Single-Molecule Three-Color FRET with Both Negligible Spectral Overlap and Long Observation Time

Full understanding of complex biological interactions frequently requires multi-color detection capability in doing single-molecule fluorescence resonance energy transfer (FRET) experiments. Existing single-molecule three-color FRET techniques, however, suffer from severe photobleaching of Alexa 488, or its alternative dyes, and have been limitedly used for kinetics studies. In this work, we developed a single-molecule three-color FRET technique based on the Cy3-Cy5-Cy7 dye trio, thus providing enhanced observation time and improved data quality. Because the absorption spectra of three fluorophores are well separated, real-time monitoring of three FRET efficiencies was possible by incorporating the alternating laser excitation (ALEX) technique both in confocal microscopy and in total-internal-reflection fluorescence (TIRF) microscopy.

• Publication year

  2010

• Venue

  PLoS ONE

• Publication date

  2010-08-19

• Fields of study

  Biology, Medicine, Physics, Chemistry

• Identifiers
  DOI 10.1371/journal.pone.0012270PMID 20808851PMCID 2924373
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Minute negative superhelicity is sufficient to induce the B-Z transition in the presence of low tension

  2010cited by this paper
• A practical guide to single-molecule FRET

  2008cited by this paper
• Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids

  2008cited by this paper
• An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments.

  2008cited by this paper
• Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances.

  2007cited by this paper
• Multicolor single-molecule spectroscopy with alternating laser excitation for the investigation of interactions and dynamics.

  2007cited by this paper
• Fluorescence-Force Spectroscopy Maps Two-Dimensional Reaction Landscape of the Holliday Junction

  2007cited by this paper
• Detecting force-induced molecular transitions with fluorescence resonant energy transfer.

  2007cited by this paper
• Nonblinking and long-lasting single-molecule fluorescence imaging

  2006cited by this paper
• Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes.

  2006cited by this paper
• Biocompatible force sensor with optical readout and dimensions of 6 nm3.

  2005cited by this paper
• Three-color single-molecule fluorescence resonance energy transfer.

  2005cited by this paper
• Exploring rare conformational species and ionic effects in DNA Holliday junctions using single-molecule spectroscopy.

  2004cited by this paper
• Single-molecule three-color FRET.

  2004cited by this paper
• Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules

  2004cited by this paper
• Structural dynamics of individual Holliday junctions

  2003cited by this paper
• Single-molecule fluorescence resonance energy transfer.

  2001cited by this paper
• Crystal structure of a DNA Holliday junction

  1999cited by this paper
• Probing the interaction between single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor

  1996cited by this paper
• Fluorescence resonance energy transfer and nucleic acids.

  1992cited by this paper
• Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules

  1989cited by this paper
• The structure of the Holliday junction, and its resolution.

  1988cited by this paper

• A FRET-based toolkit for quantifying lipid incorporation into nanoparticles.

  2026cites this paper
• Programmable DNA Framework-Based Logic-Gated Dual-Distance Regulation for Photoelectrochemical Multiplex and Sensitive Detection of Waterborne Viruses.

  2026cites this paper
• Three-color single-molecule maximum likelihood analysis of folding and binding of diffusing molecules.

  2025cites this paper
• An intrinsically disordered region of Ubp10 regulates its binding and activity on ubiquitinated histone substrates

  2025cites this paper
• The use of single-molecule FRET for the characterization of Holliday junctions containing human telomeric DNA: a methodological approach for nanoscale distance, mobility, and stability measurements

  2024influential citation
• Single-mode termination of phage transcriptions, disclosing bacterial adaptation for facilitated reinitiations

  2024influential citation
• Trends in Single-Molecule Total Internal Reflection Fluorescence Imaging and Their Biological Applications with Lab-on-a-Chip Technology

  2023influential citation
• GAGA Factor Overcomes 1D Diffusion Barrier by 3D Diffusion in Search of Nucleosomal Targets

  2023cites this paper
• Transcriptional pause extension benefits the stand-by rather than catch-up Rho-dependent termination

  2023cites this paper
• Controlling Three-Color Förster Resonance Energy Transfer in an Optical Fabry–Pérot Microcavity at Low Mode Order

  2023cites this paper
• GAGA zinc finger transcription factor searches chromatin by 1D–3D facilitated diffusion

  2023cites this paper
• Multipair Förster Resonance Energy Transfer via Spectrally Resolved Single-Molecule Detection

  2022cites this paper
• Rho-dependent transcription termination proceeds via three routes

  2022cites this paper
• Choosing the Probe for Single-Molecule Fluorescence Microscopy

  2022cites this paper
• Multicolor single-molecule FRET for DNA and RNA processes.

  2021cites this paper
• Multi-parameter photon-by-photon hidden Markov modeling

  2021cites this paper
• Single-Molecule Fluorescence Techniques for Membrane Protein Dynamics Analysis

  2021cites this paper
• Coordinated DNA and histone dynamics drive accurate histone H2A.Z exchange

  2021cites this paper
• Photochemically Switchable Interconnected Microcavities for All‐Organic Optical Logic Gate

  2021cites this paper
• Illuminating amyloid fibrils: Fluorescence-based single-molecule approaches

  2021cites this paper
• Comparative analysis of the coordinated motion of Hsp70s from different organelles observed by single-molecule three-color FRET

  2021cites this paper
• FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices

  2021cites this paper
• Combining Rapid Microfluidic Mixing and Three-Color Single-Molecule FRET for Probing the Kinetics of Protein Conformational Changes.

  2021cites this paper
• The FRET-based structural dynamics challenge -- community contributions to consistent and open science practices

  2020cites this paper
• Slow diffusion co-assembly as an efficient tool to tune colour emission in alkynyl benzoazoles

  2020cites this paper
• Single-Molecule FRET of Intrinsically Disordered Proteins.

  2020cites this paper
• A Self‐Assembled Plasmonic Substrate for Enhanced Fluorescence Resonance Energy Transfer

  2020cites this paper
• Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection

  2020cites this paper
• Chemistry Single-Molecule FRET of Intrinsically Disordered Proteins

  2020cites this paper
• Quantification of purified endogenous miRNAs with high sensitivity and specificity

  2020cites this paper
• Real-time observation of Cas9 postcatalytic domain motions

  2020cites this paper
• Application of FRET Biosensors in Mechanobiology and Mechanopharmacological Screening

  2020cites this paper
• PCNA monoubiquitination is regulated by diffusion of Rad6/Rad18 complexes along RPA filaments

  2020cites this paper
• Recent advances in single-molecule fluorescence microscopy render structural biology dynamic.

  2020cites this paper
• Single-molecule analysis of i-motif within self-assembled DNA duplexes and nanocircles

  2019cites this paper
• Dynamics of the DEAD-box ATPase Prp5 RecA-like domains provide a conformational switch during spliceosome assembly

  2019cites this paper
• Resolving the Sequence of Binding events in the self-assembly process of DNA Origami

  2019cites this paper
• Deriving a sub-nanomolar affinity peptide from TAP to enable smFRET analysis of RNA polymerase II complexes.

  2019cites this paper
• Direct observation of coordinated DNA movements on the nucleosome during chromatin remodelling

  2019cites this paper
• Two-Dimensional Electronic Spectroscopy Reveals the Spectral Dynamics of Förster Resonance Energy Transfer

  2019cites this paper
• Multi-parameter measurements of conformational dynamics in nucleic acids and nucleoprotein complexes.

  2019cites this paper
• Probing dynamics in single molecules

  2019cites this paper
• A Bayesian Nonparametric Approach to Single Molecule Förster Resonance Energy Transfer.

  2018cites this paper
• Simulation of FRET dyes allows quantitative comparison against experimental data.

  2018cites this paper
• The Protein Microenvironment Governs the Suitability of Labeling Sites for Single-Molecule Spectroscopy of RNP Complexes.

  2018cites this paper
• Quantitative single-molecule three-color Förster resonance energy transfer by photon distribution analysis

  2018cites this paper
• Investigation of sliding DNA clamp dynamics by single-molecule fluorescence, mass spectrometry and structure-based modeling

  2018cites this paper
• From organelle over cell to tissue: Development of fluorescence-based methods to monitor the fate of model transporting structures

  2018cites this paper
• Methylation-Dependent DNA Condensation: Its Implications in Chromosome Organization and Epigenetic Gene Control

  2017cites this paper
• Single-Molecule Fluorescence Energy Transfer Assays for the Characterization of Reaction Pathways of miRNA-Argonaute Complex.

  2017cites this paper
• Ligand Recognition Mechanism of Thiamine Pyrophosphate Riboswitch Aptamer

  2017cites this paper
• Single-molecule FRET studies on the cotranscriptional folding of a thiamine pyrophosphate riboswitch

  2017cites this paper
• Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET

  2017cites this paper
• Orientation-dependent FRET system reveals differences in structures and flexibilities of nicked and gapped DNA duplexes

  2017cites this paper
• Revealing Nucleic Acid Mutations Using Förster Resonance Energy Transfer-Based Probes

  2016cites this paper
• FRET-based cyanine probes for monitoring ligation reactions and their applications to mechanistic studies and catalyst screening.

  2016cites this paper
• Active Control of Repetitive Structural Transitions between Replication Forks and Holliday Junctions by Werner Syndrome Helicase.

  2016cites this paper
• Single-Molecule Multicolor FRET Assay for Studying Structural Dynamics of Biomolecules.

  2016cites this paper
• Single-molecule fluorescence resonance energy transfer in molecular biology.

  2016cites this paper
• Characterization of Ternary Protein Systems In Vivo with Tricolor Heterospecies Partition Analysis.

  2016cites this paper
• A Quantitative Theoretical Framework For Protein-Induced Fluorescence Enhancement–Förster-Type Resonance Energy Transfer (PIFE-FRET)

  2016cites this paper
• Interactions of RecQ-Family Helicases with G-quadruplex Structures at the Single Molecule Level

  2016cites this paper
• Reading the Epigenetic State of Chromatin Alters its Accessibility

  2016cites this paper
• Preparation of mononucleosomal templates for analysis of transcription with RNA polymerase using spFRET.

  2015cites this paper
• Human Argonaute 2 Has Diverse Reaction Pathways on Target RNAs.

  2015cites this paper
• MMP-2/9-Specific Activatable Lifetime Imaging Agent

  2015cites this paper
• Assembly, translocation, and activation of XerCD-dif recombination by FtsK translocase analyzed in real-time by FRET and two-color tethered fluorophore motion

  2015cites this paper
• Single Molecule FRET Studies of Reverse Transcription and Chromatin Remodeling

  2015cites this paper
• Orthogonal Functionalization of Ferritin via Supramolecular Re-Assembly

  2015cites this paper
• Studies on roles of histone H1 in nucleosome condensation

  2015cites this paper
• Maximizing information content of single-molecule FRET experiments: multi-color FRET and FRET combined with force or torque.

  2014cites this paper
• Characterization of a far-red analog of ghrelin for imaging GHS-R in P19-derived cardiomyocytes.

  2014cites this paper
• The bright future of single-molecule fluorescence imaging.

  2014cites this paper
• Alternating-laser excitation: single-molecule FRET and beyond.

  2014cites this paper
• Monitoring DNA triplex formation using multicolor fluorescence and application to insulin‐like growth factor I promoter downregulation

  2014cites this paper
• Studying RNA Using Single Molecule Fluorescence Resonance Energy Transfer

  2014cites this paper
• The Power of Single-Molecule FRET Microscopy Applied to DNA Nanotechnology

  2014cites this paper
• Computational Assessment of Single-Molecule Protein Sequencing

  2014cites this paper
• Computational Assessment of Single-Molecule Protein Sequencing

  2014influential citation
• A Starting Point for Fluorescence-Based Single-Molecule Measurements in Biomolecular Research

  2014cites this paper
• Probing Protein Multidimensional Conformational Fluctuations by Single-Molecule Multiparameter Photon Stamping Spectroscopy

  2014cites this paper
• Structure-based elucidation of the regulatory mechanism for aminopeptidase activity.

  2013cites this paper
• SOLVING SINGLE BIOMOLECULES BY ADVANCED FRET-BASED SINGLE-MOLECULE FLUORESCENCE TECHNIQUES

  2013cites this paper
• Single‐Molecule Applications

  2013cites this paper
• High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence

  2013cites this paper
• TripleFRET measurements in flow cytometry

  2013cites this paper
• A single-molecule dissection of ligand binding to a protein with intrinsic dynamics.

  2013cites this paper
• An optical trap combined with three-color FRET.

  2013cites this paper
• Helicase-mediated changes in RNA structure at the single-molecule level

  2013cites this paper
• Dynamic anchoring of the 3'-end of the guide strand controls the target dissociation of Argonaute-guide complex.

  2013cites this paper
• Single-molecule nanometry for biological physics

  2013cites this paper
• Simple method to enhance the photostability of the fluorescence reporter R6G for prolonged single-molecule studies.

  2013cites this paper
• Development of a robust model system of FRET using base surrogates tethering fluorophores for strict control of their position and orientation within DNA duplex.

  2013cites this paper
• Advances in Quantitative FRET‐Based Methods for Studying Nucleic Acids

  2012cites this paper
• Autofocusing system based on optical astigmatism analysis of single-molecule images.

  2012cites this paper
• Single-molecule analysis of SSB dynamics on single-stranded DNA.

  2012cites this paper
• Linking single-molecule blinking to chromophore structure and redox potentials.

  2012cites this paper
• Multicolour Single Molecule Imaging in Cells with Near Infra-Red Dyes

  2012cites this paper
• Versatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics.

  2011cites this paper
• Single-molecule FRET ruler based on rigid DNA origami blocks.

  2011cites this paper