tetramethylrhodamine and 6-carboxyfluorescein

Using 6-carboxyfluorescein (FAM) and tetramethyl rhodamine (TAMRA) as fluorescent signals a ratiometric fluorescent three-dimensional (3D) DNA walker based on a catalytic hairpin assembly (CHA) reaction for microRNA-122 detection was constructed. This method uses CHA reaction triggered indirectly by the target to mediate the 3D DNA walker operation to amplify the signal. The dual emission ratio fluorescent signal with a single excitation wavelength was used as the signal output. This strategy combines DNA walker with CHA reaction and proportional fluorescence signal output methods, which can effectively reduce the background fluorescence signal and the risk of generating false-positive signals. Thus, the impact of environmental factors on the experiment is reduced, thereby obtaining reliable and stable experimental results. It uses the fluorescence excitation wavelength of 488 nm and the maximum fluorescence emission wavelength of 520 nm and 580 nm, respectively. It has a good linear response at a microRNA concentration range of 156.0 pM ~ 7.00 nM and a detection limit of 42.94 pM. This strategy has been successfully applied to detect microRNAs in spiked serum samples. Graphical abstract Schematic representation of three-dimensional (3D) DNA walker constructed using catalytic hairpin self-assembly reaction (CHA)-assisted amplification and ratiometric fluorescence signal output for the detection of miRNA-122 closely related to hepatitis.

Topics: DNA; DNA Probes; Fluoresceins; Fluorescent Dyes; Gold; Humans; Inverted Repeat Sequences; Limit of Detection; Metal Nanoparticles; MicroRNAs; Nucleic Acid Hybridization; Rhodamines; Spectrometry, Fluorescence

Membrane fusion is broadly envisioned as a two- or three-step process proceeding from contacting bilayers through one or two semistable, nonlamellar lipidic intermediate structures to a fusion pore. A true fusion event requires mixing of contents between compartments and is monitored by the movement of soluble molecules between trapped compartments. We have used poly(ethylene glycol) (PEG) to rapidly generate an ensemble aggregated state A that proceeds sequentially through intermediates (I₁ and/or I₂) to a final fusion pore state (FP) with rate constants k₁, k₂, and k₃. Movement of moderately sized solutes (e.g., Tb³⁺/dipicolinic acid) has been used to detect pores assigned to intermediate states as well as to the final state (FP). Analysis of ensemble kinetic data has required that mixing of contents occurs with defined probabilities (αi) in each ensemble state, although it is unclear whether pores that form in different states are different. We introduce here a simple new assay that employs fluorescence resonance energy transfer (FRET) between a 6-carboxyfluorescein (donor) and tetramethylrhodamine (acceptor), which are covalently attached to complementary sequences of 10 bp oligonucleotides. Complementary sequences of fluorophore-labeled oligonucleotides were incorporated in vesicles separately, and the level of FRET increased in a simple exponential fashion during PEG-mediated fusion. The resulting rate constant corresponded closely to the slow rate constant of FP formation (k₃) derived from small molecule assays. Additionally, the total extent of oligonucleotide mixing corresponded to the fraction of content mixing that occurred in state FP in the small molecule assay. The results show that both large "final pores" and small (presumably transient) pores can form between vesicles throughout the fusion process. The implications of this result for the mechanism of membrane fusion are discussed.

Topics: Cell Membrane Structures; Fluoresceins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Indicators and Reagents; Kinetics; Membrane Fusion; Oligonucleotides; Phosphatidylcholines; Phosphatidylethanolamines; Picolinic Acids; Polyethylene Glycols; Porosity; Rhodamines; Solubility; Sphingomyelins; Surface Properties; Terbium

For the detection of bioanalytes, there is an ongoing search for synthetic sensors to replace enzyme-based assays which are sensitive to contaminants or suboptimal storage conditions. Lipopolysaccharide (LPS), a bacteria-borne endotoxin that may lead to life-threatening conditions such as septic shock, is one such case. Fluorescently labeled analogues of two peptide variants derived from the putative ligand-binding domain of the LPS-binding protein CD14 were developed that detect and discriminate LPS and lipids down to the submicromolar concentration range. Peptides are terminally labeled with carboxyfluorescein and tetramethylrhodamine. For one given peptide, sensitivity and specificity for the detection of LPS and discrimination from other lipids are achieved by spectral signatures that combine changes in the fluorescence resonance energy transfer (FRET) between both dyes and the total emission of tetramethylrhodamine. Alternatively, specificity is obtained by combining the FRET efficiencies of both peptide variants. In comparison to published synthetic LPS sensors, the CD14-derived sensors yield an increase in sensitivity by about 3 orders of magnitude and exhibit specificity for analytes for which the design of synthetic recognition elements is a challenging task. Moreover, one of the sensors enabled the detection of LPS in the presence of up to 50% fetal calf serum, thereby demonstrating the feasibility of this peptide-based approach for clinically relevant samples.

Topics: Binding Sites; Biosensing Techniques; Fluoresceins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Ligands; Lipids; Lipopolysaccharides; Peptides; Rhodamines

Opening of permeability transition (PT) pores in the mitochondrial inner membrane causes the mitochondrial permeability transition (MPT) and leads to mitochondrial swelling, membrane depolarization, and release of intramitochondrial solutes. Here, our aim was to develop high-throughput assays using a fluorescence plate reader to screen potential inducers and blockers of the MPT. Isolated rat liver mitochondria (0.5 mg/ml) were incubated in multiwell plates with tetramethylrhodamine methyl ester (TMRM, 1 microM), a potential-indicating fluorophore, and Fluo-5N (1 microM), a low-affinity Ca(2+) indicator. Incubation led to mitochondrial polarization, as indicated by uncoupler-sensitive quenching of the red TMRM fluorescence. CaCl(2) (100 microM) addition led to ruthenium red-sensitive mitochondrial Ca(2+) uptake, as indicated by green Fluo-5N fluorescence. After Ca(2+) accumulation, mitochondria depolarized, released Ca(2+) into the medium, and began to swell. This swelling was monitored as a decrease in light absorbance at 620 nm. Swelling, depolarization, and Ca(2+) release were prevented by cyclosporin A (1 microM), confirming that these events represented the MPT. Measurements of Ca(2+), mitochondrial membrane potential, and swelling could be made independently from the same wells without cross interference, and all three signals could be read from every well of a 48-well plate in about 1 min. In other experiments, mitochondria were ester-loaded with carboxydichlorofluorescein (carboxy-DCF) during the isolation procedure. Release of carboxy-DCF after PT pore opening led to an unquenching of green carboxy-DCF fluorescence occurring simultaneously with swelling. By combining measurements of carboxy-DCF release, Ca(2+) uptake, membrane potential, and swelling, MPT inducers and blockers can be distinguished from uncouplers, respiratory inhibitors, and blockers of Ca(2+) uptake. This high-throughput multiwell assay is amenable for screening panels of compounds for their ability to promote or block the MPT.

Topics: Animals; Calcium; Fluoresceins; Fluorescence; Intracellular Membranes; Ion Channels; Male; Membrane Potentials; Membrane Proteins; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Swelling; Permeability; Rats; Rats, Sprague-Dawley; Rhodamines

Fluorescence resonance energy transfer has been used to study the aggregation and organization of pardaxin and its analogues within lipid membranes. Peptide molecules labeled with 5- (and 6-) carboxyfluorescein at their N-terminal amino acid served as donors in these energy transfer measurements, whereas peptides labeled with 5- (and 6-) carboxytetramethylrhodamine at either their N- or C-terminal amino acid, served as acceptors. The membrane-permeating activity of the native molecule was maintained in the labeled peptides. Upon aggregation of the labeled peptides, fluorescence energy transfer was detected as a quenching of the donor fluorescence (520 nm), as well as an enhancement of the acceptor fluorescence (575 nm). Correlation exists between self-aggregation of the different analogues within membranes and their poreforming abilities. A comparison of the degrees of fluorescence energy transfer from N1-donor-labeled pardaxin to N1-acceptor-labeled pardaxin with the transfer efficiency observed in the interaction between the same donor and C1-acceptor-labeled pardaxin suggests that aggregates are formed in an ordered manner, with a preferentially parallel orientation of monomers within the aggregate. The extent of hetero-oligomer formation, i.e. complexes composed of two different analogue species, revealed that complementary charges contribute to peptide-peptide recognition within the lipid bilayer. Taken together, these results provide further support for the barrel stave model, involving parallel organization of monomers within the aggregate, as a description of the pore formation mechanism of pardaxin and its analogues.

Topics: Amino Acid Sequence; Chromatography, High Pressure Liquid; Coloring Agents; Fish Venoms; Fluoresceins; Fluorescence; Lipid Bilayers; Liposomes; Molecular Sequence Data; Peptides; Phospholipids; Rhodamines