and changes of APP expression in the damaged MBN are still elusive. Hence, we performed multiple-labeling immunocytochemistry for choline-acetyltransferase (ChAT), neuron-specific nuclear protein (NeuN) and APP 4, 24, and 48 h after NMDA infusion in the MBN. Whereas all cholinergic neurons were immunoreactive for NeuN, this neuronal marker also labeled a population of ChAT-immunonegative non-cholinergic neurons. Both neuron populations exhibited a similar degree of sensitivity to NMDA excitotoxicity that became evident as early as 4 h post-lesion. Cholinergic MBN neurons showed abundant APP immunoreactivity (approximately 90%), while only a fraction (approximately 20-30%) of non-cholinergic neurons expressed the protein. Remarkably, cholinergic but not non-cholinergic neurons retained their APP immunoreactivity after NMDA infusion. In conclusion, cholinergic MBN neurons are not preferentially sensitive to short-term excitotoxicity, but are one of the major sources of APP in the basal forebrain.

Topics: Acetylcholine; Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Basal Nucleus of Meynert; Biomarkers; Carbocyanines; Cell Count; Choline O-Acetyltransferase; Cholinergic Fibers; Down-Regulation; Fluorescent Dyes; Immunoglobulin G; Immunohistochemistry; Male; N-Methylaspartate; Neurons; Neurotoxins; Nuclear Proteins; Rats; Rats, Wistar; Receptor, Nerve Growth Factor

Fluorescence resonance energy transfer studies allow to determine global shape properties of nucleic acids and nucleoprotein complexes. In many DNA-protein complexes, the DNA is more or less bent and the degree of bending can be obtained by FRET. For example, the DNA in complex with the integration host factor (IHF) is kinked by approximately 160 degrees building a U-shaped structure. The two DNA helix ends come close to one another in space in a distance range easily measurable by FRET. The global DNA structure of this complex can be mimicked by introducing two regions with unpaired bases ('bulges') into the DNA each producing a sharp kink of approximately 80 degrees. These U-shaped DNA constructs were used to measure the electrostatic interaction of the two nearly parallel negatively charged DNA helix arms. The electrostatic repulsion between the helix arms, and as a consequence their distance, decreases with growing salt concentration of mono- or divalent cations. This experimental approach also allows the sensitive study of the local structure of DNA sequences positioned between the two bulges.

Topics: Acrylic Resins; Carbocyanines; DNA; Fluorescein; Nucleic Acid Conformation; Protein Binding; Spectrometry, Fluorescence

The use of microarrays to monitor gene expression has become a standard research tool at both academic and industrial research institutions. Quality control of common printing defects during DNA deposition onto glass substrates is critical to maintaining data integrity and preventing the needless consumption of precious RNA, labeling reagents, and time. Here we demonstrate a nondestructive method for monitoring the quality of every spot on every chip of a microarray production run. We have identified many common manufacturing defects, while not perturbing the attachment of our oligonucleotide target to the substrate or altering further hybridization. This protocol is simple, fast, and inexpensive.

Topics: Carbocyanines; Fluorescent Dyes; Hybridomas; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Quality Control; T-Lymphocytes

We are using DNA microarray-based gene expression profiling to classify temporal patterns of gene expression during the development of maize embryos, to understand mRNA-level control of embryogenesis and to dissect metabolic pathways and their interactions in the maize embryo. Genes involved in carbohydrate, fatty acid, and amino acid metabolism, the tricarboxylic acid (TCA) cycle, glycolysis, the pentose phosphate pathway, embryogenesis, membrane transport, signal transduction, cofactor biosynthesis, photosynthesis, oxidative phosphorylation and electron transfer, as well as 600 random complementary DNA (cDNA) clones from maize embryos, were arrayed on glass slides. DNA arrays were hybridized with fluorescent dye-labeled cDNA probes synthesized from kernel and embryo poly(A)(+)RNA from different stages of maize seed development. Several characteristic developmental patterns of expression were identified and correlated with gene function. Patterns of coordinated gene expression in the TCA cycle and glycolysis were analyzed in detail. The steady state level of poly(A)(+) RNA for many genes varies dramatically during maize embryo development. Expression patterns of genes coding for enzymes of fatty acid biosynthesis and glycolysis are coordinately regulated during development. Genes of unknown function may by assigned a hypothetical role based on their patterns of expression resembling well characterized genes. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s10142-002-0046-6.

Topics: Blotting, Northern; Carbocyanines; Citric Acid Cycle; DNA Probes; DNA, Complementary; Expressed Sequence Tags; Fatty Acids; Gene Expression Profiling; Gene Expression Regulation, Plant; Gene Frequency; Gene Library; Glycolysis; Oligonucleotide Array Sequence Analysis; Plant Leaves; Plant Roots; Zea mays

We have studied the correlation between structural dynamics and function of the hairpin ribozyme. The enzyme-substrate complex exists in either docked (active) or undocked (inactive) conformations. Using single-molecule fluorescence methods, we found complex structural dynamics with four docked states of distinct stabilities and a strong memory effect where each molecule rarely switches between different docked states. We also found substrate cleavage to be rate-limited by a combination of conformational transitions and reversible chemistry equilibrium. The complex structural dynamics quantitatively explain the heterogeneous cleavage kinetics common to many catalytic RNAs. The intimate coupling of structural dynamics and function is likely a general phenomenon for RNA.

Topics: Carbocyanines; Catalysis; Enzymes, Immobilized; Fluorescence; Hydrogen Bonding; Kinetics; Nepovirus; Nucleic Acid Conformation; RNA, Catalytic; RNA, Satellite; RNA, Viral; Spectrometry, Fluorescence; Thermodynamics

Oligonucleotide-based DNA microarrays are becoming increasingly useful for the analysis of gene expression and single nucleotide polymorphisms. Here we report a systematic study of the sensitivity, specificity and dynamic range of microarray signals and their dependence on the labeling and hybridization conditions as well as on the length, concentration, attachment moiety and purity of the oligonucleotides. Both a controlled set of in vitro synthesized transcripts and RNAs from biological samples were used in these experiments. An algorithm is presented that allows the efficient selection of oligonucleotides able to discriminate a single nucleotide mismatch. Critical parameters for various applications are discussed based on statistical analysis of the results. These data will facilitate the design and standardization of custom-made microarrays applicable to gene expression profiling and sequencing analyses.

Topics: Animals; Carbocyanines; Cell Line; Formamides; HeLa Cells; Humans; Molecular Weight; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Oligonucleotides; RNA-Binding Proteins; RNA, Antisense; RNA, Messenger; Sensitivity and Specificity; Software; Temperature

Topics: Carbocyanines; Deoxycytosine Nucleotides; DNA, Complementary; Oligonucleotide Array Sequence Analysis

We have performed a comprehensive analysis of the expression profiles in 25 adult and 4 fetal human tissues by means of a cDNA microarray consisting of 23,040 human genes. This study revealed a number of genes that were expressed specifically in each of those tissues. Among the 29 tissues examined, 4,080 genes were highly expressed (at least a five-fold expression ratio) in one or only a few tissues and 1,163 of those were expressed exclusively (more than a ten-fold higher expression ratio) in a particular tissue. Expression of some of the genes in the latter category was confirmed by northern analysis. A hierarchical clustering analysis of gene-expression profiles in nerve tissues (adult brain, fetal brain, and spinal cord), lymphoid tissues (bone marrow, thymus, spleen, and lymph node), muscle tissues (heart and skeletal muscle), or adipose tissues (mesenteric adipose and mammary gland) identified a set of genes that were commonly expressed among related tissues. These data should provide useful information for medical research, especially for efforts to identify tissue-specific molecules as potential targets of novel drugs to treat human diseases.

Topics: Adult; Carbocyanines; Fetus; Gene Expression Profiling; Genome, Human; Humans; Oligonucleotide Array Sequence Analysis; Organ Specificity

Microarray expression analysis demands large amounts of RNA that are often not available. RNA amplification techniques have been developed to overcome this prcblem, but limited data are available regarding the reproducibility and maintenance of original transcript ratios. We optimized and validated two amplification techniques: a modified in vitro transcription for the linear amplification of 3 microg total RNA and a SMART PCR-based technique for the exponential amplification of 50 ng total RNA. To determine bias between transcript ratios, we compared the expression profiles in mouse testis versus spleen between the two amplification methods and a standard labeling protocol, using microarrays containing 4596 cDNAs spotted in duplicate. With each method, replicate hybridizations were highly reproducible. However, when comparing the amplification methods to standard labeling, correlation coefficients were lower. Twelve genes that exhibited inconsistent or contradictory expression ratios among the three methods were verified by quantitative RT-PCR. The amplification methods showed slightly more discrepancies in the expression ratios when compared to quantitative RT-PCR results but were more sensitive in terms of detecting expressed genes. In conclusion, although amplification methods introduce slight changes in the transcript ratios compared to standard labeling, they are highly reproducible. For small sample size, in vitro transcription is the preferred method, but one should never combine different labeling strategies within a single study.

Topics: Blotting, Southern; Carbocyanines; Gene Expression Profiling; Nucleic Acid Amplification Techniques; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Statistics as Topic

We derived a theoretical model that explains certain biases observed in the two-color microarray hybridization experiments reported in the literature. We show that true competition is achieved only when the hybridization kinetics of the two differentially labeled probes are the same. If the hybridization kinetics of the two differentially labeled probes is different, which can occur when the labeling and hybridization conditions for the two probes are dissimilar, then differential expression observed becomes a function of the amount of the target (i.e., DNA spotted on the slide). We use this model to validate the microarray methodology by determining the differential expression of four select Arabidopsis genes and two human genes (beta-actin and GAPDH) as a function of the amount of target arrayed. We show through both modeling and experiments that the rate constants for Cy5- and Cy3-labeled probes are the same under our exrimental conditions. Therefore, the target concentrations need not greatly exceed the probe concentration. It is obvious from the data presented that a simple treatment of an individual hybridization rate calculation does notfully describe what is occuring in today's complex, multispecies experiments. The method of validation is easily implemented to ensure data reliability by two-color microarray.

Topics: Arabidopsis; Carbocyanines; Kinetics; Oligonucleotide Array Sequence Analysis

Non denaturing gradient gel electrophoresis (GGE) is commonly used to analyze the size distribution of lipoprotein particles. Its relatively low sensitivity and linear dynamic range limit use of GGE to quantify protein content of lipoproteins. We demonstrate a new high sensitivity method for analysis and quantitation of biotinylated apolipoprotein B (apoB)-containing lipoproteins using a fluorescent streptavidin-Cy3 conjugate and non covalent preelectrophoretic binding. Forty-four lipoprotein subfractions spanning the VLDL and LDL particle spectrum subfractions (11 each from four human subjects) were prepared by density gradient ultracentrifugation. An aliquot of each sample was biotinylated and GGE was performed. Gels also were stained for lipid with Oil Red O (32 samples) and for protein with Coomassie Brilliant Blue (30 samples). There was a significant relationship between the Cy3 fluorescent label area under the curve and the mass of apoB (P < 0.02-0.004) and total cholesterol (P < 0.03-0.004). Particle diameters of each absorbence/fluorescent peak were comparable between Oil-Red O and streptavidin-Cy3 treated biotinylated lipoproteins (+/-3.54 A, P = 0.3). Biotinylation and prestaining of lipoprotein particle with streptavidin-Cy3 provides a new fluorescence-based method for detection and quantitative analysis of lipoprotein subspecies by gradient gel electrophoresis.

Topics: Apolipoproteins B; Azo Compounds; Biotinylation; Carbocyanines; Fluorescent Dyes; Humans; Lipoproteins; Rosaniline Dyes; Streptavidin

In previous studies, we showed that peptide nucleic acid (PNA) probes have significant advantages over conventional synthetic RNA or DNA probes in FISH procedures for detecting telomeric and trinucleotide repeat sequences. Here, we report that directly labeled PNA probes recognizing chromosome-specific repeat sequences are also powerful tools for detecting and enumerating specific chromosomes in interphase and metaphase cells. This is illustrated by multicolor FISH experiments with cells from normal individuals and patients with numerical sex chromosome aberrations.

Topics: Carbocyanines; Chromatography, High Pressure Liquid; Chromosomes, Human; Female; Fibroblasts; Fluorescent Dyes; Humans; In Situ Hybridization, Fluorescence; Lymphocytes; Male; Nucleic Acid Probes; Peptide Nucleic Acids

Systemic administration of a recombinant adenovirus encoding the human interferon-beta gene (H5.110CMVhIFN-beta) results in transduction of hepatocytes and detectable circulating levels of IFN-beta protein. In preclinical studies in mice, we noticed a distinctly nonlinear dose response, with low levels of virus (1-3 x 10(10) viral particles) yielding barely detectable levels of IFN-beta but with a higher viral dose (1 x 10(11) particles) resulting in disproportionately high IFN-beta levels. Further studies showed that transgene expression levels from low viral doses could be dramatically enhanced by coadministering an unrelated recombinant adenovirus (H5.110CMVlacZ), suggesting that there was a viral dose threshold effect for efficient viral transduction and/or IFN-beta expression. This enhancement of reporter expression by a nonreporter adenovirus, effective upon coadministration, was further enhanced by preadministration of H5.110CMVlacZ (up to 8 h), but was ineffective if the helper virus was administered as little as 5 min after the H5.110CMVhIFN-beta reporter virus. Our data suggest that the reticuloendothelial system plays a role in this threshold effect, such that low doses of virus are efficiently taken up by the RES/Kupffer cells without leading to appreciable transgene expression, whereas high doses saturate these cells and are able to productively transduce hepatocytes. A better understanding of this phenomenon could have an impact on gene therapy clinical trial safety and efficacy.

Topics: Adenoviridae; alpha 1-Antitrypsin; Animals; beta-Galactosidase; Carbocyanines; Dose-Response Relationship, Drug; Endothelium; Enzyme-Linked Immunosorbent Assay; Fluorescent Dyes; Genes, Reporter; Genetic Vectors; Hepatocytes; Humans; Interferon-beta; Kupffer Cells; Liver; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Nude; Species Specificity; Tissue Distribution; Transduction, Genetic; Transgenes; Tumor Cells, Cultured

Class A scavenger receptors (SR-A) mediate microglial interaction with fibrillar beta-amyloid (fAbeta). We report here that neonatal microglia from SR-A knockout mice (SR-A-/-) adhere to surface-bound fAbeta, and produce reactive oxygen species (ROS) as efficiently as wildtype microglia; that both wildtype and SR-A-/- microglia express SR-BI; that antibodies against SR-BI do not affect adhesion or ROS production by wildtype microglia, but inhibit adhesion and ROS production of SR-A-/- microglia to immobilized fAbeta by approximately 40%. Adhesion to fAbeta-coated surfaces, and uptake of fAbeta by both wildtype and SR-A-/- microglia was almost completely inhibited by incubation with fucoidan. Thus SR-BI and SR-A mediate similar effector functions in neonatal microglia, which suggests that SR-BI plays as important a role as SR-A, and can maintain the wildtype phenotype in SR-A-/- microglia.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Animals, Newborn; Antineoplastic Agents; Brain; Carbocyanines; CD36 Antigens; Cell Adhesion; Cells, Cultured; Fluorescent Dyes; Gene Expression; Liver; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Knockout; Microglia; Peptide Fragments; Polysaccharides; Reactive Oxygen Species; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Scavenger; Scavenger Receptors, Class A; Scavenger Receptors, Class B

Previous immunohistochemical studies failed to reveal neurokinin (NK)(1) tachykinin receptors on intestinal muscle, despite convincing pharmacologic data indicating their presence. This study aimed to apply optimal immunohistochemical methods to reveal the receptors.. NK(1)-receptor immunoreactivity was examined by confocal microscopy in tissue incubated with or without 10(-7) mol/L substance P (SP), 10(-7) mol/L SP plus 10(-6) mol/L NK(1) receptor antagonist (CP99994), or with fluorescent cyanine 3.18 (Cy3) SP.. Without incubation, NK(1)-receptor immunoreactivity was strong on muscle of the rectum and distal colon and weak in proximal colon and small intestine. NK(1) receptor was located on the surface of muscle cells in all gut regions. Exposure to SP increased the intensity of immunoreactivity, and the receptor moved into the cytoplasm. Mobilization of the receptor by SP was blocked by the NK(1)-receptor antagonist CP99994. Cy3-SP was internalized by muscle cells and colocalized with the receptor. NK(1)-receptor immunoreactivity occurred on crypt epithelial cells in the small intestine and the base of glands in the proximal colon.. The NK(1) receptor occurs on the external muscle throughout the small and large intestines. SP binds and triggers NK(1)-receptor aggregation and internalization in the muscle.

Topics: Animals; Carbocyanines; Colon; Endocytosis; Epithelial Cells; Female; Fluorescent Dyes; Guinea Pigs; Immunohistochemistry; Intestinal Mucosa; Intestine, Small; Male; Muscle, Smooth; Nerve Fibers; Neurokinin-1 Receptor Antagonists; Piperidines; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Rectum; Substance P

The contact with the postsynaptic target induces structural and functional modifications in the serotonergic cell C1 of Helix pomatia. In previous studies we have found that the presence of a non-physiological target down-regulates the number of presynaptic varicosities formed by cultured C1 neurons and has a strong inhibitory effect on the action potential-evoked Ca(2+) influx and neurotransmitter release at C1 terminals. Since a large body of experimental evidence implicates the synapsins in the development and functional maturation of synaptic connections, we have investigated whether the injection of exogenous synapsin I into the presynaptic neuron C1 could affect the inhibitory effect of the wrong target on neurotransmitter release. C1 neurons were cultured with the wrong target neuron C3 for three to five days and then injected with either dephosphorylated or Ca(2+)/calmodulin-dependent protein kinase II-phosphorylated Cy3-labeled synapsin I. The subcellular distribution of exogenous synapsin I, followed by fluorescence videomicroscopy, revealed that only synapsin I phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II diffused in the cytoplasm and reached the terminal arborizations of the axon, while the dephosphorylated form did not diffuse beyond the cell body. Evoked neurotransmitter release was measured during C1 stimulation using a freshly dissociated neuron B2 (sniffer) micromanipulated in close contact with the terminals of C1. A three-fold increase in the amplitude of the sniffer depolarization with respect to the pre-injection amplitude (190+/-29% increase, n=10, P<0.006) was found 5 min after injection of Ca(2+)/calmodulin-dependent protein kinase II-phosphorylated synapsin I that lasted for about 30 min. No significant change was observed after injection of buffer or dephosphorylated synapsin I. These data indicate that the presence of synapsin I induces a fast increase in neurotransmitter release that overcomes the inhibitory effect of the non-physiological target and suggest that the expression of synapsins may play a role in the modulation of synaptic strength and neural connectivity.

Topics: Actins; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Carbocyanines; Cell Communication; Cells, Cultured; Fluorescent Antibody Technique; Ganglia, Invertebrate; Helix, Snails; Intracellular Fluid; Models, Animal; Neural Pathways; Neurotransmitter Agents; Phosphorylation; Presynaptic Terminals; Serotonin; Synapsins; Synaptic Vesicles

The hairpin ribozyme in its natural context consists of two loops in RNA duplexes that are connected as arms of a four-way helical junction. Magnesium ions induce folding into the active conformation in which the two loops are in proximity. In this study, we have investigated nucleotides that are important to this folding process. We have analyzed the folding in terms of the cooperativity and apparent affinity for magnesium ions as a function of changes in base sequence and functional groups, using fluorescence resonance energy transfer. Our results suggest that the interaction between the loops is the sum of a number of component interactions. Some sequence variants such as A10U, G+1A, and C25U exhibit loss of cooperativity and reduced affinity of apparent magnesium ion binding. These variants are also very impaired in ribozyme cleavage activity. Nucleotides A10, G+1, and C25 thus appear to be essential in creating the conformational environment necessary for ion binding. The double variant G+1A/C25U exhibits a marked recovery of both folding and catalytic activity compared to either individual variant, consistent with the proposal of a triple-base interaction among A9, G+1, and C25 [Pinard, R., Lambert, D., Walter, N. G., Heckman, J. E., Major, F., and Burke, J. M. (1999) Biochemistry 38, 16035-16039]. However, substitution of A9 leads to relatively small changes in folding properties and cleavage activity, and the double variant G+1DAP/C25U (DAP is 2,6-diaminopurine), which could form an isosteric triple-base interaction, exhibits folding and cleavage activities that are both very impaired compared to those of the natural sequence. Our results indicate an important role for a Watson--Crick base pair between G+1 and C25; this may be buttressed by an interaction with A9, but the loss of this has less significant consequences for folding. 2'-Deoxyribose substitution leads to folding with reduced magnesium ion affinity in the following order: unmodified RNA > dA9 > dA10 > dC25 approximately dA10 plus dC25. The results are interpreted in terms of an interaction between the ribose ring of C25 and the ribose and base of A10, in agreement with the proposal of Ryder and Strobel [Ryder, S. P., and Strobel, S. A. (1999) J. Mol. Biol. 291, 295-311]. In general, there is a correlation between the ability to undergo ion-induced folding and the rate of ribozyme cleavage. An exception to this is provided by G8, for which substitution with uridine leads to severe impai

Topics: 2-Aminopurine; Adenine; Base Composition; Base Sequence; Carbocyanines; Cytosine; Deoxyribonucleosides; Energy Transfer; Fluorescein; Fluorescent Dyes; Guanine; Hydrolysis; Molecular Sequence Data; Nepovirus; Nucleic Acid Conformation; Oligonucleotides; Point Mutation; Purine Nucleotides; Ribose; RNA, Catalytic; Spectrometry, Fluorescence; Uridine

To characterize the receptors involved in binding fibrillar amyloid A-beta (fA beta), we compared the uptake of fA beta in microglia from wildtype (MSR-A+/+) and MSR-A knockout (MSR-A-/-) mice. On average, there was a 60% reduction in the uptake of Cy3-fA beta in microglia from the MSR-A-/- mice. Cy3-fA beta uptake in the MSR-A-/- mice was still competable by scavenger receptor ligands, including acetylated low-density lipoprotein (Ac-LDL) and fucoidan. This indicates that uptake by MSR-B and/or other MSRs is also involved in the uptake of fA beta by microglia. However, the significant reduction in the uptake of fA beta in the MSR-A-/- microglia suggests that fA beta gets internalized mostly by MSR-As in microglia. Uptake of modified fA beta (ClqA beta) was similar in the MSR-A-/- microglia as in the wildtype indicating that the uptake of the opsonized fA beta is independent of MSR-A.

Topics: Amyloid beta-Peptides; Animals; Animals, Newborn; Carbocyanines; Cell Separation; Cells, Cultured; Fluorescent Dyes; Immunoglobulin G; Mice; Mice, Knockout; Microglia; Receptors, Immunologic; Receptors, Scavenger

1. Transcytosis of albumin, involving the 60 kDa albumin-binding glycoprotein, gp60, was studied in cultured type II alveolar epithelial cells obtained from rat lungs. 2. Type II cells internalized the interfacial fluorescent dye RH 414, which marks for plasmalemma vesicles. Fluorescent forms of albumin and anti-gp60 antibody colocalized in the same plasmalemma vesicles. 3. Antibody (100 microg ml(-1)) cross-linking of gp60 for brief periods (15 min) markedly stimulated vesicular uptake of fluorescently tagged albumin. The caveolar disrupting agent, filipin (10 nM), abolished the stimulated internalization of albumin. 4. The vast majority of plasmalemmal vesicles carrying albumin also immunostained for caveolin-1; however, lysosomes did not stain for caveolin-1. Filipin depleted the epithelial cells of the caveolin-1-positive, albumin-transporting plasmalemma vesicles. 5. Prolonged (> 1 h) stimulation of type II cells with cross-linking anti-gp60 antibody produced loss of cell-surface gp60 and abolished endocytic albumin uptake. 6. Transalveolar transport of albumin was also studied in the isogravimetric rat lung preparation perfused at 37 degrees C. (125)I-labelled albumin was instilled into distal airspaces of lungs, and the resulting (125)I-labelled albumin efflux into the vascular perfusate was determined. 7. Unlabelled albumin (studied over a range of 0-10 g (100 instilled ml)(-1)) inhibited 40 % of the transport of labelled albumin ((5.7 +/- 0.4) x 10(5) counts (instilled ml)(-1)) with an IC(50) value of 0.34 g (100 ml)(-1). 8. Filipin blocked the displacement-sensitive component of (125)I-labelled albumin transport, but had no effect on the transport of the paracellular tracer (3)[H]mannitol. 9. Displacement-sensitive (125)I-labelled albumin transport had a significantly greater Q(10) (27-37 degrees C) than the non-displaceable component. 10. Cross-linking of gp60 by antibody instillation stimulated only the displacement-sensitive (125)I-labelled albumin transalveolar transport in intact rat lungs. 11. To estimate the transport capacity of the displacement-sensitive system, the percentage of instilled (125)I-labelled albumin counts remaining in lung tissue was compared in lungs treated with instillates containing either 0.05 g (100 ml)(-1) unlabelled albumin or 5 g (100 ml)(-1) unlabelled albumin. Approximately 25 % of instilled (125)I-labelled albumin was cleared from the lung preparations per hour by the displacement-sensitive transport pathway.

Topics: Animals; Anti-Bacterial Agents; Biological Transport; Carbocyanines; Caveolin 1; Caveolins; Cells, Cultured; Diuretics, Osmotic; Endocytosis; Epithelial Cells; Filipin; Fluorescent Dyes; Glycoproteins; Iodine Radioisotopes; Male; Mannitol; Pulmonary Alveoli; Pyridinium Compounds; Rats; Rats, Sprague-Dawley; Serum Albumin; Specific Pathogen-Free Organisms; Temperature; Tritium

Hepatitis B virus (HBV) is a major risk factor for the development of hepatocellular carcinoma (HCC). HBV encodes the potentially oncogenic HBx protein, which mainly functions as a transcriptional co-activator involving in multiple gene deregulations. However, mechanisms underlying HBx-mediated oncogenicity remain unclear. To determine the role(s) of HBx in the early genesis of HCC, we utilized the NCI Oncochip microarray that contains 2208 human cDNA clones to examine the gene expression profiles in either freshly isolated normal primary adult human hepatocytes (Hhep) or an HCC cell line (SK-Hep-1) ecotopically expressing HBx via an adenoviral system. The gene expression profiles also were determined in liver samples from HBV-infected chronic active hepatitis patients when compared with normal liver samples. The microarray results were validated through Northern blot analysis of the expression of selected genes. Using reciprocally labeling hybridizations, scatterplot analysis of gene expression ratios in human primary hepatocytes expressing HBx demonstrates that microarrays are highly reproducible. The comparison of gene expression profiles between HBx-expressing primary hepatocytes and HBV-infected liver samples shows a consistent alteration of many cellular genes including a subset of oncogenes (such as c-myc and c-myb) and tumor suppressor genes (such as APC, p53, WAF1 and WT1). Furthermore, clustering algorithm analysis showed distinctive gene expression profiles in Hhep and SK-Hep-1 cells. Our findings are consistent with the hypothesis that the deregulation of cellular genes by oncogenic HBx may be an early event that favors hepatocyte proliferation during liver carcinogenesis.

Topics: Adult; Blotting, Northern; Carbocyanines; Carcinoma, Hepatocellular; Fluorescent Dyes; Freezing; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Hepatitis B virus; Hepatitis B, Chronic; Hepatocytes; Humans; Liver; Liver Neoplasms; Oligonucleotide Array Sequence Analysis; Staining and Labeling; Trans-Activators; Tumor Cells, Cultured; Viral Regulatory and Accessory Proteins

The double helix is known to form as a result of hybridization of complementary nucleic acid strands in aqueous solution. In the helix the negatively charged phosphate groups of each nucleic acid strand are distributed helically on the outside of the duplex and are available for interaction with cationic groups. Cation-coated glass surfaces are now widely used in biotechnology, especially for covalent attachment of cDNAs and oligonucleotides as surface-bound probes on microarrays. These cationic surfaces can bind the nucleic acid backbone electrostatically through the phosphate moiety. Here we describe a simple method to fabricate DNA microarrays based upon adsorptive rather than covalent attachment of oligonucleotides to a positively charged surface. We show that such adsorbed oligonucleotide probes form a densely packed monolayer, which retains capacity for base pair-specific hybridization with a solution state DNA target strand to form the duplex. However, both strand dissociation kinetics and the rate of DNase digestion suggest, on symmetry grounds, that the target DNA binds to such adsorbed oligonucleotides to form a highly asymmetrical and unwound duplex. Thus, it is suggested that, at least on a charged surface, a non-helical DNA duplex can be the preferred structural isomer under standard biochemical conditions.

Topics: Carbocyanines; Deoxyribonuclease I; DNA, Single-Stranded; Fluorescent Dyes; Glass; Nucleic Acid Conformation; Nucleic Acid Denaturation; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Oligonucleotides; Silanes; Surface Properties

The goal of this study was to develop a DNA micro array procedure for molecular human leukocyte antigen (HLA) typing of a large number of samples. DNA was isolated from peripheral blood samples and polymerase chain reaction (PCR) amplified for HLA-A, -B, and -DRB. Amplified DNA samples were spotted on silane-treated glass slides using a micro array spotter. The spotter was capable of spotting multiple slides with up to 9216 samples per slide or 2304 samples in quadruplicate. The allele specific oligo nucleotide probes for HLA-A, -B, and -DRB were labeled with the fluorescent dye Cy3, while a control probe, to quantitate the total amount of PCR product in a sample, was labeled with Cy5. Each slide was hybridized with a mixture of an allele specific Cy3 probe plus the control Cy5 probe. Following hybridization and wash, the amount of probe hybridizing to each DNA sample on the slide was measured with a micro array scanner. A computer program was used for image analysis, to calculate the average Cy3/Cy5 ratios and to identify the positive and negative samples. In turn, this information was used to determine the HLA phenotype of each sample. There was very good concordance between the results obtained for all three loci using Cy-labeled probes as compared with those previously obtained by chemiluminescent detection of alkaline phosphatase labeled probes. This methodology has the potential of greatly simplifying HLA molecular typing of large number of samples.

Topics: Alleles; Carbocyanines; DNA; Electronic Data Processing; Histocompatibility Testing; HLA-A Antigens; HLA-B Antigens; HLA-DR Antigens; Humans; In Situ Hybridization, Fluorescence

Topics: Carbocyanines; Fluorescent Dyes; Genome, Human; Humans; Oligonucleotide Array Sequence Analysis; RNA, Messenger

Laboratories use different laser-based scanners to scan microarray images. To assess whether results from different scanners are comparable, and thus whether data from different laboratories can be compared, we scanned the same microarray slide with three commercial scanners that use different imaging techniques. After the acquisition of the microarray images produced by the three scanners, the images were quantified using a single imaging software package and protocol. The results were compared, and we found that the data obtained from the three scanners were comparable and that the variations caused by the use of different instruments were negligible, in spite of the fact that the scanners were based on different optical imaging techniques.

Topics: Carbocyanines; Fluorescent Dyes; Image Processing, Computer-Assisted; Lasers; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Quality Control; RNA Probes; Sensitivity and Specificity; Software; Ultraviolet Rays

The postsynaptic density (PSD) at excitatory dendritic synapses comprises a protein complex of glutamate receptors, scaffolding elements, and signaling enzymes. For example, NMDA receptors (NMDARs) are linked to several proteins in the PSD, such as PSD-95, and are also tethered via binding proteins such as alpha-actinin directly to filamentous actin of the cytoskeleton. Depolymerization of the cytoskeleton modulates the activity of NMDARs, and, in turn, strong activation of NMDARs can trigger depolymerization of actin. Myosin, the motor protein of muscular contraction and nonmuscle motility, is also associated with NMDARs and the PSD. We show here that constitutively active myosin light chain kinase (MLCK) enhances NMDAR-mediated whole-cell and synaptic currents in acutely isolated CA1 pyramidal and cultured hippocampal neurons, whereas inhibitors of MLCK depress these currents. This MLCK-dependent regulation was observed in cell-attached patches but was lost after excision to inside-out patches. Furthermore, the enhancement induced by constitutively active MLCK and the depression of MLCK inhibitors were eliminated after depolymerization of the cytoskeleton. NMDARs and MLCK did not colocalize in clusters on the dendrites of cultured hippocampal neurons, further indicating that the effects of MLCK are mediated indirectly via actomyosin. Our results suggest that MLCK enhances actomyosin contractility to either increase the membrane tension on NMDARs or to alter physical relationships between the actin cytoskeleton and the linker proteins of NMDARs.

Topics: Actins; Animals; Calcium; Carbocyanines; Cell Separation; Cells, Cultured; Dendrites; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Fluorescent Dyes; Hippocampus; Mice; Myosin-Light-Chain Kinase; N-Methylaspartate; Neurons; Patch-Clamp Techniques; Pyramidal Cells; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate

Single-molecule imaging techniques were used to reveal the binding of individual cyclic adenosine 3',5'-monophosphate molecules to heterotrimeric guanine nucleotide-binding protein coupled receptors on the surface of living Dictyostelium discoideum cells. The binding sites were uniformly distributed and diffused rapidly in the plane of the membrane. The probabilities of individual association and dissociation events were greater for receptors at the anterior end of the cell. Agonist-induced receptor phosphorylation had little effect on any of the monitored properties, whereas G protein coupling influenced the binding kinetics. These observations illustrate the dynamic properties of receptors involved in gradient sensing and suggest that these may be polarized in chemotactic cells.

Topics: Animals; Carbocyanines; Cell Membrane; Chemotaxis; Cyclic AMP; Dictyostelium; Diffusion; Guanosine Diphosphate; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Kinetics; Microscopy, Fluorescence; Mutation; Phosphorylation; Pseudopodia; Receptors, Cyclic AMP; Signal Transduction

The field of DNA microarray technology has necessitated the cooperative efforts of interdisciplinary scientific teams to achieve its primary goal of rapidly measuring global gene expression patterns. A collaborative effort was established to produce a chemically reactive surface on glass slide substrates to which unmodified DNA will covalently bind for improvement of cDNA microarray technology. Using the p-aminophenyl trimethoxysilane (ATMS)/diazotization chemistry that was developed, microarrays were fabricated and analyzed. This immobilization method produced uniform spots containing equivalent or greater amounts of DNA than commercially available immobilization techniques. In addition, hybridization analyses of microarrays made with ATMS/diazotization chemistry showed very sensitive detection of the target sequence, two to three orders of magnitude more sensitive than the commercial chemistries. Repeated stripping and re-hybridization of these slides showed that DNA loss was minimal, allowing multiple rounds of hybridization. Thus, the ATMS/diazotization chemistry facilitated covalent binding of unmodified DNA, and the reusable microarrays that were produced showed enhanced levels of hybridization and very low background fluorescence.

Topics: Adsorption; Carbocyanines; DNA; DNA Probes; DNA, Fungal; Fluorescence; Fluorescent Dyes; Gene Expression Profiling; Glass; Neurospora crassa; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Polylysine; Reproducibility of Results; Silanes

Junction-resolving enzymes are nucleases that are specific for the structure of the four-way DNA junction. The binding of RuvC of Escherichia coli and Hjc of Sulfolobus solfataricus can be followed by an increase in the fluorescence anisotropy of Cy3 terminally attached to one of the helical arms of a four-way junction. By contrast, there was no change in fluorescein anisotropy with the binding of single dimers of these proteins. Fluorescence resonance energy transfer has therefore been used between fluorescein and Cy3 fluorophores attached to the ends of helical arms to analyse the global structure of the junction on protein binding. The results indicate that both enzymes induce a marked change in the global DNA conformation on the binding of a single dimer. The structure of the protein-junction complexes is independent of the presence or absence of divalent metal ions, unlike that of the protein-free junction. The structures of the RuvC and Hjc complexes are different, but both represent a significant opening of the structure compared to the stacked X-structure of the protein-free junction in the presence of magnesium ions. This protein-induced opening is likely to be important in the function of these enzymes.

Topics: Bacterial Proteins; Carbocyanines; Dimerization; DNA; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Endodeoxyribonucleases; Energy Transfer; Escherichia coli; Escherichia coli Proteins; Fluorescein; Fluorescence Polarization; Fluorescent Dyes; Fluorometry; Holliday Junction Resolvases; Models, Molecular; Nucleic Acid Conformation; Protein Binding; Recombination, Genetic; Sulfolobus

A flexible, non-gel-based single nucleotide polymorphism (SNP) detection method is described. The method adopts thermostable ligation for allele discrimination and rolling circle amplification (RCA) for signal enhancement. Clear allelic discrimination was achieved after staining of the final reaction mixtures with Cybr-Gold and visualisation by UV illumination. The use of a compatible buffer system for all enzymes allows the reaction to be initiated and detected in the same tube or microplate well, so that the experiment can be scaled up easily for high-throughput detection. Only a small amount of DNA (i.e. 50 ng) is required per assay, and use of carefully designed short padlock probes coupled with generic primers and probes make the SNP detection cost effective. Biallelic assay by hybridisation of the RCA products with fluorescence dye-labelled probes is demonstrated, indicating that ligation-RCA (L-RCA) has potential for multiplexed assays.

Topics: Alleles; Avena; Bacillus Phages; Carbocyanines; DNA Primers; DNA Probes; DNA-Directed DNA Polymerase; Fluorescent Dyes; Genotype; Intramolecular Transferases; Plants; Point Mutation; Polymerase Chain Reaction; Polymorphism, Single Nucleotide

Cholinergic neurons of the basal forebrain form one of the neuron populations that are susceptible to excitotoxic injury. Whereas neuropharmacological studies have aimed at rescuing cholinergic neurons from acute excitotoxic attacks, the short-term temporal profile of excitotoxic damage to cholinergic nerve cells remains largely elusive. The effects of N-methyl-D-aspartate (NMDA) infusion on cytochemical markers of cholinergic neurons in rat magnocellular nucleus basalis were therefore determined 4, 24 and 48 h post-lesion. Additionally, the influence of excitotoxic damage on the efficacy of in vivo labelling of cholinergic neurons with carbocyanine 3-192IgG was investigated. Carbocyanine 3-192IgG was unilaterally injected in the lateral ventricle. Twenty-four hours later, NMDA (60 nM/microl) was infused in the right magnocellular nucleus basalis, while control lesions were performed contralaterally. Triple immunofluorescence labelling for carbocyanine 3-192IgG, NMDA receptor 2A and B subunits and choline-acetyltransferase (ChAT) was employed to determine temporal changes in NMDA receptor immunoreactivity on cholinergic neurons. The extent of neuronal degeneration was studied by staining with Fluoro-Jade. Moreover, changes in the numbers of ChAT or p75 low-affinity neurotrophin receptor immunoreactive neurons, and the degree of their co-labelling with carbocyanine 3-192IgG were determined in basal forebrain nuclei. The effects of NMDA-induced lesions on cortical projections of cholinergic nucleus basalis neurons were studied by acetylcholinesterase (AChE) histochemistry. Characteristic signs of cellular damage, as indicated by decreased immunoreactivity for NMDA receptors, ChAT and p75 low-affinity neurotrophin receptors, were already detected at the shortest post-lesion interval investigated. Fluoro-Jade at 4 h post-lesion only labelled the core of the excitotoxic lesion. Longer survival led to enhanced Fluoro-Jade staining, and to the decline of ChAT immunoreactivity reaching a maximum 24 h post-surgery. Significant loss of p75 low-affinity neurotrophin receptor immunoreactivity and of cortical AChE-positive projections only became apparent 48 h post-lesion. Carbocyanine 3-192IgG labelling in the ipsilateral basal forebrain exceeded that of the contralateral hemisphere at all time points investigated and progressively declined in the damaged magnocellular nucleus basalis up to 48 h after NMDA infusion. The present study indicates that excitotoxic lesion

Topics: Acetylcholine; Animals; Basal Nucleus of Meynert; Carbocyanines; Choline O-Acetyltransferase; Excitatory Amino Acid Agonists; Fluoresceins; Fluorescent Dyes; Immunoglobulin G; Immunohistochemistry; Injections, Intraventricular; Male; Microscopy, Confocal; N-Methylaspartate; Nerve Degeneration; Neural Pathways; Neuroglia; Neurons; Neurotoxins; Organic Chemicals; Rats; Rats, Wistar; Receptor, Nerve Growth Factor; Receptors, N-Methyl-D-Aspartate; Sensitivity and Specificity

Spectral properties of carbocyanine dye 3-methyl-2-[3-methyl-2-(3-methyl-2,3-dihydro-1,3-benzothiazole-2-iliden)-1- butenyl]-1,3-benzothiazole-3-il iodide (Cyan betaiPr) in water solution, as well as in the presence of different types of double stranded DNA have been studied. While in water solution of 'free' dye Cyan betaiPr stays mainly in monomeric form, in the presence of DNA the dye molecules form J-aggregates. The molecular structure of these J-aggregates causes the Davydov splitting of their absorption band, corresponding to the first electronic transition. A study of site-specificity showed that in the presence of poly (dA/dT) the majority of Cyan betaiPr molecules form J-aggregates, while in the presence of poly (dGC/dGC) dye molecules stay mainly in monomeric form and in presence of chicken erythrocytes DNA both J-aggregate and monomeric forms of dye are present. We suppose that Cyan betaiPr molecules aggregate in DNA groove, which serves as a template for J-aggregate forming. An increase of ionic strength of solution leads to the release of dye molecules from DNA grooves and prevents J-aggregates formation.

Topics: Absorption; Carbocyanines; DNA; Fluorescence; Fluorescent Dyes; Molecular Structure; Sodium Chloride; Solutions; Spectrometry, Fluorescence

Metallothioneins (MTs) are low molecular weight, stress-activated proteins that protect cells against heavy metals, oxidants, and some electrophilic drugs. Both nuclear and cytoplasmic MT phenotypes have been observed in cells even though MTs (6 kDa) are well below the size exclusion limit for diffusion through the nuclear envelope. To study the factors controlling MT subcellular partitioning, we covalently linked MTII to a fluorescent label and examined its subcellular distribution in response both to pharmacologic and physical perturbations. Fluorescent MTII localized to the nucleus of digitonin-permeabilized human SCC25 carcinoma cells, consistent with its endogenous distribution in these cells. Nuclear sequestration of the fluorescent MTII was inhibited by a 100-fold molar excess of unlabeled MTII and by wheat germ agglutinin, indicating a saturable binding mechanism and the involvement of one or more glycoproteins, respectively. Depletion of adenosine triphosphate (ATP) inhibited MTII nuclear localization, implying energy-dependent nuclear translocation or retention of MT. Neither chilling nor the absence of cytosolic extracts inhibited nuclear sequestration of MTII, supporting diffusion-based entry mechanism. In situ biochemical extractions of the nuclear MTII revealed at least two distinct binding activities. Collectively, these data indicate that MTII diffuses into the nucleus of SCC25 cells, where it is selectively and actively retained by nuclear binding factors, imparting its localization phenotype.

Topics: Adenosine Triphosphate; Animals; Biological Transport; Cadmium; Calmodulin; Carbocyanines; Cell Extracts; Cell Line; Cell Membrane Permeability; Cell Nucleus; Cold Temperature; Cytosol; Diffusion; Energy Metabolism; Fluorescent Dyes; Glycosylation; Humans; Metallothionein; Mice; Protein Binding; Wheat Germ Agglutinins

Numerous experimental and clinical studies have been completed regarding the effects of carbogen and nicotinamide on tumor oxygenation and radiosensitivity. The current study incorporates three physiological measurement techniques to further define spatial variations in oxygen availability and development of hypoxia after single- and multifraction irradiation in KHT murine fibrosarcomas. Distances to anatomical and perfused blood vessels were measured using immunohistochemical and fluorescent staining, intravascular oxygen levels were determined cryospectrophotometrically, and tumor hypoxia was quantified using uptake of EF5, a marker of hypoxia. Carbogen, nicotinamide, and the combination of both all increased intravascular oxygen availability compared to controls. While nicotinamide had no effect on the number of perfused blood vessels in nonirradiated tumors, carbogen produced a substantial closing of vessels. After a single dose of 4 Gy, only the combination of nicotinamide and carbogen produced significant improvements in oxygen availability, while numbers of perfused vessels were significantly increased for nicotinamide, unchanged for the combination of nicotinamide and carbogen, and significantly decreased for carbogen. After 4 x 4-Gy fractions, oxygen availability was increased substantially with the combination of nicotinamide and carbogen, somewhat with carbogen, and not at all with nicotinamide. Tumor oxygenation changes were estimated by EF5/Cy3 intensity distributions, which demonstrated that manipulative agents could produce disparate effects on tumor hypoxia when combined with either single- or multifraction irradiation.

Topics: Animals; Carbocyanines; Carbon Dioxide; Cell Hypoxia; Combined Modality Therapy; Dose Fractionation, Radiation; Etanidazole; Female; Fibrosarcoma; Fluorescent Dyes; Hydrocarbons, Fluorinated; Indicators and Reagents; Mice; Mice, Inbred C3H; Niacinamide; Oxygen; Oxygen Consumption; Radiation-Sensitizing Agents; Tumor Cells, Cultured

We have investigated the infectious entry pathway of adeno-associated virus (AAV) and recombinant AAV vectors by assessing AAV-mediated gene transfer and by covalently conjugating fluorophores to AAV and monitoring entry by fluorescence microscopy. We examined AAV entry in HeLa cells and in HeLa cell lines which inducibly expressed a dominant interfering mutant of dynamin. The data demonstrate that AAV internalizes rapidly by standard receptor-mediated endocytosis from clathrin-coated pits (half-time <10 min). The lysosomotropic agents ammonium chloride and bafilomycin A(1) prevent AAV-mediated gene transfer when present during the first 30 min after the onset of endocytosis, indicating that AAV escapes from early endosomes yet requires an acidic environment for penetration into the cytosol. Following release from the endosome, AAV rapidly moves to the cell nucleus and accumulates perinuclearly beginning within 30 min after the onset of endocytosis. We present data indicating that escape of AAV from the endosome and trafficking of viral particles to the nucleus are unaffected by the presence of adenovirus, the primary helper virus for a productive AAV infection. Within 2 h, viral particles could be detected within the cell nucleus, suggesting that AAV enters the nucleus prior to uncoating. Interestingly, the majority of the intracellular virus particles remain in a stable perinuclear compartment even though gene expression from nuclear AAV genomes can be detected. This suggests that the process of nuclear entry is rate limiting or that AAV entry involves multiple pathways. Nevertheless, these data establish specific points in the AAV infectious entry process and have allowed the generation of a model for future expansion to specific cell types and AAV vector analysis in vivo.

Topics: Acids; Adenoviridae; Biological Transport; Carbocyanines; Cell Line, Transformed; Cell Nucleus; Clathrin; Coated Pits, Cell-Membrane; Dependovirus; Endocytosis; Endosomes; Fluorescent Dyes; Genetic Vectors; HeLa Cells; Humans

Topics: Carbocyanines; Cells, Cultured; Dimerization; Epidermal Growth Factor; ErbB Receptors; Fluorescent Dyes; Humans; Microscopy, Fluorescence; Phosphorylation; Signal Transduction; Time Factors

The early events in signal transduction from the epidermal growth factor (EGF) receptor (EGFR) are dimerization and autophosphorylation of the receptor, induced by binding of EGF. Here we observe these events in living cells by visualizing single molecules of fluorescent-dye-labelled EGF in the plasma membrane of A431 carcinoma cells. Single-molecule tracking reveals that the predominant mechanism of dimerization involves the formation of a cell-surface complex of one EGF molecule and an EGFR dimer, followed by the direct arrest of a second EGF molecule, indicating that the EGFR dimers were probably preformed before the binding of the second EGF molecule. Single-molecule fluorescence-resonance energy transfer shows that EGF-EGFR complexes indeed form dimers at the molecular level. Use of a monoclonal antibody specific to the phosphorylated (activated) EGFR reveals that the EGFR becomes phosphorylated after dimerization.

Topics: Antibodies, Monoclonal; Calcium; Carbocyanines; Carcinoma; Cell Membrane; Dimerization; Energy Transfer; Epidermal Growth Factor; ErbB Receptors; Fluorescent Dyes; Humans; Intracellular Fluid; Microscopy, Fluorescence; Phosphorylation; Rhodamines; Signal Transduction; Tumor Cells, Cultured

Membrane-associated and secreted proteins are an important class of proteins and include receptors, transporters, adhesion molecules, hormones and cytokines. Although algorithms have been developed to recognize potential amino-terminal membrane-targeting signals or transmembrane domains in protein sequences, their accuracy is limited and they require knowledge of the entire coding sequence, including the N terminus, which is not currently available for most of the genes in most organisms, including human. Several experimental approaches for identifying secreted and membrane proteins have been described, but none have taken a comprehensive genomic approach. Furthermore, none of these methods allow easy classification of clones from arrayed cDNA libraries, for which large-scale gene-expression data are now becoming available through the use of DNA microarrays. We describe here a rapid and efficient method for identifying genes that encode secreted or membrane proteins. mRNA species bound to membrane-associated polysomes were separated from other mRNAs by sedimentation equilibrium or sedimentation velocity. The distribution of individual transcripts in the 'membrane-bound' and 'cytosolic' fractions was quantitated for thousands of genes by hybridization to DNA microarrays. Transcripts known to encode secreted or membrane proteins were enriched in the membrane-bound fractions, whereas those known to encode cytoplasmic proteins were enriched in the fractions containing mRNAs associated with free and cytoplasmic ribosomes. On this basis, we identified over 275 human genes and 285 yeast genes that are likely to encode previously unrecognized secreted or membrane proteins.

Topics: Carbocyanines; Cell Membrane; Humans; Jurkat Cells; Membrane Proteins; Nuclear Proteins; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Saccharomyces cerevisiae; Subcellular Fractions

Fluorescence resonance energy transfer provides valuable long-range distance information about macromolecules in solution. Fluorescein and Cy3 are an important donor-acceptor pair of fluorophores; the characteristic Förster length for this pair on DNA is 56 A, so the pair can be used to study relatively long distances. Measurement of FRET efficiency for a series of DNA duplexes terminally labeled with fluorescein and Cy3 suggests that the Cy3 is close to the helical axis of the DNA. An NMR analysis of a self-complementary DNA duplex 5'-labeled with Cy3 shows that the fluorophore is stacked onto the end of the helix, in a manner similar to that of an additional base pair. This provides a known point from which distances calculated from FRET measurements are measured. Using the FRET efficiencies for the series of DNA duplexes as restraints, we have determined an effective position for the fluorescein, which is maximally extended laterally from the helix. The knowledge of the fluorophore positions can now be used for more precise interpretation of FRET data from nucleic acids.

Topics: Base Sequence; Carbocyanines; DNA; Energy Transfer; Fluorescein; Fluorescent Dyes; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Spectrometry, Fluorescence

Myosin V is an unconventional myosin thought to move processively along actin filaments. To have hard evidence for the high processivity, we sought to observe directly the movement by individual native chick brain myosin V (BMV) molecules with fluorescent calmodulin. Single BMV molecules did exhibit highly processive movement along actin filaments fixed to a coverslip. BMV continued to move up to the barbed end of its actin track, and did not readily detach from action. The barbed end, therefore, got brighter with time, because of a constant stream of BMV traffic. The maximum speed of the processive movement was 1 microm/s, and the maximum actin-activated ATPase rate was 2.4 s(-1). These values apparently imply that BMV travels a great distance, 400 nm, per an ATPase cycle.

Topics: Actins; Adenosine Triphosphatases; Animals; Brain; Calmodulin; Calmodulin-Binding Proteins; Carbocyanines; Chickens; Enzyme Activation; Fluorescent Dyes; Kinetics; Microscopy, Video; Molecular Motor Proteins; Movement; Myosin Type V; Nerve Tissue Proteins; Osmolar Concentration; Potassium Chloride; Protein Binding; Protein Denaturation

Recently developed methods for the selective labelling of cholinergic basal forebrain neurons containing the low-affinity neurotrophin receptor p75 (p75(NTR)) in vivo and in vitro are based on carbocyanine 3 (Cy3)-tagged antibodies directed against p75(NTR). The present study focuses on the maintenance of this neuronal label after injection of such fluorescent antibodies into the cerebral ventricle. One, 3, and 10 days after injection this marker exclusively stains neurons immunoreactive for the cholinergic markers choline acetyltransferase and vesicular acetylcholine transporter in the rat medial septum, diagonal band and nucleus basalis. Thirty days after injection the in vivo labelling was nearly abolished. Predominant labelling of lysosomes was shown by electron microscopic analysis following photoconversion of the Cy3-label to an electron-dense reaction product. The pre-labelling of cholinergic neurons might facilitate pharmacological and electrophysiological approaches in living slices and cell culture systems as well as detailed investigations focused on the transport of neurotrophins in vivo and in animals with experimentally altered p75(NTR) expression.

Topics: Acetylcholinesterase; Animals; Antibodies; Carbocyanines; Choline O-Acetyltransferase; Cholinergic Fibers; Fluorescent Dyes; Microscopy, Confocal; Microscopy, Fluorescence; Microscopy, Immunoelectron; Oxygen; Photochemistry; Prosencephalon; Rats; Rats, Wistar; Receptor, Nerve Growth Factor

Orientation dependence of single-fluorophore intensity was exploited in order to videotape conformational changes in a protein machine in real time. The fluorophore Cy3 attached to the central subunit of F(1)-ATPase revealed that the subunit rotates in the molecule in discrete 120 degrees steps and that each step is driven by the hydrolysis of one ATP molecule. These results, unlike those from the previous study under a frictional load, show that the 120 degrees stepping is a genuine property of this molecular motor. The data also show that the rate of ATP binding is insensitive to the load exerted on the rotor subunit.

Topics: Adenosine Triphosphate; Bacillus; Carbocyanines; Fluorescence; Fluorescent Dyes; Proton-Translocating ATPases

Using fluorescence microscopy, we studied the catalysis by and folding of individual Tetrahymena thermophila ribozyme molecules. The dye-labeled and surface-immobilized ribozymes used were shown to be functionally indistinguishable from the unmodified free ribozyme in solution. A reversible local folding step in which a duplex docks and undocks from the ribozyme core was observed directly in single-molecule time trajectories, allowing the determination of the rate constants and characterization of the transition state. A rarely populated docked state, not measurable by ensemble methods, was observed. In the overall folding process, intermediate folding states and multiple folding pathways were observed. In addition to observing previously established folding pathways, a pathway with an observed folding rate constant of 1 per second was discovered. These results establish single-molecule fluorescence as a powerful tool for examining RNA folding.

Topics: Animals; Biotinylation; Carbocyanines; Catalysis; Fluorescent Dyes; Guanosine; Kinetics; Microscopy, Fluorescence; Models, Molecular; Nucleic Acid Conformation; Oligoribonucleotides; RNA, Catalytic; RNA, Protozoan; Tetrahymena thermophila

A method for analyzing combinatorial DNA arrays using oligonucleotide-modified gold nanoparticle probes and a conventional flatbed scanner is described here. Labeling oligonucleotide targets with nanoparticle rather than fluorophore probes substantially alters the melting profiles of the targets from an array substrate. This difference permits the discrimination of an oligonucleotide sequence from targets with single nucleotide mismatches with a selectivity that is over three times that observed for fluorophore-labeled targets. In addition, when coupled with a signal amplification method based on nanoparticle-promoted reduction of silver(I), the sensitivity of this scanometric array detection system exceeds that of the analogous fluorophore system by two orders of magnitude.

Topics: Base Pair Mismatch; Base Pairing; Carbocyanines; Fluorescent Dyes; Gold; Microscopy, Confocal; Microscopy, Fluorescence; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes; Sensitivity and Specificity; Temperature

The uses of multiplex detection methodologies are dramatically increasing as a means to increase sample throughput and to demonstrate quantitative differences between multiple targets in gene or protein expression analysis. In this study, we investigate the application of multiplex fluorescent detection for three proteins on the same Western blot using a laser-scanning imaging system, the Bio-Rad Molecular Imager FX. We show that independent detection and quantitation of multiple targets is achievable with little or no correction for fluorescent crosstalk by using fluorescent tags preferentially excited with different laser lines and detected at wavelengths that minimize fluorescence crosstalk. We demonstrate that the use of fluorescent detection methods can provide a tenfold greater quantifiable range but with two- to fourfold less sensitivity than chemiluminescent detection methodologies. Two examples of three-color multiplex detection using FITC-, Cy3- and Cy5-conjugated probes on Western blots are provided to demonstrate applications of this approach.

Topics: Actins; Animals; Apoproteins; Apoptosis; Blotting, Western; Carbocyanines; Caspase 8; Caspase 9; Caspases; Cyclic AMP-Dependent Protein Kinases; Fluorescein-5-isothiocyanate; Fluorescent Dyes; HeLa Cells; Humans; Jurkat Cells; Lasers; Proteins; Spectrometry, Fluorescence; Spectrophotometry; Transferrin

To improve signal detection on cDNA microarrays, we adapted a fluorescent oligonucleotide dendrimeric signal amplification system to microarray technology. This signal detection method requires 16-fold less RNA for probe synthesis, does not depend on the incorporation of fluorescent dNTPs into a reverse transcription reaction, generates a high signal-to-background ratio, and can be used to allow for multichannel detection on a single chip. Furthermore, since the dendrimers can be detected individually, it may be possible, by employing dendrimer-binding standards, to calculate the numbers of bound cDNAs can be estimated. These features make the dendrimer signal detection reagent ideal for high-throughput functional genomics research.

Topics: Biotechnology; Carbocyanines; DNA Primers; DNA, Complementary; Fluorescent Dyes; Gene Expression Profiling; Genomics; Nucleic Acid Conformation; Nucleic Acid Hybridization; Oligodeoxyribonucleotides; Oligonucleotide Array Sequence Analysis; Reference Standards; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Substrate Specificity; Transcription, Genetic

Evidence for a new signaling mechanism consisting of ligand-independent lateral propagation of receptor activation in the plasma membrane is presented. We visualized the phosphorylation of green fluorescent protein (GFP)-tagged ErbB1 (ErbB1-GFP) receptors in cells focally stimulated with epidermal growth factor (EGF) covalently attached to beads. This was achieved by quantitative imaging of protein reaction states in cells by fluorescence resonance energy transfer (FRET) with global analysis of fluorescence lifetime imaging microscopy (FLIM) data. The rapid and extensive propagation of receptor phosphorylation over the entire cell after focal stimulation demonstrates a signaling wave at the plasma membrane resulting in full activation of all receptors.

Topics: Arsenicals; Carbocyanines; Cell Membrane; Diffusion; Dimerization; Endocytosis; Energy Transfer; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Fluorescence; Fluorescent Dyes; Green Fluorescent Proteins; Humans; Immunoglobulin Fab Fragments; Ligands; Luminescent Proteins; Microscopy, Confocal; Microscopy, Fluorescence; Microspheres; Phosphorylation; Phosphotyrosine; Protein Tyrosine Phosphatases; Signal Transduction; Tumor Cells, Cultured

The ability to analyze multiple polymorphic/mutation sites rapidly and accurately is pivotal in all areas of genetic analysis. We have applied single nucleotide primer extension (SNE) for detection of multiple point mutations in a micro-array format using two-color, fluorescent dye-tagged dideoxynucleoside triphosphate terminators (ddNTPs). The oligonucleotide primer ending one nucleotide short of the mutation site being probed is bound to the slide and single-base extended in place with two different Cy5/Cy3 dye-tagged terminators using solution-phase, locus-specific, single-stranded complementary templates generated by PCR from genomic DNA. The composite fluorescence produced contains peaks of distinct wave lengths corresponding to each Cy dye-tagged terminator incorporated, resulting in a fluorescent 'fingerprint' for each DNA target. DNA polymerase-catalyzed incorporation of Cy dye-tagged dideoxynucleoside triphosphates was dependent on the particular dyes, the specific ddNTP, the DNA target concentration, sequence of the template, on-slide temperature cycling and washing conditions. Results from analysis of mutations in the human hemochromatosis and connexin 26 genes show that this approach has several advantages over existing methods and is simple, rapid, robust, cost effective and accurate with potential applications in many areas of genetic analysis.

Topics: Amino Acid Substitution; Carbocyanines; Connexin 26; Connexins; DNA Mutational Analysis; DNA Primers; DNA Probes; Fluorescent Dyes; Hemochromatosis Protein; Histocompatibility Antigens Class I; HLA Antigens; Humans; Membrane Proteins; Point Mutation; Reproducibility of Results; Sensitivity and Specificity; Sequence Deletion

Total internal reflection fluorescence (TIRF) microscopy, used in conjunction with flash photolysis, provides a useful way of investigating the kinetics of macromolecular interactions. We compare different TIRF optical geometries to establish an optimal combination. Excitation light was introduced via four different arrangements: (1) a prism positioned on the microscope optical axis, (2) an offset prism with propagation through a silica slide trans to the objective lens, (3) an offset prism with propagation through a silica coverslip cis to a water-immersion objective lens and (4) a prismless arrangement using a high NA oil-immersion objective lens. Photolysis was achieved using a Xe flash lamp and a customised silica condenser lens. Single myosin molecules labelled with a Cy3 fluorophore were used as a test sample. Although the offset trans prism gave the best signal-to-background ratio, a customised thin rhombic prism incorporated, on axis, into the flash condenser assembly was almost as good and was more practical for scanning multiple fields. An oil-immersion lens gave the brightest image for sample depths < 30 micrometer but above this limit, a water-immersion lens was better. The prismless arrangement may offer advantages in other situations but it is important to check the actual numerical aperture of the objective lens.

Topics: Animals; Carbocyanines; Fluorescent Dyes; Microscopy, Fluorescence; Myosins; Optics and Photonics; Photolysis; Rabbits

A continuous load of 100 g (approximately 1 N) was applied externally to the mandible of rats for three hours and transferred to the alveolar bone via a molar interocclusal rubber pad. The animals were perfusion fixed and undecalcified sagittal molar slices approximately 150 microns thick were immunolabelled with endothelin-1 (ET-1) and alpha-smooth muscle actin (alpha-SMA) antibodies conjugated with CY5 and CY3 fluorophores, respectively. A confocal laser scanning microscope was used to capture the images as digital files in stacked layers for 3-D reconstructions. Blood vessel endothelium ET-1 immunolabelling occurred at disparate sites in the microvascular bed of normal and loaded alveolar bone. Three principal patterns of immunolabelling occurred: vessels labelled with either ET-1 or alpha-SMA, and vessels labelled with both antibodies. Short-term continuous loading produced upregulation of ET-1 immunofluorescence in the blood vessel endothelium, the surface cells of Haversian canals and bone marrow spaces. Pericytes were numerous along arterioles and postcapillary-sized venules.

Topics: Actins; Alveolar Process; Animals; Antibodies; Arterioles; Bone Marrow; Carbocyanines; Endothelin-1; Endothelium, Vascular; Fluorescent Antibody Technique, Direct; Fluorescent Dyes; Haversian System; Image Processing, Computer-Assisted; Imaging, Three-Dimensional; Immunohistochemistry; Male; Microcirculation; Microscopy, Confocal; Molar; Pericytes; Rats; Rats, Sprague-Dawley; Stress, Mechanical; Tooth; Venules; Weight-Bearing

Investigations of fluorescence lifetimes of the dye 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoic acid (NBD-HA) and of DNA M13 primers labeled with NBD-HA, Cy3, rhodamine green and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3-propionic acid (BODIPY-FL) dyes in polyacrylamide gels of various degrees of crosslinking and different crosslinkers, and in a cellulose sieving buffer with different organic modifiers, are described. The dependence of fluorescence lifetime on gel matrix and on experimental conditions was studied in order to identify which factors may be important for optimization of multiplex fluorescence lifetime detection. Lifetimes were determined in both batch solution and on-the-fly, on-column in CE. Results show that lifetimes of the primer-attached dyes remain constant in gels of different composition. Additionally, multiexponential fluorescence decays are observed for primer-attached dyes in batch solutions of the cellulose sieving buffers but are reduced to monoexponential decays when measured on-the-fly, on-column in CE. Lifetime detectability can be improved by addition of an organic modifier to the gel matrix.

Topics: Acrylic Resins; Boron Compounds; Buffers; Carbocyanines; Cellulose; DNA Primers; Electrophoresis, Capillary; Fluorescence; Fluorescent Dyes; Gels; Time

Following receptor binding and internalization, intracellular trafficking of adenovirus (Ad) among subgroups B and C is different, with significant amounts of Ad serotype 7 (Ad7) (subgroup B) virions found in cytoplasm during the initial hours of infection while Ad5 (subgroup C) virions rapidly translocate to the nucleus. To evaluate the role of the fiber in these differences, we examined intracellular trafficking of Ad5, Ad7, and Ad5f7 (a chimeric vector composed of the Ad5 capsid with the fiber replaced by the Ad7 fiber) by conjugating Ad capsids directly with Cy3 fluorescent dye, permitting the trafficking of the capsids to be examined by fluorescence microscopy. The human lung carcinoma cell line A549 was infected with Cy3-conjugated viruses for 10 min followed by a 1-h incubation. Ad5 virions rapidly translocated to the nucleus (within 1 h of infection), while Ad7 virions were widely distributed in the cytoplasm at the same time point. Interestingly, chimeric Ad5f7 virions behaved similarly to Ad7 but not Ad5. In this regard, the percentages of nuclear localization of Ad5, Ad7, and Ad5f7 at 1 h following infection were 72% +/- 4%, 32% +/- 6%, and 38% +/- 2%, respectively. Consistent with these observations, fluorescence in situ hybridization demonstrated that most of the Ad5 DNA was detected at the nucleus after 1 h, but at the same time point, DNA of Ad7 and Ad5f7 was distributed in both the nucleus and cytoplasm. Quantification of the kinetics of Ad genomic DNA delivery to the nucleus using a fluorogenic probe-based PCR assay (TaqMan PCR) demonstrated that the percentages of nuclear association of Ad5 DNA and Ad5f7 DNA at 1 h postinfection were 80% +/- 13% and 43% +/- 1%, respectively. Although it has been generally accepted that Ad fiber protein mediates attachment of virions to cells and that fibers dissociate during endocytic uptake, these data suggest that in addition to mediating binding to the cell surface, fiber likely modulates intracellular trafficking as well.

Topics: Adenoviruses, Human; Biological Transport; Capsid; Capsid Proteins; Carbocyanines; Cell Nucleus; DNA, Viral; Fluorescent Dyes; Genes, Viral; Genetic Vectors; Genome, Viral; Humans; Intracellular Fluid; Kinetics; Serotyping; Tumor Cells, Cultured

Human erythrocyte spectrin was labelled with the probe 5, 5'-disulfato-1-(6-hexanoic acid N-hydroxysuccinimide ester)-1'-ethyl-3,3,3',3'-tetramethylindocarbocyanine (Cy3). Cy3-spectrin was bound to the outer surface of dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles and its diffusion measured by fluorescence recovery after photobleaching (FRAP). It was found that at 30 degrees C, above the lipid gel to liquid-crystalline phase transition of the lipids, Cy3-spectrin had an unexpectedly high diffusion coefficient D=(2.1+/-0.6)x10(-7)) cm2/s. At the phase transition, diffusion of Cy3-spectrin was only slightly lower; D=(1.3+/-0.3)x10(-7) cm2/s, whereas at 14 degrees C, well below the lipid phase transition, diffusion was found to be much slower with D=(3.1+/-0.12)x10(-9) cm2/s. The fast diffusion of Cy3-spectrin on the lipid surface implies that the individual bonds which bind spectrin to the lipid surface must rapidly be made and broken. In the light of these results, spectrin-lipid interactions alone appear unlikely to have any significant role in supporting the cell membrane. Probably, the interactions serve only to localise the spectrin at the inner lipid surface in order to facilitate formation of the cytoskeleton.

Topics: Carbocyanines; Diffusion; Dimyristoylphosphatidylcholine; Fluorescent Dyes; Lipids; Liposomes; Spectrin; Surface Properties; Temperature

Turnover is important for the maintenance and remodeling of the cytoskeleton during the processes of cell morphogenesis, mitosis and motility. Microtubule (MT) turnover is thought to occur by dynamic instability, growth and shortening at distal (plus) ends. Recent observation of MT release from the centrosome and depolymerization from proximal (minus) ends indicates the existence of a minus end pathway. To evaluate the relative contributions of plus and minus end pathways to turnover, we analyzed MT dynamics in a model system, the fish melanophore, a large non-motile cell with a regular radial array of long MTs. MT ends were tracked in digital fluorescence time-lapse sequences and life histories of individual MTs were analyzed using random walk theory generalized to the case of diffusion with drift. Analysis of plus end dynamics gave an apparent diffusion coefficient of D=7.5 microm2/minute. The random walk model predicts that the half-time for turnover driven solely by plus end dynamics will depend strongly on position in the cell. Based on the experimentally determined value of D, turnover of MTs near the center of a typical melanophore of radius 70 microm was calculated to require over 5 hours, a paradoxically long time. To examine MT behavior deep in the cytoplasm, we developed a novel, sequential subtraction mode of image analysis. This analysis revealed a subpopulation of MTs which shortened from their minus ends, presumably after constitutive release from the centrosome. Given the relative slowness of plus end dynamics to turn over the root of a long MT, the turnover of MTs near the cell center is determined primarily by the minus-end pathway. MTs released from the centrosome become replaced by newly nucleated ones. The relative contributions of plus and minus end pathways was estimated from the diffusion coefficient, D, for the plus end, the length distribution of MTs, t he frequency of free minus ends, and the rate of minus-end shortening. We conclude that, in large animal cells with a centrosomally focussed array of MTs, turnover occurs by a combination of plus and minus end pathways, the plus end dominating at the cell periphery and the minus end dominating near the cell center.

Topics: Animals; Carbocyanines; Cell Differentiation; Cell Movement; Fishes; Kinetics; Melanophores; Microscopy, Fluorescence; Microtubules; Mitosis; Movement

Topics: Animals; Blood Vessels; Carbocyanines; Deoxycholic Acid; Deoxyribonucleases; Detergents; Edetic Acid; Epoxy Compounds; Epoxy Resins; Fluorescent Dyes; Immersion; Microscopy, Confocal; Octoxynol; Rats; Specimen Handling; Tissue Fixation

The ends of chromosomes (telomeres) are important to maintain chromosome stability, and the loss of telomere repeat sequences has been implicated in cellular senescence and genomic instability of cancer cells. The traditional method for measuring the length of telomeres (Southern analysis) requires a large number of cells (>10(5)) and does not provide information on the telomere length of individual chromosomes. Here, we describe a digital image microscopy system for measurements of the fluorescence intensity derived from telomere repeat sequences in metaphase cells following quantitative fluorescence in situ hybridization (Q-FISH).. Samples are prepared for microscopy using Q-FISH with Cy3 labeled peptide nucleic acid probes specific for (T(2)AG(3))(n) sequences and the DNA dye DAPI. Separate images of Cy3 and DAPI fluorescence are acquired and processed with a dedicated computer program (TFL-TELO). With the program, the integrated fluorescence intensity value for each telomere, which is proportional to the number of hybridized probes, is calculated and presented to the user.. Indirect tests of our method were performed using simulated as well as defined tests objects. The precision and consistency of human telomere length measurements was then analyzed in a number of experiments. It was found that by averaging the results of less than 30 cells, a good indication of the telomere length (SD of 10-15%) can be obtained.. We demonstrate that accurate and repeatable fluorescence intensity measurements can be made from Q-FISH images that provide information on the length of telomere repeats at individual chromosomes from limited number of cells.

Topics: Algorithms; Calibration; Carbocyanines; DNA; Humans; In Situ Hybridization, Fluorescence; Indoles; Karyotyping; Lymphocytes; Male; Metaphase; Microscopy, Fluorescence; Particle Size; Repetitive Sequences, Nucleic Acid; Signal Processing, Computer-Assisted; Telomere

Tyramide signal amplification has successfully been applied to enhance detection limits of both immunological reactions and in situ hybridization methods. The technique uses short-range deposition of activated tyramide mediated by horseradish peroxidase. We have adapted this method to fluorescence in situ hybridization on embryonic tissue sections using fluorophore-labeled tyramide. The sensitivity of the procedure was sufficient to analyze the embryonic expression of mRNAs encoding both transcription factors and structural proteins. Combining fluorescence in situ hybridization and immunofluorescence with confocal microscopy allows the simultaneous detection of distinct mRNA species or of mRNAs together with proteins on the cellular level. Thus, the cell types expressing a particular gene at a given developmental stage can be studied even if no antibody to the gene product of interest is available. Moreover, the technique allows to study in situ the combinatorial marker expression that characterizes progenitor stages of a given cell lineage.

Topics: Animals; Carbocyanines; DNA-Binding Proteins; Embryonic and Fetal Development; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Gene Expression; In Situ Hybridization, Fluorescence; Lung; Mice; Sensitivity and Specificity; Transcription Factors

Adenovirus (Ad) vectors used for gene therapy are efficient in entering the infected cell and targeting their genome to the nucleus. To study the mechanism of the interaction between Ad and the nuclear envelope we have established an in vitro assay using rat liver nuclei incubated with serotype 5 Ad vector. Binding of either fluorophore (Cy3)-labeled Ad5 (Cy3-AdGFP) or [3H]Ad5 was blocked by excess unlabeled Ad5, indicating that the interaction was specific. Binding reached equilibrium within 30 min, exhibited temperature dependence with more binding occurring at 37 degrees C than at 4 degrees C and appeared to be irreversible. Prior treatment of nuclei with glutaraldehyde or proteolysis of nuclei with trypsin inhibited the Cy3-AdGFP association with nuclei, and pretreatment of Cy3-AdGFP with human anti-Ad5 serum, but not naive human serum, inhibited Cy3-AdGFP, suggesting a requirement for direct interaction between Ad5 and nuclei. Addition of excess unlabeled Ad serotype 2 or Ad serotype 7 competed for binding with Cy3-AdGFP, indicating that the capsid determinant of nuclear binding was conserved among group B and C Ad serotypes. These data suggest that the Ad capsid and nuclear envelope contain specific domains that mediate binding of the two entities and that binding mechanisms to the nuclear envelope might be a common final pathway of different Ad serotypes.

Topics: Adenoviridae; Animals; Capsid; Carbocyanines; Fluorescent Dyes; Genetic Vectors; Humans; In Vitro Techniques; Liver; Microscopy, Fluorescence; Nuclear Envelope; Rats

The localisation of NK(3) tachykinin receptors in guinea-pig ileum was studied using the fluorescently labelled agonists, Cy3. 5-neurokinin A and Cy3.5-kassinin. Binding to nerve cell bodies in the myenteric and submucosal plexuses was visualised using confocal microscopy. Binding to NK(1) receptors was blocked by the NK(1) receptor antagonist, CP-99994. NK(3) receptors, demonstrated by binding in the presence of CP-99994, occurred in 72% of myenteric and 38% of submucosal neurons. Colocalisation with other markers was examined to deduce the classes of neurons with NK(3) receptors. In myenteric ganglia, NK(3) receptors were present on the following: 73% of calbindin-immunoreactive (IR) intrinsic primary afferent neurons, 63% of calretinin-IR excitatory motor neurons and ascending interneurons, 63% of nitric oxide synthase-IR inhibitory motor neurons and descending interneurons, 79% of strongly neuropeptide Y (NPY)-IR secretomotor neurons, 67% of weakly NPY-IR descending interneurons and motor neurons, and 46% of NK(1) receptor-IR neurons. In submucosal ganglia, NK(3) receptors were on 65% of calretinin-IR secretomotor/vasodilator neurons, 81% of NPY-IR cholinergic secretomotor neurons, 2% of vasoactive intestinal peptide-IR non-cholinergic secretomotor neurons and were completely absent from substance P-IR intrinsic primary afferent neurons. The results support physiological studies suggesting that NK(3) receptors mediate tachykinin transmission between myenteric sensory neurons and to interneurons and/or motor neurons in descending inhibitory and ascending excitatory pathways.

Topics: Animals; Carbocyanines; Cell Count; Female; Fluorescent Dyes; Guinea Pigs; Ileum; In Vitro Techniques; Kassinin; Male; Microscopy, Confocal; Myenteric Plexus; Neurokinin A; Neurons; Piperidines; Receptors, Neurokinin-3; Submucous Plexus

Conjugates between anti-tetanus F(ab')2 fragments and the (37-72) fragment of the HIV Tat protein were taken up by chromaffin cells, NG108-15 neurohybridoma cells and Rev-2-T-6 lymphoma cells. The uptake could not be inhibited by competition with (37-72)Tat, but was reduced in the presence of metabolic inhibitors or at low temperature. The disulfide as well as the thioether conjugate were translocated to the cytoplasmic space, but only the disulfide conjugate moderately restored the stimulated transmitter release inhibited by tetanus toxin. Therefore, disulfide conjugates are more promising than thioethers for the neutralization of intracellular antigens. These conjugates provide new tools to study neuroprotection against bacterial neurotoxins.

Topics: Animals; Antibodies; Carbocyanines; Cattle; Chromaffin Cells; Disulfides; Exocytosis; Gene Products, tat; Immunoglobulin Fab Fragments; Microscopy, Fluorescence; Norepinephrine; Peptide Fragments; Sulfides; tat Gene Products, Human Immunodeficiency Virus; Tetanus Toxin; Tumor Cells, Cultured

How seizures arise and recur in epilepsy is unknown. Recent genetic, pharmacological and electrophysiological data indicate a significant but undisclosed role for voltage-dependent calcium channels. Since the contribution such channels make to nerve function reflects the targeting of discrete subtypes to distinct cellular regions, we hypothesized that epilepsy reflects alterations in their spatiotemporal patterns of expression at the cell surface. To test this possibility, we examined the expression and distribution of hippocampal N-type calcium channels in an animal seizure model: kindling. Confocal microscopy of N-type calcium channels labeled with a new fluorescent ligand, coupled with a novel technique for analysing multiple images, revealed a 20-40% increase in their expression in CA1 and CA3 within 24 h post-seizure. These increases persisted in the dendritic fields of CA1, but had dissipated in CA3 by 28 days post-seizure. Such changes correlate poorly with cell number or synaptogenesis, but are consistent with increased N-type calcium channel expression on presynaptic terminals or, more likely, dendrites. These data rationalize recent electrophysiology and in situ hybridization data, and suggest that kindling alters N-type calcium channel trafficking mechanisms to cause a persistent, local, remodeling of their distributions in CA1 dendrites. The persistent induction of N-type calcium channels may be part of a mechanism for, and a hallmark of, synaptic plasticity, in which kindling represents a reinforcement of synapses en masse.

Topics: Animals; Calcium Channels, N-Type; Carbocyanines; Fluorescent Dyes; Hippocampus; Image Processing, Computer-Assisted; In Vitro Techniques; Kindling, Neurologic; Male; Microscopy, Confocal; omega-Conotoxins; Rats; Rats, Long-Evans; Time Factors

Human chromosomes terminate in a series of T2AG3 repeats, which, together with associated proteins, are essential for chromosome stability. In somatic cells, these sequences are known to be gradually lost through successive cells divisions; however, information about changes on specific chromosomes is not available. Individual telomeres could mediate important biological effects as was shown in yeast, in which loss of a single telomere results in cell-cycle arrest and chromosome loss. We now demonstrate by quantitative fluorescence in situ hybridization (Q-FISH; ref. 7) that the number of T2AG3 repeats on specific chromosome arms is very similar in different tissues from the same donor and varies only to some extent between donors. In all sixteen individuals studied, telomeres on chromosome 17p were shorter than the median telomere length--a finding confirmed by analysis of terminal restriction fragments from sorted chromosomes. These observations provide evidence of chromosome-specific factors regulating the number of T2AG3 repeats in individual telomeres and raise the possibility that the relatively short telomeres on chromosome 17p contribute to the frequent loss of 17p alleles in human cancers.

Topics: Adult; Bone Marrow Cells; Carbocyanines; Cells, Cultured; Chromosomes, Human, Pair 17; Fibroblasts; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; In Situ Hybridization, Fluorescence; Indoles; Metaphase; Repetitive Sequences, Nucleic Acid; Telomere

The transport of axonal microtubules in growing neurites has been a controversial issue because of clear but conflicting results obtained with fluorescence-marking techniques. We have attempted to resolve the discordance via analysis of the relationship between apparent microtubule translocation and cell adhesion. Neuronal cultures were prepared from Xenopus embryos 1 d after injection of Cy3-conjugated tubulin into one of the blastomeres of two-cell-stage embryos. Anterograde translocation of axonal microtubules was observed in neurons cultured on a laminin-coated surface, in agreement with previously published data for Xenopus embryonic neurons. However, when neuronal cultures were prepared on a concanavalin A-treated surface, the axonal microtubules were stationary, as reported for all other neurons investigated previously. Neuronal cultures prepared on laminin- and concanavalin A-coated surfaces also demonstrated dramatic differences in the pattern of axonal growth, dynamics of axonal microtubules, and response to brefeldin A treatment. Our findings suggest that transport and dynamics of axonal microtubules may be directly affected by the mechanical tension produced by growth cone activity.

Topics: Animals; Axons; Brefeldin A; Carbocyanines; Cell Adhesion; Cell Culture Techniques; Cell Membrane; Cells, Cultured; Concanavalin A; Cyclopentanes; Fluorescent Dyes; Laminin; Microinjections; Microtubules; Neurons; Photochemistry; Protein Synthesis Inhibitors; Stress, Mechanical; Swine; Tubulin; Xenopus

Stem cell factor (SCF) binding to the c-kit receptor triggers homodimerization and intermolecular tyrosine phosphorylation of the c-kit receptor, thus initiating signal transduction. Receptor dimerization is a critical early step in this process. Prior biochemical studies of c-kit receptor dimerization have mainly used affinity cross-linking techniques, which are beset with problems including low efficiency of cross-linking and the usual requirement for radiolabeled SCF to detect the cross-linked complex. We used the fluorescence resonance energy transfer (FRET) technique to examine the effects of SCF and other hematopoietic cytokines on c-kit receptor dimerization. The nonneutralizing anti-c-kit receptor monoclonal antibody 104D2 was directly conjugated to fluorescein isothiocyanate (FITC) or to the carbocyanine dye Cy3 and used to label cytokine-responsive human hematopoietic cell lines. The ability of SCF to induce c-kit receptor dimerization was assessed by flow cytometric analysis of FRET between the donor fluorochrome FITC and the acceptor fluorochrome Cy3. SCF induced a dose-dependent increase in c-kit receptor dimerization that correlated well with the concentrations of SCF required to stimulate cell proliferation. Receptor dimerization was detectable within 3 minutes after the addition of SCF and was maximal 30 minutes after the addition of SCF. Confocal microscopy showed redistribution of the c-kit receptor (from a diffuse distribution on the cell surface to "caps" at one end of the cell) within 3 minutes after SCF addition, followed by receptor internalization. Reappearance of the c-kit receptor on the cell surface required new protein synthesis, suggesting that the c-kit receptor is not recycled to the cell surface after internalization. Finally, erythropoietin (Epo), but not the structurally and functionally related cytokine thrombopoietin (Tpo), stimulated c-kit receptor dimerization detectable by FRET, and tyrosine phosphorylation of the c-kit receptor. These results suggest that exposure to Epo can activate the c-kit receptor and provide further evidence for cross-talk between the Epo and c-kit receptors in human hematopoietic cell lines. Studies with progeny of burst-forming unit-erythroid (BFU-E) suggest that the FRET technique is sufficiently sensitive to detect c-kit receptor dimerization on normal human hematopoietic cells.

Topics: Carbocyanines; Cell Division; Cell Line; Cytokines; Dimerization; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Hematopoietic Stem Cells; Humans; Kinetics; Microscopy, Confocal; Proto-Oncogene Proteins c-kit; Spectrometry, Fluorescence; Stem Cell Factor

The pathogenic agent, adenovirus (Ad), has taken on a new role as a vector for gene transfer in both laboratory and clinical settings. To help understand the intracellular pathways and fate of Ad gene transfer vectors, we covalently conjugated fluorophores to E1-, E3- Ad vectors and used quantitative fluorescence microscopy to assess essential steps of Ad vector gene transfer to the A549 human epithelial lung cell line including binding, internalization, escape from endosomes, translocation to the nucleus, dissociation of capsids and gene expression. The data demonstrate that Ad internalizes with a t1/2 2.5 min, breaks out of endosomes early, likely prior to endosome-endosome fusion, exhibits sustained, intracellular velocities averaging 0.58 microm/sec, and translocates to the nucleus with >80% of internalized fluorophore demonstrating nuclear localization within 60 min of infection. Interestingly, 24 hr after infection, half of the initially internalized fluorescence was detected but lacked nuclear localization, suggesting that the capsid is released from the nucleus and is likely degraded. Fluorescent labeling of virions provides a novel quantitative, morphological strategy to characterize the interaction of gene transfer vectors with the intracellular environment.

Topics: Adenoviridae; Carbocyanines; Cell Nucleus; Cytosol; Endosomes; Fluorescent Dyes; Gene Transfer Techniques; Genetic Vectors; Humans; Hydrogen-Ion Concentration; Time Factors; Tumor Cells, Cultured; Virion

Monoclonal antibodies to two different targetable antigens were conjugated to each of four commercially available cyanine fluorochromes. Equal amounts of all four antibodies were coinjected into tumor-bearing animals and imaged. Small, superficial tumors were adequately labeled using all four fluorochromes. Large tumors were labeled well only by Cy7, probably due to self-masking and dilution effects. Cy7 was superior to other cyanine fluorochromes for visualizing structures located deep within the animal.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Benzothiazoles; Carbocyanines; Fluorescent Dyes; Humans; Mice; Mice, Inbred BALB C; Nucleolin; Phosphoproteins; RNA-Binding Proteins; Teratocarcinoma; Tumor Cells, Cultured

Sensitization of RBL-2H3 mast cells with monomeric fluorescein-5-isothiocyanate (FITC)-labeled immunoglobulin E (IgE) results in slow but highly efficient accumulation of labeled IgE fragments in a pool of acidic peripheral vesicles that are visible by fluorescence microscopy after raising endosomal pH with ammonium chloride. Stimulation of cells containing these FITC-IgE fragments by aggregation of high affinity receptors for IgE (FcepsilonRI) or by Ca2+ ionophore and phorbol 12-myristate 13-acetate results in release of FITC fluorescence from the cells, which can be monitored continuously with a spectrofluorometer. The fluorescence release process corresponds to cellular degranulation: it is prevented under conditions that prevent stimulated beta-hexosaminidase release, and these two processes exhibit the same antigen dose-dependence and kinetics. Pulse-chase labeling reveals that aggregation of FITC-IgE bound to FcepsilonRI at the cell surface causes internalization and delivery to the regulated secretory vesicles with a high efficiency similar to monomeric IgE-FcepsilonRI, but more rapidly. Binding of Cy3-modified IgE to FcepsilonRI results in labeling of the same secretory vesicles as in FITC-IgE-sensitized cells, and these Cy3-labeled vesicles can be observed by fluorescence microscopy without neutralization of intracellular compartments. Simultaneous three-photon microscopy of serotonin fluorescence and two-photon microscopy of Cy3 fluorescence reveals that these Cy3-labeled vesicles coincide with serotonin-labeled secretory granules. After stimulation of the cells via aggregation of IgE-FcepsilonRI or addition of Ca2+ ionophore and phorbol 12-myristate 13-acetate, depletion of the Cy3 label from the intracellular vesicles is observed with confocal microscopy. These results provide strong evidence for the lysosomal nature of secretory granules in these cells. In addition, they provide the basis for a direct, real-time method for monitoring single cell degranulation.

Topics: Animals; Carbocyanines; Cell Degranulation; Cytoplasmic Granules; Endocytosis; Exocytosis; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Immunoglobulin E; Immunoglobulin Fragments; Ionophores; Mast Cells; Microscopy, Confocal; Microscopy, Fluorescence; Rats; Receptors, IgE; Serotonin; Tetradecanoylphorbol Acetate

The aims of this work were to determine whether cells that are similar to the interstitial cells of Cajal (ICC) and have immunoreactivity for the neurokinin 1 (NK1) receptor are indeed ICC; to determine whether the agonist, substance P, binds to and activates the receptor on presumptive ICC; and to investigate the relationship between substance P-immunoreactive nerve fibres and ICC. ICC at the level of the myenteric plexus and in the deep muscular plexus in the duodenum and ileum of the guinea-pig were investigated. Immunoreactivities for the ICC marker, Kit, and the NK1 receptor were colocalised in ICC of the myenteric and deep muscular plexuses. In tissue fixed immediately after its removal from the animal, NK1 receptor-immunoreactive ICC were found at the level of the myenteric plexus in the duodenum, but not in the ileum, and in the deep muscular plexus in the duodenum and ileum. The majority of receptor immunoreactivity was on the cell surface. ICC were exposed to substance P (10(-7) M), initially at 4 degrees C for 1 h to allow the agonist to bind, followed by incubation at 37 degrees C to allow receptor internalisation to proceed. Exposure to substance P caused the NK1 receptor immunoreactivity to aggregate in clumps in the cytoplasm of ICC of the myenteric and deep muscular plexuses, including the ICC of the myenteric plexus of the ileum, where NK1 receptor immunoreactivity was not seen if tissue was not exposed to substance P. Substance P, to which the fluorescent label, cyanine 3.18 (Cy-3), was coupled, bound to the ICC. The Cy-3-substance P was internalised with the receptor following warming to 37 degrees C. Many, but not all, ICC were closely apposed by nerve fibres with immunoreactivity for substance P. It is concluded that the NK1 receptor immunoreactivity on ICC represents receptor that is functional in the sense that it binds the natural agonist substance P and undergoes agonist-induced internalisation. ICC are likely to receive excitatory innervation from the close approaches of tachykinin-containing nerve fibres.

Topics: Animals; Carbocyanines; Cell Membrane; Duodenum; Enteric Nervous System; Female; Fluorescent Antibody Technique; Fluorescent Dyes; Guinea Pigs; Ileum; Intestine, Small; Male; Microscopy, Fluorescence; Monensin; Myenteric Plexus; Nerve Fibers; Proto-Oncogene Proteins c-kit; Receptors, Neurokinin-1; Substance P

Topics: Animals; Caenorhabditis elegans; Carbocyanines; Fluorescent Dyes; Green Fluorescent Proteins; Kinesins; Luminescent Proteins; Male; Maleimides; Microscopy, Fluorescence; Microscopy, Video; Molecular Motor Proteins; Movement; Mutagenesis, Site-Directed; Protein Biosynthesis; Protein Isoforms; Recombinant Fusion Proteins; Sea Urchins; Sperm Tail

Fluorescence correlation microscopy (FCM), the combination of fluorescence correlation spectroscopy (FCS) and digital microscopy (Brock and Jovin, 1998. Cell. Mol. Biol. 44:847-856), has been implemented for measuring molecular diffusion and association in living cells with explicit consideration of autocorrelations arising from autofluorescence. Autofluorescence excited at 532 nm colocalizes with mitochondria, has flavin-like spectral characteristics, exhibits relaxation times characteristic for the diffusion of high-molecular-weight proteins, and depends on the incubation conditions of the cells. These time- and location-dependent properties preclude the assignment of universal background parameters. The lower limit for detection of microinjected dextran molecules labeled with the carboxymethylindocyanine dye Cy3 was a few thousand molecules per cell, and the diffusion constant of 1.7 x 10(-7) cm2/s agreed well with values measured with other methods. Based on the fluorescence signal per molecule (fpm) and the molecule number derived from autocorrelation analysis, a new method is devised to define intracellular association states. We conclude that FCM is a powerful, noninvasive method for probing molecular interactions in femtoliter volume elements within defined subcellular locations in living cells.

Topics: 3T3 Cells; Animals; Carbocyanines; Dextrans; Diffusion; Fluorescent Dyes; Humans; Lasers; Mice; Microinjections; Microscopy, Fluorescence; Mitochondria; Time Factors; Tumor Cells, Cultured

We describe fluorescence immunostaining of four different antigens in the same section. The fluorochrome conjugates used show highest emission in the blue, green, yellow, and red regions of the visible light spectrum, respectively. Specially designed single fluorochrome filter combinations allow selective visualization of each fluorochrome label in turn, without visible crosstalk with the others.

Topics: Adrenocorticotropic Hormone; Animals; Carbocyanines; Cattle; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique, Direct; Fluorescent Dyes; Humans; Microscopy, Fluorescence; Pituitary Gland; Rats; Rhodamines; Swine; Tranexamic Acid

We demonstrate that the CAtalyzed Reporter Deposition method (CARD), utilizing the novel fluorescent reporter Cy3.29-tyramide, is successful in the Fluorescent in Situ Hybridization (FISH) detection of RNA and single-copy DNA. Histone 4 expression is detected in RNA extracts of 5-phase, synchronized HeLa cells by dot-blot analysis. Gene expression of histone 4 in HeLa cells is demonstrated by FISH via CARD, utilizing oligonucleotide probes. Fluorescence intensity measurements on CARD-amplified histone 4 RNA detection showed (a) a 25-fold amplification of the signal brightness by biotinylated oligonucleotide probes and (b) a sixfold amplification of the signal brightness by horseradish peroxidase (HRP)-labeled histone 4 probes vs the directly stained control. The sensitivity of the CARD method is demonstrated by the FISH detection of single-copy DNA on human corneal fibroblast and HeLa S5 interphase nuclei. Chromosomal localization of the single copy DNA is demonstrated on HeLa S3 metaphase chromosome spreads.

Topics: Avidin; Biotin; Carbocyanines; Cell Nucleus; Cornea; DNA; Fibroblasts; Fluorescent Dyes; Gene Expression; HeLa Cells; Histones; Horseradish Peroxidase; Humans; Immunoenzyme Techniques; In Situ Hybridization, Fluorescence; Interphase; RNA; S Phase; Tyramine

In order to resolve multiple fluorophores by their lifetimes in discrete tissue domains, the labeling intensity must be sufficiently strong and the intensity-difference between the labels must not be too large, the rate of fading should be similar for all fluorophores, and the lifetimes of the fluorophores should be sufficiently discrete. We could readily distinguish Cyanine-3.18 (Cy-3), Lissamine Rhodamine (LRSC), and Texas Red when they were not colocalized in tissue profiles. Colocalization of Cy-3 and LRSC, as well as Cy3 and Texas Red, could also be distinguished, while the combination of LRSC and Texas Red was more difficult. We have used fluorescence lifetime recordings in confocal microscopy to detect different neuropeptides in neurons. We demonstrate that somatostatin and galanin are colocalized in axon profiles of the spinal cord dorsal horn.

Topics: Animals; Biotechnology; Calcitonin Gene-Related Peptide; Carbocyanines; Computer Simulation; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Ganglia, Spinal; Microscopy, Confocal; Neurons, Afferent; Rats; Rats, Sprague-Dawley; Rhodamines; Serotonin; Somatostatin; Spinal Cord; Synaptophysin; Xanthenes

In this work, we studied the fluorescence and hybridization of multiply-labeled DNA probes which have the hydrophilic fluorophore 1-(straightepsilon-carboxypentynyl)-1'-ethyl- 3,3,3', 3'-tetramethylindocarbocyanine-5,5'-disulfonate (Cy3) attached via either a short or long linker at the C-5 position of deoxyuridine. We describe the effects of labeling density, fluorophore charge and linker length upon five properties of the probe: fluorescence intensity, the change in fluorescence upon duplex formation, the quantum yield of fluorescence (Phif), probe-target stability and specificity. For the hydrophilic dye Cy3, we have demonstrated that the fluorescence intensity andPhifare maximized when labeling every 6th base using the long linker. With a less hydrophilic dye, a labeling density this high could not be achieved without serious quenching of the fluorescence. The target specificity of multiply-labeled DNA probes was just as high as compared to the unmodified control probe, however, a less stable probe-target duplex is formed that exhibits a lower melting temperature. A mechanism that accounts for this destabilization is proposed which is consistent with our data. It involves dye-dye and dye-nucleotide interactions which appear to stabilize a single-stranded conformation of the probe.

Topics: Carbocyanines; DNA Probes; Fluorescence; Fluorescent Dyes; Molecular Structure; Sensitivity and Specificity

Fluorescent labelling methods for detecting microorganisms in water have limited sensitivity partly due to the natural autofluorescence from environmental particles. The aim of this study was to examine the autofluorescence of water samples to determine the optimal excitation source and fluorescent labels for minimising background autofluorescence and therefore enhancing sensitive detection of Cryptosporidium oocysts. Particles concentrated from water were examined using fluorimetry at a wide range of excitation wavelengths to determine their autofluorescent properties. Two major peaks were identified emitting at 390 to 510 nm and at 640 to 700 nm. Flow cytometry was used to define the optical properties of oocysts immunofluorescently labelled with a range of fluorochromes. Concentrated water samples were analysed using flow cytometry and the number of particles with fluorescence and light scatter properties similar to the fluorescently labelled oocysts recorded. Fluorescein isothiocyanate exited at 488 nm was the most suitable label for oocysts in untreated water with less than 70 particles having optical properties similar to labelled oocysts, detected in 10 litre concentrates. The fluorochromes CY3, phycoerythrin (PE), and tetramethylrhodamine B thioisocyanate (TRITC) excited at 542 nm were the most suitable labels for oocysts in drinking water with less than 40 particles having optical properties similar to labelled oocysts, detected in 100 litre concentrates.

Topics: Animals; Carbocyanines; Cryptosporidium parvum; Environmental Pollutants; Evaluation Studies as Topic; Flow Cytometry; Fluorescence; Fluorescent Antibody Technique; Fluorescent Dyes; Fresh Water; Microscopy; Phycoerythrin; Rhodamines; Water Microbiology; Water Pollutants

We have refined prismless total internal reflection fluorescence microscopy with extremely low background to visualize single fluorophores attached to protein molecules interacting with a filamentous biopolymer labelled with different colour fluorophores. By using Stokes and anti-Stokes fluorescence, two different colour fluorescences from two different colour fluorophores excited with a single wavelength laser can be observed simultaneously. This microscopy was applied to visualize motor proteins, actin and myosin molecules. Single myosin molecules labelled with a tetramethylrhodamine-5-iodoacetamide interacting with a BODIPY FL-labelled actin filament, a filamentous polymer of actin molecules, were observed clearly and simultaneously in aqueous solution. Individual hydrolysis reactions of Cy3-labelled ATP by single myosin molecules and sliding of a BODIPY FL-labelled actin filament along the myosin molecules could also be observed simultaneously. Thus, this technique is useful for observing single molecular processes of proteins interacting with a biological macromolecule such as an actin filament and a DNA.

Topics: Actins; Actomyosin; Adenosine Triphosphate; Boron Compounds; Carbocyanines; Fluorescent Dyes; Lasers; Microscopy, Fluorescence; Microscopy, Video; Myosin Subfragments; Rhodamines; Spectrometry, Fluorescence

Progress has been made in improving the immunohistochemical detection of antigens for imaging and flow cytometry. We report the synthesis of a novel fluorescent horseradish peroxidase substrate, Cy3.29-tyramide, and its application in an enzyme-based signal amplification system, catalyzed reporter deposition (CARD). The catalyzed deposition of Cy3.29-tyramide was used to detect cell surface markers such as CD8 and CD25 on tonsil tissue and human lymphocytes. We compared the fluorescence CARD method to standard indirect immunofluorescence detection methods and found that an amplification of up to 15-fold was possible with CARD. The detection of the intracellular protein myosin II in fibroblastic cells and rabbit serum proteins blotted onto nitrocellulose was also improved. Thus, fluorescent CARD is a simple modification that can be made to standard immunofluorescence staining protocols to enhance significantly the detection of antigens.

Topics: 3T3 Cells; Animals; Antigens, Surface; Biomarkers; Carbocyanines; Collodion; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Fluorescent Dyes; Horseradish Peroxidase; Humans; Lymphocytes; Mice; Molecular Structure; Myosin Type II; Reproducibility of Results; Tyramine

Synthesis of fluorochrome-modified deoxyribonucleotides has been carried out mostly by linking the fluorochrome molecule to the C-5 position of dUTP via an allylamine spacer, similar to the modification of allylamine-dUTP with the haptens biotin and digoxigenin. Recently, a new series of fluorescent nucleotides has been prepared by using an alkynyl bridge between the uracil moiety and the fluorochrome. Here we report the qualitative and quantitative analysis of fluorescence in situ hybridization results obtained on interphase cells and chromosomes with a variety of highly repetitive and single-copy DNA probes that were modified by nick translation with such alkynyl dUTPs. A qualitative comparison was made of the alkynyl dUTPs conjugated to the fluorochromes fluorescein, the cyanine dye Cy3, tetramethylrhodamine, Lissamine and Texas Red. With the exception of tetramethylrhodamine, all fluorochromes performed satisfactorily. The cyanine dye Cy3 provided the highest sensitivity, i.e., cosmid and YAC probes could easily be visualized by conventional fluorescence microscopy. In a quantitative assay, different nick translation conditions were tested using a human chromosome 1 satellite III probe (pUC1.77) and alkynyl dUTPs labeled with fluorescein and Cy3. Using these two nucleotides, FISH signal intensities on interphase nuclei from human lymphocytes were quantitated by digital imaging microscopy. The strongest signals were obtained when during nick translation the ratio between dTTP and fluorescein-dUTP or Cy3-dUTP was 1:5.

Topics: Carbocyanines; Chromosomes, Artificial, Yeast; Chromosomes, Human, Pair 1; Cosmids; Deoxyuracil Nucleotides; DNA Probes; Evaluation Studies as Topic; Fluorescein; Fluoresceins; Fluorescent Dyes; Humans; In Situ Hybridization, Fluorescence; Leukocytes; Sensitivity and Specificity

Upon permeabilization of Swiss mouse 3T3 fibroblasts, an isoform of phosphatidylinositol transfer protein (PI-TP) was preferentially retained, a major part of which was associated with the perinuclear Golgi system (K. J. de Vries, A. Momchilova-Pankova, G. T. Snoek, and K. W. A. Wirtz, Exp. Cell Res. 215, 109-113, 1994). In the present study, the intracellular localization of this isoform (PI-TP beta) and the regular form (PI-TP alpha) was investigated in fetal bovine heart endothelial cells by microinjection of fluorescently labeled analogs followed by confocal laser scanning microscopy. The PI-TP alpha and PI-TP beta used were purified from bovine brain cytosol and covalently labeled with sulfoindocyanine dyes. By this novel method it was found that PI-TP beta was preferentially associated with perinuclear membrane structures whereas PI-TP alpha was predominantly present in the nucleus and in the cytoplasm. This intracellular localization was confirmed by indirect immunofluorescence indicating that the fluorescently labeled PI-TP alpha and PI-TP beta were targeted to the same sites as their endogeneous counterparts.

Topics: Animals; Aorta; Brain Chemistry; Carbocyanines; Carrier Proteins; Cattle; Cytoplasm; Endothelium, Vascular; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Membrane Proteins; Microinjections; Microscopy, Confocal; Nuclear Envelope; Phosphatidylinositols; Phospholipid Transfer Proteins

We report the first demonstration of directly recording fluorescence spectra of single cells in flow cytometry. An intensified, 512-element photodiode array was used in conjunction with a dispersing prism to capture the fluorescence emission spectra of Coulter ImmunoCheck calibration beads and Dictyostelium discoideum spores stained with the indocarbocyanine derivative CY3, fluorescein isothiocyanate, or R-phycoerythrin. The demonstration was made feasible by enhancing the signal-to-noise ratio of the detection process by using a fast gating technique applied to the detector. Results show that the complete fluorescence spectra of individual stained cells contain information that is normally not captured by conventional flow cytometers. By using the spectrographic flow cytometer, all this information is recorded, allowing small features and shifts in the fluorescence spectra of labelled particles to be studied.

Topics: Animals; Carbocyanines; Dictyostelium; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescence; Fluorescent Dyes; Lasers; Phycoerythrin

Peptides labelled with the fluorophore cyanine 3 were used to study naturally expressed neuropeptide receptors by confocal microscopy in continuous cell lines, primary cultures, and unfixed tissue. Swiss 3T3 fibroblasts bound cyanine 3-gastrin-releasing peptide at 4 degrees C, and internalized the peptide after 10 min at 37 degrees C. Internalization was specific, since it was blocked by incubation with unlabelled peptide. Primary cultures of myenteric neurons of the guinea pig incubated with cyanine 3-substance P at 4 degrees C had specific surface labelling. After 30 s at 37 degrees C, the peptide was internalized into vesicles in both the soma and neurites. Direct observation of live neurons showed movement of fluorescent vesicles to a perinuclear region after 30 min. Endocytosis was associated with a loss of surface binding sites. Unfixed whole mounts of guinea pig and rat ileum were incubated with cyanine 3-neurokinin A at 4 degrees C. After 5 min at 37 degrees C, Cy3-neurokinin A was specifically internalized in neurons and smooth muscle cells. After 30 min, a perinuclear labelling occurred in some cells. Labelling in rat neurons was diminished by the NK3-R antagonist SR142801. Thus, cyanine 3-neuropeptides are valuable tools to study expression and endocytosis of naturally expressed receptors.

Topics: 3T3 Cells; Animals; Carbocyanines; Endocytosis; Fluorescent Dyes; Mice; Microscopy, Confocal; Neurokinin A; Neurons; Neuropeptides; Rats; Receptors, Bombesin; Receptors, Tachykinin; Substance P

We labeled substance P (SP), neurokinin A (NKA), and [Lys0]gastrin-releasing peptide-27 (GRP) with cyanine 3.18 (cy3). Cy3-peptides purified by HPLC were fully active, determined by [Ca2+]i mobilization. Binding was specific because it was abolished by unlabeled peptides and receptor antagonists. Transfected cells yielded a log-fold greater cy3 intensity than control cells by FACS. Confocal microscopy of transfected cells and cultured enteric neurons showed that cy3-SP bound to surface receptors and was internalized. Internalization was observed in living cells by capture of sequential images. Recovery of surface binding sites was monitored by flow cytometry using cy3-SP. Thus, cy3 neuropeptides can be used to isolate and study receptor-bearing cells.

Topics: Animals; Carbocyanines; Cell Line, Transformed; Cells, Cultured; Chromatography, High Pressure Liquid; Computer Systems; Endocytosis; Flow Cytometry; Fluorescent Dyes; Gastrin-Releasing Peptide; Guinea Pigs; Neurokinin A; Peptides; Rats; Receptors, Neuropeptide; Substance P; Transfection

Utilization of a class of fluorescent cyanine dyes (Cy3) for the assay of gap junctional communication by the dye transfer method was examined. When compared with Lucifer Yellow (LY), a commonly used tracer, microinjected Cy3 dye was found to yield similar degrees of cell coupling. Blockade of the transfer of both tracers by 12-O-tetradecanoylphorbol-13 acetate (TPA), which is known to cause closure of communicating channels, confirmed gap junctional mediation of dye movement. The fixability of a microinjected amine derivative of Cy3 dye demonstrated its compatibility with immunostaining protocols involving fluorescein isothiocyanate (FITC)-conjugated reagents. These results together with the brilliant fluorescence of Cy3 dyes suggest the potential of Cy3 reagents as additional tools to study gap junction function.

Topics: Animals; Astrocytes; Carbocyanines; Cells, Cultured; Fluorescein-5-isothiocyanate; Fluorescence; Gap Junctions; Immunohistochemistry

We developed a new chromosome banding method by in situ hybridization of human Cot-1 DNA as a probe. Clear banding was produced on metaphase chromosomes of lymphoblastoid cells after probe detection with a fluorescent dye Cy3. Comparison with the known banding patterns revealed a similarity to the R-banding with some significant differences: some centromeric heterochromatin regions show Cot-1 positive bands. This suggests that some repetitive sequences from the heterochromatin regions constitute a major component of Cot-1 DNA. This unique chromosome banding method, Cot-1 banding, may be used as a supplement to the conventional karyotype analysis. Scanning analysis of the fluorescence intensities of Cot-1 banding and Q-banding are useful for objectively analyzing the banding pattern including a detection of chromosome aberrations. The Cot-1 banding with Cy3 is particularly powerful when applied for the gene mapping by fluorescence in situ hybridization (FISH) because red fluorescence of Cy3 for chromosome staining can be readily distinguished from green fluorescence of fluorescein isothiocyanate (FITC) for probe labeling. Using this novel method, we mapped a 4 kb-DNA fragment from myelin protein zero (MPZ) gene on the chromosome 1q22 to q23.

Topics: Carbocyanines; Chromosome Banding; Chromosomes, Human, Pair 1; DNA Probes; Fluorescent Dyes; Humans; In Situ Hybridization, Fluorescence; Myelin P0 Protein; Repetitive Sequences, Nucleic Acid

A highly-sensitive flourescence method, capable of detecting cytokine receptors present at low concentrations (around 100 molecules per cell) by flow cytometry, was adapted for use on tissue sections. This method was used to examine the expression of several cytokine receptors in lymphoid tissues. IL-2 receptors were distributed broadly, with higher concentrations in T cell areas. IL-1 receptor Type 1 was detected in T cell areas and in the follicular mantle, and was strongly expressed on vascular endothelium. IL-6 receptor was found at very low concentration, both within and outside germinal centres. The gp 130 molecule, which is involved in the functional receptor complex for IL-6 and several other cytokines, was present at higher concentrations, particularly in the germinal centre. Analysis of receptor expression in secondary lymphoid tissue provides evidence bearing on the physiological roles of cytokines, as these tissues contain cells at various stages of physiological activation located in well-defined functional zones.

Topics: Antibodies, Monoclonal; Carbocyanines; Flow Cytometry; Fluorescent Antibody Technique; Fluorescent Dyes; Humans; Lymphoid Tissue; Microscopy, Fluorescence; Phycoerythrin; Receptors, Cytokine; Sensitivity and Specificity; Staining and Labeling

Directly labeled fluorescent DNA probes have been made by nick translation and PCR using dUTP attached to the fluorescent label, Cy3, with different length linkers. With preparation of probes by PCR we find that linker length affects the efficiency of incorporation of Cy3-dUTP, the yield of labeled probe, and the signal intensity of labeled probes hybridized to chromosome target sequences. For nick translation and PCR, both the level of incorporation and the hybridization fluorescence signal increased in parallel when the length of the linker arm is increased. Under optimal conditions, PCR yielded more densely labeled probes, however, the yield of PCR labeled probe decreased with greater linear density of labeling. By using a Cy3-modified dUTP with the longest linker under optimal conditions it was possible to label up to 28% of the possible substitution sites on the target DNA with reasonable yield by PCR and 18% by nick translation. A mechanism involving steric interactions between the polymerase, cyanine-labeled sites on template and extending chains and the modified dUTP substrate is proposed to explain the inverse correlation between the labeling efficiency and the yield of DNA probe synthesis by PCR.

Topics: Carbocyanines; Deoxyuracil Nucleotides; DNA Probes; Fluorescent Dyes; In Situ Hybridization, Fluorescence; Nucleic Acid Hybridization; Polymerase Chain Reaction