carbocyanines and thiazole-orange

For decades the detection of nucleic acids and their interactions at low abundances has been a challenging task that has thus far been solved by enzymatic target amplification. In this work we aimed at developing efficient tools for amplification-free nucleic acid detection, which resulted in the synthesis of new fluorescent nanoparticles. Here, the fluorescent nanoparticles were made by simple and inexpensive radical emulsion polymerization of butyl acrylate in the presence of fluorescent dyes and additional functionalization reagents. This provided ultra-bright macrofluorophores of 9-84 nm mean diameter, modified with additional alkyne and amino groups for bioconjugation. By using click and NHS chemistries, the new nanoparticles were attached to target-specific DNA probes that were used in fluorimetry and fluorescence microscopy. Overall, these fluorescent nanoparticles and their oligonucleotide derivatives have higher photostability, brighter fluorescence and hence dramatically lower limits of target detection than the individual organic dyes. These properties make them useful in approaches directed towards ultrasensitive detection of nucleic acids, in particular for imaging and in vitro diagnostics of DNA.

Topics: Acrylates; Aminoacridines; Azides; Benzothiazoles; Carbocyanines; Click Chemistry; DNA Probes; Dynamic Light Scattering; Fluorescent Dyes; Fluorometry; Limit of Detection; MicroRNAs; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Nanoparticles; Particle Size; Perylene; Polymerization; Propylamines; Quinolines; Ultraviolet Rays

A convenient approach for the direct synthesis of meso-substituted thiazole orange (meso-TO) analogues has been unprecedentedly developed through the AlCl3-catalyzed reaction of parent TO with benzyl alcohol derivatives. Single-crystal X-ray structures show that the prepared new meso-TO analogues are nonplanar, forming a sharp contrast to planar TO. The spectral properties show that nonplanar meso-TO analogues do not aggregate, existing in monomer form (M) in PBS buffer, and have little effect of solvatochromism in different solvents. In comparison with the parents, meso-TO analogues exhibit a large Stokes shift, excellent light fastness, and inertness to singlet oxygen. A cellular association study demonstrates that incorporation of a benzyl group at the meso position methine of parent TO can decrease the cytotoxicity, change staining area in cells, and emit long-wavelength fluorescence for an extended time, which are useful for the development of smarter TOs for imaging in biological science.

Topics: Animals; Benzothiazoles; Carbocyanines; Crystallography, X-Ray; Fluorescence; Humans; Light; Magnetic Resonance Spectroscopy; MCF-7 Cells; Microscopy, Confocal; Models, Molecular; Molecular Structure; Photochemical Processes; Protein Domains; Quinolines; Rats; Singlet Oxygen; Solvents

RNA G-quadruplexes (G4s) are one of the key components of the transcriptome that act as efficient post-transcriptional regulatory elements in living cells. To conduct further studies of the unique biological functions of RNA G4s, techniques need to be developed that can efficiently recognize RNA G4 structures under various conditions, in fixed cells and living cells, as well as in vitro. This paper presents the development of such a method, a new technique using a cyanine dye called CyT, which can detect both canonical and non-canonical RNA G4 structures from test tubes to living human cells. The ability of CyT to distinguish between G4 and nonG4 RNA offers a promising tool for future RNA G4-based biomarker discovery and potential diagnostic applications.

Topics: Benzothiazoles; Carbocyanines; Cell Line; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; G-Quadruplexes; Humans; Microscopy, Confocal; Quinolines; RNA

A series of abasic site-binding ligands conjugated with cyanine dyes have been developed for "off-on" fluorescence sensing of an orphan nucleobase in DNA duplexes and DNA-RNA hybrids.

Topics: Benzothiazoles; Carbocyanines; DNA; Fluorescent Dyes; Ligands; Naphthyridines; Nucleic Acid Conformation; Quinolines; RNA; Spectrometry, Fluorescence

Exciton interactions between thiazole orange and thiazole red as nucleotide substitutes in DNA hairpins interfere with efficient energy transfer and fluorescence color change as readout. This interference can be tuned by two structural parameters that control the hairpin duplex stability.

Topics: Benzothiazoles; Carbocyanines; DNA Probes; Energy Transfer; Fluorescent Dyes; Nucleic Acid Conformation; Photochemical Processes; Quinolines; Spectrometry, Fluorescence; Spectrophotometry

The combination of thiazole orange and thiazole red as an internal energy transfer-based fluorophore pair in oligonucleotides provides an outstanding analytical tool to follow DNA/RNA hybridization through a distinct fluorescence color change from red to green. Herein, we demonstrate that this concept can be applied to small interfering RNA (siRNA) to monitor RNA integrity in living cells in real time with a remarkable dynamic range and excellent contrast ratios in cellular media. Furthermore, we show that our siRNA-sensors still possess their gene silencing function toward the knockdown of enhanced green fluorescent protein in CHO-K1 cells.

Topics: Animals; Base Sequence; Benzothiazoles; Biosensing Techniques; Carbocyanines; CHO Cells; Cricetinae; Cricetulus; Fluorescent Dyes; Gene Knockdown Techniques; Gene Silencing; Green Fluorescent Proteins; HeLa Cells; Humans; Microscopy, Confocal; Nucleic Acid Hybridization; Quinolines; RNA, Small Interfering; Transfection

The combination of thiazole orange (TO) and thiazole red (TR) as an internal pair of fluorescent DNA base surrogates ("DNA traffic lights") allows us to follow at least two consecutive DNA strand displacements in real time through a distinct fluorescence colour change from green to red and vice versa.

Topics: Benzothiazoles; Carbocyanines; Color; DNA; Fluorescence; Fluorescent Dyes; Molecular Structure; Quinolines; Spectrometry, Fluorescence; Staining and Labeling

The insertion of cyanine dye azides as energy donor dyes via postsynthetic "click"-type cycloaddition chemistry with e.g. a new thiazole orange azide combined with thiazole red yields dual emitting DNA probes with good fluorescence readout properties.

Topics: Benzothiazoles; Carbocyanines; Click Chemistry; Cycloaddition Reaction; DNA Probes; Energy Transfer; Fluorescent Dyes; Quinolines; Spectrophotometry, Ultraviolet

Analysis of (1)H and (13)C NMR and mass spectral data for the fluorescent nucleic acid stain SYBR Safe indicates that it contains a cyanine-based cationic core structure identical to thiazole orange. The difference between these two compounds is the type of N-substitution on the quinolinium ring system (SYBR Safe, n-Pr; thiazole orange, Me). The (1)H and (13)C NMR resonances for both compounds were assigned on the basis of one- and two-dimensional (COSY, ROESY, HSQC, and HMBC) experiments. The preferred conformation of these compounds was computed by ab initio methods and found to be consistent with the NMR data.

Topics: Benzothiazoles; Carbocyanines; Carbon Isotopes; Coloring Agents; Magnetic Resonance Spectroscopy; Molecular Structure; Quinolines

We have synthesized fluorescent DNA duplexes featuring multiple thiazole orange (TO) intercalating dyes covalently attached to the DNA via a triazole linkage. The intercalating dyes stabilize the duplex against thermal denaturation and show bright fluorescence in the green region of the spectrum. The emission color can be changed to orange or red by addition of energy-accepting Cy3 or Cy5 dyes attached covalently to the DNA duplex. The dye-modified DNA duplexes were then attached to a secondary antibody for intracellular fluorescence imaging of centrosomes in Drosophila embryos. Bright fluorescent foci were observed at the centrosomes in both the donor (TO) and acceptor (Cy5) channels, because the energy transfer efficiency is moderate. Monitoring the Cy5 emission channel significantly minimized the background signal because of the large shift in emission wavelength allowed by energy transfer.

Topics: Animals; Benzothiazoles; Carbocyanines; Centrosome; Diagnostic Imaging; DNA; Drosophila; Energy Transfer; Fluorescent Dyes; Immunoconjugates; Intercalating Agents; Quinolines

Subnuclear poly(A)(+) RNA localization in living mammalian cells was visualized by ratiometric analysis using hybridization-sensitive fluorescent oligonucleotide probes. Probes were oligonucleotides, which contained a Cy5 fluorescent dye at the strand end and a thiazole orange double-labeled nucleotide inside strand. A ratiometric analysis using poly(A)-targeting probes revealed a distribution of the probe itself as red fluorescence and localization of the target RNA sequence in cell nuclei as green fluorescence. The fluorescence of the subnuclear poly(A)(+) RNA hybridized with the poly(A)-targeting probes was observed as puncta in interchromatin areas.

Topics: Benzothiazoles; Carbocyanines; Fluorescent Dyes; Nucleic Acid Hybridization; Oligonucleotide Probes; Poly A; Quinolines; RNA

Kinetics and equilibria of cyanine dyes thiazole orange (TO) and benzothiazole orange (BO) self-aggregation and binding to CT-DNA are investigated in aqueous solution at 25 degrees C and pH 7. Absorbance spectra and T-jump experiments reveal that BO forms J-aggregates while TO forms more stable H-aggregates. Fluorescence and absorbance titrations show that TO binds to DNA more tightly than BO. TO stacks externally to DNA for low polymer-to-dye concentration ratios (C(P)/C(D)) while dye intercalation occurs for high values of C(P)/C(D). T-jump and stopped-flow experiments performed at high C(P)/C(D) agree with reaction scheme D+S <=> D,S <=> DS(I) <=> DS(II) where the precursor complex D,S evolves to a partially intercalated complex DS(I) which converts to the more stable intercalate DS(II). Non-electrostatic forces play a major role in D,S stabilization. Last step is similar for both dyes suggesting accommodation of the common benzothiazole residue between base pairs. Experiments using poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) confirm base pair preference for TO.

Topics: Benzothiazoles; Binding Sites; Carbocyanines; Computer Simulation; Crystallization; Dimerization; DNA; Fluorescent Dyes; Intercalating Agents; Kinetics; Models, Chemical; Quinolines

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)