Cyanine fluorophore derivatives with enhanced photostability

Altman, Roger B.; Terry, Daniel S.; Zhou, Zhou; Zheng, Qinsi; Geggier, Peter; Kolster, Rachel; Zhao, Yongfang; Javitch, Jonathan A.; Warren, Joel; Blanchard, Scott C.

doi:10.1038/nmeth.1774

Cited by 288 publications

(370 citation statements)

References 19 publications

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“…2). The signal-to-noise ratios (SNRs) (22,23) for continuous single-molecule fluorescence of the four dyes on the custom RS are comparable with the SNRs measured on our in-house built TIRF (3.18 for Cy3, 4.90 for Cy3.5, 2.64 for Cy5, and 3.05 for Cy5.5) under our measurement conditions ( Fig. 3 and SI Materials and Methods).…”

Section: Significancesupporting

confidence: 76%

High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence

Chen

Dalal

Petrov

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

138

162

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Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community.D etermining the molecular details of the time evolution of complex multicomponent biological systems requires analysis at the single-molecule level because of their stochastic and heterogeneous nature. Ideally, such experiments would track simultaneously the composition of a biological system (bound ligands, factors, and cofactors) and the conformation of the individual molecules in real time. Single-molecule fluorescence methods, such as total internal reflection fluorescence (TIRF) microscopy, allow the observations of the compositional dynamics (through arrival of fluorescently labeled ligands, factors, or cofactors) and conformational dynamics (through FRET) of single-molecular species. However, these traditional singlemolecule methods are hindered by limitations in maximal fluorescent component concentrations (up to 50 nM) (1), limited simultaneous detection (two to three colors) (2-6), and low throughput (a few hundred molecules at most per experiment) (7). As such, the full potential of single-molecule fluorescence to investigate a range of biological problems under physiologically relevant conditions has not yet been harnessed.Zero-mode waveguides (ZMWs) are small metallic apertures patterned on glass substrates that overcome the concentration restrictions by optically limiting background excitation (8). Each ZMW consists of an ∼150-nm-diameter metallic aperture that restricts the excitation light to a zeptoliter volume, making possible experiments with near-physiological concentrations (up to 20 μM) of fluorescently labeled ligands (1). Previous advances in nanofabrication (9), surface chemistry (10), and detection instrumentation (11) have led to ZMW-based instrumentation capable of the direct observation of DNA polymerization (12), reverse transcription (13), processive myosin motion (14), and translation by the ribosome (15, 16) with multicolor single-molecule detection. However, this sophisticated technology has not been broadly available to the scientific community. Despite multiple efforts to develop ZMW instrumentation, the combined difficulties in fabrica...

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Section: Significancesupporting

confidence: 76%

High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence

Chen

Dalal

Petrov

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

138

162

View full text Add to dashboard Cite

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“…Akin to earlier steady state measurements of ternary complex formation (16), the fluorescence-based assays described here are based on changes in relative fluorescence intensity. However, unlike earlier work, the approach we describe is based on the environmentally sensitive Cy3 fluorophore (43,58) at a distinct site of tRNA attachment, the naturally occurring modified nucleotide acp 3 U present at position 47 in E. coli tRNA Phe . Although this modification is not as ubiquitous as the s 4 U residue utilized previously, tRNA molecules modified at this site have been shown to be fully functional in all aspects of the translation process, including aminoacylation, tRNA selection, and translocation (43,46,60).…”

Section: Discussionmentioning

confidence: 99%

Elongation Factor Ts Directly Facilitates the Formation and Disassembly of the Escherichia coli Elongation Factor Tu·GTP·Aminoacyl-tRNA Ternary Complex

Burnett

Altman

Ferrao

et al. 2013

Journal of Biological Chemistry

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Background: Aminoacyl-tRNA (aa-tRNA) enters the ribosome in a ternary complex with the G-protein elongation factor Tu (EF-Tu) and GTP. Results: EF-Tu⅐GTP⅐aa-tRNA ternary complex formation and decay rates are accelerated in the presence of the nucleotide exchange factor elongation factor Ts (EF-Ts). Conclusion: EF-Ts directly facilitates the formation and disassociation of ternary complex. Significance: This system demonstrates a novel function of EF-Ts.

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“…Nevertheless, these fluorescent probes suffer from some defects such as photobleaching of fluorescent dye, high toxicity and expensive cost of noble metal sources, [18][19][20][21] which has further restricted their widespread application. Fortunately, it has been reported that the poly(amide amine) (PAMAM) dendrimers can emit strong fluorescence under suitable conditions, 22,23 and represent a new class of competitive material because of their autofluorescence and other prominent properties including favorable water solubility, biocompatibility, and nonimmunogenic and environmental friendliness.…”

Section: Introductionmentioning

confidence: 99%

Autofluorescent Hyperbranched Poly(amide amine) as Effective Fluorescent Probe for Label-free Detection of Copper(II) Ions

Wang

2017

ANAL. SCI.

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A label-free fluorescent probe based on autofluorescent hyperbranched poly(amide amine) (HPAMAM) for copper ions was designed. HPAMAM is a cationic polymer containing many amino groups, which could bind Cu 2+ ions to form cupric amine complexes, leading to a selective quenching of the fluorescence intensity of HPAMAM via inner filter effect. The fluorescence intensity of HPAMAM decreased with increasing concentration of Cu 2+ ions and the linear response ranged from 0.05 to 25 μM (R 2 = 0.995), with the corresponding detection limit (3σ/k) of 17.15 nM. The HPAMAM fluorescent probe provided a simple, rapid, selective and sensitive fluorometric method for detecting Cu 2+ ions, which could be also applied for detection of Cu 2+ ions in real water samples.

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Cyanine fluorophore derivatives with enhanced photostability

Cited by 288 publications

References 19 publications

High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence

High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence

Elongation Factor Ts Directly Facilitates the Formation and Disassembly of the Escherichia coli Elongation Factor Tu·GTP·Aminoacyl-tRNA Ternary Complex

Autofluorescent Hyperbranched Poly(amide amine) as Effective Fluorescent Probe for Label-free Detection of Copper(II) Ions

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