Abstract
Fluorescence resonance energy transfer (FRET) is a powerful spectroscopic method for measuring distances in the 2–8 nm range. Often, conformational changes and molecular interactions are difficult or impossible to synchronize, or too rare or transient to detect using ensemble FRET. Single-molecule FRET (smFRET) opens new opportunities to probe biomolecular conformational changes or interactions that are missing in static snapshots provided by traditional structural biology tools, as well as to measure the kinetics of these dynamics on various timescales and under physiological conditions, including inside cells. Advances in labelling technologies, combining smFRET with optical and magnetic tweezers and Bayesian inference-based and information theory-based analysis tools are revealing rich biomolecular dynamics. We also discuss the challenges and opportunities in integrating dynamics into traditionally static structural biology approaches, extending smFRET into cells and tissues, advancing technical innovations and democratizing the practice of smFRET.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 1 digital issues and online access to articles
$99.00 per year
only $99.00 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Stryer, L. & Haugland, R. P. Energy transfer: a spectroscopic ruler. Proc. Natl Acad. Sci. USA 58, 719–726 (1967).
Ha, T. et al. Probing the interaction between two single molecules — fluorescence resonance energy transfer between a single donor and a single acceptor. Proc. Natl Acad. Sci. USA 93, 6264–6268 (1996).
Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nat. Methods 5, 507–516 (2008).
Lerner, E. et al. Toward dynamic structural biology: two decades of single-molecule Forster resonance energy transfer. Science 359, eaan1133 (2018).
Hohng, S. et al. Fluorescence-force spectroscopy maps two-dimensional reaction landscape of the holliday junction. Science 318, 279–283 (2007).
Tarsa, P. B. et al. Detecting force-induced molecular transitions with fluorescence resonant energy transfer. Angew. Chem. Int. Ed. 46, 1999–2001 (2007).
Shroff, H. et al. Biocompatible force sensor with optical readout and dimensions of 6 nm3. Nano Lett. 5, 1509–1514 (2005).
Lee, M., Kim, S. H. & Hong, S. C. Minute negative superhelicity is sufficient to induce the B–Z transition in the presence of low tension. Proc. Natl Acad. Sci. USA 107, 4985–4990 (2010).
Hellenkamp, B. et al. Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study. Nat. Methods 15, 669–676 (2018).
Kalinin, S. et al. A toolkit and benchmark study for FRET-restrained high-precision structural modeling. Nat. Methods 9, 1218–1225 (2012).
Hohng, S., Joo, C. & Ha, T. Single-molecule three-color FRET. Biophys. J. 87, 1328–1337 (2004).
Clamme, J. P. & Deniz, A. A. Three-color single-molecule fluorescence resonance energy transfer. Chemphyschem 6, 74–77 (2005).
Lee, J. et al. Single-molecule four-color FRET. Angew. Chem. Int. Ed. 49, 9922–9925 (2010).
Feng, X. A., Poyton, M. F. & Ha, T. Multicolor single-molecule FRET for DNA and RNA processes. Curr. Opin. Struct. Biol. 70, 26–33 (2021).
Axelrod, D., Burghardt, T. P. & Thompson, N. L. Total internal reflection fluorescence. Annu. Rev. Biophys. Bioeng. 13, 247–268 (1984).
Lerner, E. et al. FRET-based dynamic structural biology: challenges, perspectives and an appeal for open-science practices. eLife 10, e60416 (2021).
Cho, Y., An, H. J., Kim, T., Lee, C. & Lee, N. K. Mechanism of cyanine5 to cyanine3 photoconversion and its application for high-density single-particle tracking in a living cell. J. Am. Chem. Soc. 143, 14125–14135 (2021).
Mashanov, G. I., Tacon, D., Knight, A. E., Peckham, M. & Molloy, J. E. Visualizing single molecules inside living cells using total internal reflection fluorescence microscopy. Methods 29, 142–152 (2003).
Juette, M. F. et al. Single-molecule imaging of non-equilibrium molecular ensembles on the millisecond timescale. Nat. Methods 13, 341–344 (2016).
Deniz, A. A. et al. Single-pair fluorescence resonance energy transfer on freely diffusing molecules: observation of Forster distance dependence and subpopulations. Proc. Natl Acad. Sci. USA 96, 3670–3675 (1999).
Kapanidis, A. N. et al. Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules. Proc. Natl Acad. Sci. USA 101, 8936–8941 (2004).
Oikawa, H., Takahashi, T., Kamonprasertsuk, S. & Takahashi, S. Microsecond resolved single-molecule FRET time series measurements based on the line confocal optical system combined with hybrid photodetectors. Phys. Chem. Chem Phys 20, 3277–3285 (2018).
Nir, E. et al. Shot-noise limited single-molecule FRET histograms: comparison between theory and experiments. J. Phys. Chem. B 110, 22103–22124 (2006).
Muller, B. K., Zaychikov, E., Brauchle, C. & Lamb, D. C. Pulsed interleaved excitation. Biophys. J. 89, 3508–3522 (2005).
Laurence, T. A., Kong, X., Jager, M. & Weiss, S. Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins. Proc. Natl Acad. Sci. USA 102, 17348–17353 (2005).
Chung, H. S., McHale, K., Louis, J. M. & Eaton, W. A. Single-molecule fluorescence experiments determine protein folding transition path times. Science 335, 981–984 (2012).
Hohng, S., Lee, S., Lee, J. & Jo, M. H. Maximizing information content of single-molecule FRET experiments: multi-color FRET and FRET combined with force or torque. Chem. Soc. Rev. 43, 1007–1013 (2014).
Sakon, J. J. & Weninger, K. R. Detecting the conformation of individual proteins in live cells. Nat. Methods 7, 203–205 (2010).
Fessl, T. et al. Towards characterization of DNA structure under physiological conditions in vivo at the single-molecule level using single-pair FRET. Nucleic Acids Res. 40, e121 (2012).
Crawford, R. et al. Long-lived intracellular single-molecule fluorescence using electroporated molecules. Biophys. J. 105, 2439–2450 (2013).
Sustarsic, M. & Kapanidis, A. N. Taking the ruler to the jungle: single-molecule FRET for understanding biomolecular structure and dynamics in live cells. Curr. Opin. Struct. Biol. 34, 52–59 (2015).
Asher, W. B. et al. Single-molecule FRET imaging of GPCR dimers in living cells. Nat. Methods 18, 397–405 (2021).
Zhao, Z. W. et al. Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy. Proc. Natl Acad. Sci. USA 111, 681–686 (2014).
Brasselet, S., Peterman, E. J. G., Miyawaki, A. & Moerner, W. E. Single-molecule fluorescence resonant energy transfer in calcium concentration dependent cameleon. J. Phys. Chem. B 104, 3676–3682 (2000).
Hohng, S. & Ha, T. Single-molecule quantum-dot fluorescence resonance energy transfer. Chemphyschem 6, 956–960 (2005).
Poyton, M. F. et al. Coordinated DNA and histone dynamics drive accurate histone H2A.Z exchange. Sci. Adv. 8, eabj5509 (2022).
Ratzke, C., Hellenkamp, B. & Hugel, T. Four-colour FRET reveals directionality in the Hsp90 multicomponent machinery. Nat. Commun. 5, 4192 (2014).
Das, D. K. et al. Direct visualization of the conformational dynamics of single influenza hemagglutinin trimers. Cell 174, 926–937.e12 (2018).
Nikic, I. & Lemke, E. A. Genetic code expansion enabled site-specific dual-color protein labeling: superresolution microscopy and beyond. Curr. Opin. Chem. Biol. 28, 164–173 (2015).
Desai, B. J. & Gonzalez, R. L. Jr Multiplexed genomic encoding of non-canonical amino acids for labeling large complexes. Nat. Chem. Biol. 16, 1129–1135 (2020).
England, P. M. Unnatural amino acid mutagenesis: a precise tool for probing protein structure and function. Biochemistry 43, 11623–11629 (2004).
Joo, C. & Ha, T. Single-molecule FRET with total internal reflection microscopy. Cold Spring Harb. Protoc. https://doi.org/10.1101/pdb.top072058 (2012).
Paul, T., Ha, T. & Myong, S. Regeneration of PEG slide for multiple rounds of single-molecule measurements. Biophys. J. 120, 1788–1799 (2021).
Hua, B. et al. An improved surface passivation method for single-molecule studies. Nat. Methods 11, 1233–1236 (2014).
Boukobza, E., Sonnenfeld, A. & Haran, G. Immobilization in surface-tethered lipid vesicles as a new tool for single biomolecule spectroscopy. J. Phys. Chem. B 105, 12165–12170 (2001).
Cohen, A. E. & Moerner, W. E. Method for trapping and manipulating nanoscale objects in solution. Appl. Phys. Lett. 86, 093109 (2005).
Pollok, B. A. & Heim, R. Using GFP in FRET-based applications. Trends Cell Biol. 9, 57–60 (1999).
Bajar, B. T., Wang, E. S., Zhang, S., Lin, M. Z. & Chu, J. A guide to fluorescent protein FRET pairs. Sensors 16, 1488 (2016).
Konig, I. et al. Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells. Nat. Methods 12, 773–779 (2015).
Yang, S. et al. Transcription and translation contribute to gene locus relocation to the nucleoid periphery in E. coli. Nat. Commun. 10, 5131 (2019).
Stracy, M. et al. Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid. Proc. Natl Acad. Sci. USA 112, E4390–4399 (2015).
Edelstein, A. D. et al. Advanced methods of microscope control using muManager software. J. Biol. Methods 1, e10 (2014).
Seo, M. H., Park, J., Kim, E., Hohng, S. & Kim, H. S. Protein conformational dynamics dictate the binding affinity for a ligand. Nat. Commun. 5, 3724 (2014).
Aitken, C. E., Marshall, R. A. & Puglisi, J. D. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys. J. 94, 1826–1835 (2008).
Helmerich, D. A., Beliu, G., Matikonda, S. S., Schnermann, M. J. & Sauer, M. Photoblueing of organic dyes can cause artifacts in super-resolution microscopy. Nat. Methods 18, 253–257 (2021).
Zheng, Q. et al. Ultra-stable organic fluorophores for single-molecule research. Chem. Soc. Rev. 43, 1044–1056 (2014).
Zosel, F., Mercadante, D., Nettels, D. & Schuler, B. A proline switch explains kinetic heterogeneity in a coupled folding and binding reaction. Nat. Commun. 9, 3332 (2018).
Preus, S., Hildebrandt, L. L. & Birkedal, V. Optimal background estimators in single-molecule FRET microscopy. Biophys. J. 111, 1278–1286 (2016).
Deschout, H. et al. Precisely and accurately localizing single emitters in fluorescence microscopy. Nat. Methods 11, 253–266 (2014).
Borner, R. et al. Simulations of camera-based single-molecule fluorescence experiments. PLoS ONE 13, e0195277 (2018).
Holden, S. J. et al. Defining the limits of single-molecule FRET resolution in TIRF microscopy. Biophys. J. 99, 3102–3111 (2010).
Preus, S., Noer, S. L., Hildebrandt, L. L., Gudnason, D. & Birkedal, V. iSMS: single-molecule FRET microscopy software. Nat. Methods 12, 593–594 (2015).
Thomsen, J. et al. DeepFRET, a software for rapid and automated single-molecule FRET data classification using deep learning. eLife 9, e60404 (2020).
Li, J., Zhang, L., Johnson-Buck, A. & Walter, N. G. Automatic classification and segmentation of single-molecule fluorescence time traces with deep learning. Nat. Commun. 11, 5833 (2020).
Huisjes, N. M. et al. Mars, a molecule archive suite for reproducible analysis and reporting of single-molecule properties from bioimages. eLife 11, e75899 (2022).
Wanninger, S. et al. Deep-LASI: deep-learning assisted, single-molecule imaging analysis of multi-color DNA origami structures. Nat. Commun. 14, 6564 (2023).
McCann, J. J., Choi, U. B., Zheng, L., Weninger, K. & Bowen, M. E. Optimizing methods to recover absolute FRET efficiency from immobilized single molecules. Biophys. J. 99, 961–970 (2010).
Dimura, M. et al. Quantitative FRET studies and integrative modeling unravel the structure and dynamics of biomolecular systems. Curr. Opin. Struct. Biol. 40, 163–185 (2016).
McKinney, S. A., Joo, C. & Ha, T. Analysis of single-molecule FRET trajectories using hidden Markov modeling. Biophys. J. 91, 1941–1951 (2006).
Verma, A. R. et al. Increasing the accuracy of single-molecule data analysis using tMAVEN. Biophys. J. https://doi.org/10.1016/j.bpj.2024.01.022 (2024).
van de Meent, J. W., Bronson, J. E., Wiggins, C. H. & Gonzalez, R. L. Jr Empirical Bayes methods enable advanced population-level analyses of single-molecule FRET experiments. Biophys. J. 106, 1327–1337 (2014).
Kinz-Thompson, C. D. & Gonzalez, R. L. Jr Increasing the time resolution of single-molecule experiments with Bayesian inference. Biophys. J. 114, 289–300 (2018).
Kim, S. E., Lee, I. B., Hyeon, C. & Hong, S. C. Deciphering kinetic information from single-molecule FRET data that show slow transitions. J. Phys. Chem. B 119, 6974–6978 (2015).
Schmid, S., Gotz, M. & Hugel, T. Single-molecule analysis beyond dwell times: demonstration and assessment in and out of equilibrium. Biophys. J. 111, 1375–1384 (2016).
Hon, J. & Gonzalez, R. L. Jr Bayesian-estimated hierarchical HMMs enable robust analysis of single-molecule kinetic heterogeneity. Biophys. J. 116, 1790–1802 (2019).
Sgouralis, I. et al. A Bayesian nonparametric approach to single molecule forster resonance energy transfer. J. Phys. Chem. B 123, 675–688 (2019).
Bronson, J. E., Fei, J., Hofman, J. M., Gonzalez, R. L. Jr & Wiggins, C. H. Learning rates and states from biophysical time series: a Bayesian approach to model selection and single-molecule FRET data. Biophys. J. 97, 3196–3205 (2009).
Nagl, S. et al. Microarray analysis of protein–protein interactions based on FRET using subnanosecond-resolved fluorescence lifetime imaging. Biosens. Bioelectron. 24, 397–402 (2008).
Sisamakis, E., Valeri, A., Kalinin, S., Rothwell, P. J. & Seidel, C. A. Accurate single-molecule FRET studies using multiparameter fluorescence detection. Methods Enzymol. 475, 455–514 (2010).
Knop, J. M., Patra, S., Harish, B., Royer, C. A. & Winter, R. The deep sea osmolyte trimethylamine N-oxide and macromolecular crowders rescue the antiparallel conformation of the human telomeric G-quadruplex from urea and pressure stress. Chemistry 24, 14346–14351 (2018).
Gopich, I. V. & Szabo, A. Theory of the energy transfer efficiency and fluorescence lifetime distribution in single-molecule FRET. Proc. Natl Acad. Sci. USA 109, 7747–7752 (2012).
Margineanu, A. et al. Screening for protein–protein interactions using Forster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM). Sci. Rep. 6, 28186 (2016).
George Abraham, B. et al. Fluorescent protein based FRET pairs with improved dynamic range for fluorescence lifetime measurements. PLoS ONE 10, e0134436 (2015).
Becker, W. Fluorescence lifetime imaging—techniques and applications. J. Microsc. 247, 119–136 (2012).
Levitt, J. A. et al. Quantitative real-time imaging of intracellular FRET biosensor dynamics using rapid multi-beam confocal FLIM. Sci. Rep. 10, 5146 (2020).
Reissaus, C. A. et al. PIE-FLIM measurements of two different FRET-based biosensor activities in the same living cells. Biophys. J. 118, 1820–1829 (2020).
Torella, J. P., Holden, S. J., Santoso, Y., Hohlbein, J. & Kapanidis, A. N. Identifying molecular dynamics in single-molecule FRET experiments with burst variance analysis. Biophys. J. 100, 1568–1577 (2011).
Tomov, T. E. et al. Disentangling subpopulations in single-molecule FRET and ALEX experiments with photon distribution analysis. Biophys. J. 102, 1163–1173 (2012).
Kalinin, S., Valeri, A., Antonik, M., Felekyan, S. & Seidel, C. A. Detection of structural dynamics by FRET: a photon distribution and fluorescence lifetime analysis of systems with multiple states. J. Phys. Chem. B 114, 7983–7995 (2010).
Chung, H. S. & Eaton, W. A. Protein folding transition path times from single molecule FRET. Curr. Opin. Struct. Biol. 48, 30–39 (2018).
Pirchi, M. et al. Photon-by-photon hidden Markov model analysis for microsecond single-molecule FRET kinetics. J. Phys. Chem. B 120, 13065–13075 (2016).
Keller, B. G., Kobitski, A., Jaschke, A., Nienhaus, G. U. & Noe, F. Complex RNA folding kinetics revealed by single-molecule FRET and hidden Markov models. J. Am. Chem. Soc. 136, 4534–4543 (2014).
Felekyan, S., Sanabria, H., Kalinin, S., Kuhnemuth, R. & Seidel, C. A. Analyzing Forster resonance energy transfer with fluctuation algorithms. Methods Enzymol. 519, 39–85 (2013).
Gurunathan, K. & Levitus, M. FRET fluctuation spectroscopy of diffusing biopolymers: contributions of conformational dynamics and translational diffusion. J. Phys. Chem. B 114, 980–986 (2010).
Schuler, B. Perspective: chain dynamics of unfolded and intrinsically disordered proteins from nanosecond fluorescence correlation spectroscopy combined with single-molecule FRET. J. Chem. Phys. 149, 010901 (2018).
Nettels, D., Gopich, I. V., Hoffmann, A. & Schuler, B. Ultrafast dynamics of protein collapse from single-molecule photon statistics. Proc. Natl Acad. Sci. USA 104, 2655–2660 (2007).
Yoo, J., Kim, J. Y., Louis, J. M., Gopich, I. V. & Chung, H. S. Fast three-color single-molecule FRET using statistical inference. Nat. Commun. 11, 3336 (2020).
Ingargiola, A. et al. Multispot single-molecule FRET: high-throughput analysis of freely diffusing molecules. PLoS ONE 12, e0175766 (2017).
Ingargiola, A., Weiss, S. & Lerner, E. Monte Carlo diffusion-enhanced photon inference: distance distributions and conformational dynamics in single-molecule FRET. J. Phys. Chem. B 122, 11598–11615 (2018).
Nagy, P. et al. rFRET: a comprehensive, MATLAB-based program for analyzing intensity-based ratiometric microscopic FRET experiments. Cytometry A 89, 376–384 (2016).
Widengren, J. et al. Single-molecule detection and identification of multiple species by multiparameter fluorescence detection. Anal. Chem. 78, 2039–2050 (2006).
Lee, N. K. et al. Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation. Biophys. J. 88, 2939–2953 (2005).
Margeat, E. et al. Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes. Biophys. J. 90, 1419–1431 (2006).
Frank, J. & Gonzalez, R. L. Jr Structure and dynamics of a processive Brownian motor: the translating ribosome. Annu. Rev. Biochem. 79, 381–412 (2010).
Fan, J., Moreno, A. T., Baier, A. S., Loparo, J. J. & Peterson, C. L. H2A.Z deposition by SWR1C involves multiple ATP-dependent steps. Nat. Commun. 13, 7052 (2022).
Gamarra, N., Johnson, S. L., Trnka, M. J., Burlingame, A. L. & Narlikar, G. J. The nucleosomal acidic patch relieves auto-inhibition by the ISWI remodeler SNF2h. eLife 7, e35322 (2018).
Evans, G. W., Craggs, T. & Kapanidis, A. N. The rate-limiting step of DNA synthesis by DNA polymerase occurs in the fingers-closed conformation. J. Mol. Biol. 434, 167410 (2022).
Berezhna, S. Y., Gill, J. P., Lamichhane, R. & Millar, D. P. Single-molecule Forster resonance energy transfer reveals an innate fidelity checkpoint in DNA polymerase I. J. Am. Chem. Soc. 134, 11261–11268 (2012).
Lee, D., Oh, S., Cho, H., Yoo, J. & Lee, G. Mechanistic decoupling of exonuclease III multifunctionality into AP endonuclease and exonuclease activities at the single-residue level. Nucleic Acids Res. 50, 2211–2222 (2022).
Reid, D. A. et al. Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair. Proc. Natl Acad. Sci. USA 112, E2575–E2584 (2015).
Singh, D., Sternberg, S. H., Fei, J., Doudna, J. A. & Ha, T. Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9. Nat. Commun. 7, 12778 (2016).
Yang, M. et al. The conformational dynamics of Cas9 governing DNA cleavage are revealed by single-molecule FRET. Cell Rep. 22, 372–382 (2018).
Osuka, S. et al. Real-time observation of flexible domain movements in CRISPR–Cas9. EMBO J. 37, e96941 (2018).
Chen, J. S. et al. Enhanced proofreading governs CRISPR–Cas9 targeting accuracy. Nature 550, 407–410 (2017).
Chakraborty, A. et al. Opening and closing of the bacterial RNA polymerase clamp. Science 337, 591–595 (2012).
Song, E. et al. Rho-dependent transcription termination proceeds via three routes. Nat. Commun. 13, 1663 (2022).
Abelson, J. et al. Conformational dynamics of single pre-mRNA molecules during in vitro splicing. Nat. Struct. Mol. Biol. 17, 504–512 (2010).
Rodgers, M. L. et al. Conformational dynamics of stem II of the U2 snRNA. RNA 22, 225–236 (2016).
Roca, J., Santiago-Frangos, A. & Woodson, S. A. Diversity of bacterial small RNAs drives competitive strategies for a mutual chaperone. Nat. Commun. 13, 2449 (2022).
Chandradoss, S. D., Schirle, N. T., Szczepaniak, M., MacRae, I. J. & Joo, C. A dynamic search process underlies microRNA targeting. Cell 162, 96–107 (2015).
Fei, J., Kosuri, P., MacDougall, D. D. & Gonzalez, R. L. Jr. Coupling of ribosomal L1 stalk and tRNA dynamics during translation elongation. Mol. Cell 30, 348–359 (2008).
Lapointe, C. P. et al. eIF5B and eIF1A reorient initiator tRNA to allow ribosomal subunit joining. Nature 607, 185–190 (2022).
Ray, K. K. et al. Entropic control of the free-energy landscape of an archetypal biomolecular machine. Proc. Natl Acad. Sci. USA 120, e2220591120 (2023).
Mazal, H., Iljina, M., Riven, I. & Haran, G. Ultrafast pore-loop dynamics in a AAA+ machine point to a Brownian-ratchet mechanism for protein translocation. Sci. Adv. 7, eabg4674 (2021).
Zhuang, X. et al. Correlating structural dynamics and function in single ribozyme molecules. Science 296, 1473–1476 (2002).
Liao, T. W. et al. Linking folding dynamics and function of SAM/SAH riboswitches at the single molecule level. Nucleic Acids Res. 51, 8957–8969 (2023).
Schuler, B., Lipman, E. A. & Eaton, W. A. Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy. Nature 419, 743–747 (2002).
Kim, J. Y. & Chung, H. S. Disordered proteins follow diverse transition paths as they fold and bind to a partner. Science 368, 1253–1257 (2020).
Zamel, J. et al. Structural and dynamic insights into α-synuclein dimer conformations. Structure 31, 411–423.e6 (2023).
Galvanetto, N. et al. Extreme dynamics in a biomolecular condensate. Nature 619, 876–883 (2023).
Okafor, I. C. & Ha, T. Single molecule FRET analysis of CRISPR–Cas9 single guide RNA folding dynamics. J. Phys. Chem. B 127, 45–51 (2022).
Trucks, S., Hanspach, G. & Hengesbach, M. Eukaryote specific RNA and protein features facilitate assembly and catalysis of H/ACA snoRNPs. Nucleic Acids Res. 49, 4629–4642 (2021).
Kim, H. et al. Protein-guided RNA dynamics during early ribosome assembly. Nature 506, 334–338 (2014).
Duss, O. et al. Real-time assembly of ribonucleoprotein complexes on nascent RNA transcripts. Nat. Commun. 9, 5087 (2018).
Marzano, N. R., Paudel, B. P., van Oijen, A. M. & Ecroyd, H. Real-time single-molecule observation of chaperone-assisted protein folding. Sci. Adv. 8, eadd0922 (2022).
Chamachi, N. et al. Chaperones Skp and SurA dynamically expand unfolded OmpX and synergistically disassemble oligomeric aggregates. Proc. Natl Acad. Sci. USA 119, e2118919119 (2022).
Dahiya, V. et al. Coordinated conformational processing of the tumor suppressor protein p53 by the Hsp70 and Hsp90 chaperone machineries. Mol. Cell 74, 816–830.e7 (2019).
Schmid, S. & Hugel, T. Controlling protein function by fine-tuning conformational flexibility. eLife 9, e57180 (2020).
Levring, J. et al. CFTR function, pathology and pharmacology at single-molecule resolution. Nature 616, 606–614 (2023).
Han, S. et al. Cholesterol inhibits human voltage-gated proton channel hHv1. Proc. Natl Acad. Sci. USA 119, e2205420119 (2022).
Wang, S., Vafabakhsh, R., Borschel, W. F., Ha, T. & Nichols, C. G. Structural dynamics of potassium-channel gating revealed by single-molecule FRET. Nat. Struct. Mol. Biol. 23, 31–36 (2016).
Zhao, Y. et al. Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue. Nature 474, 109–113 (2011).
Ciftci, D. et al. Linking function to global and local dynamics in an elevator-type transporter. Proc. Natl Acad. Sci. USA 118, e2025520118 (2021).
Zhu, L. et al. Realtime observation of ATP-driven single B12 molecule translocation through BtuCD-F. Preprint at bioRxiv https://doi.org/10.1101/2022.12.02.518935 (2022).
Vafabakhsh, R., Levitz, J. & Isacoff, E. Y. Conformational dynamics of a class C G-protein-coupled receptor. Nature 524, 497–501 (2015).
Liu, T., Khanal, S., Hertslet, G. D. & Lamichhane, R. Single-molecule analysis reveals that a glucagon-bound extracellular domain of the glucagon receptor is dynamic. J. Biol. Chem. 299, 105160 (2023).
Heng, J. et al. Function and dynamics of the intrinsically disordered carboxyl terminus of β2-adrenergic receptor. Nat. Commun. 14, 2005 (2023).
Liauw, B. W., Afsari, H. S. & Vafabakhsh, R. Conformational rearrangement during activation of a metabotropic glutamate receptor. Nat. Chem. Biol. 17, 291–297 (2021).
Lecat-Guillet, N. et al. Concerted conformational changes control metabotropic glutamate receptor activity. Sci. Adv. 9, eadf1378 (2023).
Landes, C. F., Rambhadran, A., Taylor, J. N., Salatan, F. & Jayaraman, V. Structural landscape of isolated agonist-binding domains from single AMPA receptors. Nat. Chem. Biol. 7, 168–173 (2011).
Asher, W. B. et al. GPCR-mediated β-arrestin activation deconvoluted with single-molecule precision. Cell 185, 1661–1675.e16 (2022).
Diaz-Salinas, M. A. et al. Conformational dynamics and allosteric modulation of the SARS-CoV-2 spike. eLife 11, e75433 (2022).
Munro, J. B. et al. Conformational dynamics of single HIV-1 envelope trimers on the surface of native virions. Science 346, 759–763 (2014).
Ngo, T. T., Zhang, Q., Zhou, R., Yodh, J. G. & Ha, T. Asymmetric unwrapping of nucleosomes under tension directed by DNA local flexibility. Cell 160, 1135–1144 (2015).
Ngo, T. T. M. et al. Dependence of nucleosome mechanical stability on DNA mismatches and histone variants. Preprint at bioRxiv https://doi.org/10.1101/2022.11.21.517409 (2022).
Berman, H. M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
Gotz, M. et al. A blind benchmark of analysis tools to infer kinetic rate constants from single-molecule FRET trajectories. Nat. Commun. 13, 5402 (2022).
Agam, G. et al. Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins. Nat. Methods 20, 523–535 (2023).
Martens, K. J. A. et al. Visualisation of dCas9 target search in vivo using an open-microscopy framework. Nat. Commun. 10, 3552 (2019).
Ambrose, B. et al. The smfBox is an open-source platform for single-molecule FRET. Nat. Commun. 11, 5641 (2020).
Rueden, C. T. et al. ImageJ2: imageJ for the next generation of scientific image data. BMC Bioinformatics 18, 529 (2017).
Greenfeld, M. et al. Single-molecule dataset (SMD): a generalized storage format for raw and processed single-molecule data. BMC Bioinformatics 16, 3 (2015).
Vallat, B., Webb, B., Westbrook, J. D., Sali, A. & Berman, H. M. Development of a prototype system for archiving integrative/hybrid structure models of biological macromolecules. Structure 26, 894–904.e2 (2018).
Rasnik, I., McKinney, S. A. & Ha, T. Nonblinking and long-lasting single-molecule fluorescence imaging. Nat. Methods 3, 891–893 (2006).
Bates, M., Blosser, T. R. & Zhuang, X. Short-range spectroscopic ruler based on a single-molecule optical switch. Phys. Rev. Lett. 94, 108101 (2005).
Arseni, C. et al. Time-related variations of some biochemical parameters in patients with short- or long-lasting post-traumatic coma. Rom. J. Neurol. Psychiatry 28, 209–223 (1990).
Dagdas, Y. S., Chen, J. S., Sternberg, S. H., Doudna, J. A. & Yildiz, A. A conformational checkpoint between DNA binding and cleavage by CRISPR–Cas9. Sci. Adv. 3, eaao0027 (2017).
Wang, Y. et al. Real-time observation of Cas9 postcatalytic domain motions. Proc. Natl Acad. Sci. USA 118, e2010650118 (2021).
Grimm, J. B. et al. A general method to improve fluorophores using deuterated auxochromes. JACS Au 1, 690–696 (2021).
Kim, J. M. et al. Dynamic 1D search and processive nucleosome translocations by RSC and ISW2 chromatin remodelers. Preprint at bioRxiv https://doi.org/10.1101/2023.06.13.544671 (2023).
Lang, M. J., Fordyce, P. M., Engh, A. M., Neuman, K. C. & Block, S. M. Simultaneous, coincident optical trapping and single-molecule fluorescence. Nat. Methods 1, 133–139 (2004).
Gebhardt, C. et al. Labelizer: systematic selection of protein residues for covalent fluorophore labeling. Preprint at bioRxiv https://doi.org/10.1101/2023.06.12.544586 (2023).
Shi, X., Lim, J. & Ha, T. Acidification of the oxygen scavenging system in single-molecule fluorescence studies: in situ sensing with a ratiometric dual-emission probe. Anal. Chem. 82, 6132–6138 (2010).
Okumus, B., Wilson, T. J., Lilley, D. M. & Ha, T. Vesicle encapsulation studies reveal that single molecule ribozyme heterogeneities are intrinsic. Biophys. J. 87, 2798–2806 (2004).
Chung, H. S. et al. Extracting rate coefficients from single-molecule photon trajectories and FRET efficiency histograms for a fast-folding protein. J. Phys. Chem. A 115, 3642–3656 (2011).
Chen, J. et al. High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence. Proc. Natl Acad. Sci. USA 111, 664–669 (2014).
Cisse, I. I., Kim, H. & Ha, T. A rule of seven in Watson–Crick base-pairing of mismatched sequences. Nat. Struct. Mol. Biol. 19, 623–627 (2012).
Sanabria, H. et al. Resolving dynamics and function of transient states in single enzyme molecules. Nat. Commun. 11, 1231 (2020).
Hellenkamp, B., Wortmann, P., Kandzia, F., Zacharias, M. & Hugel, T. Multidomain structure and correlated dynamics determined by self-consistent FRET networks. Nat. Methods 14, 174–180 (2017).
Soranno, A. et al. Integrated view of internal friction in unfolded proteins from single-molecule FRET, contact quenching, theory, and simulations. Proc. Natl Acad. Sci. USA 114, E1833–E1839 (2017).
Chen, J. et al. The structural heterogeneity of α-synuclein is governed by several distinct subpopulations with interconversion times slower than milliseconds. Structure 29, 1048–1064.e6 (2021).
Trofymchuk, K. et al. Addressable nanoantennas with cleared hotspots for single-molecule detection on a portable smartphone microscope. Nat. Commun. 12, 950 (2021).
Holzmeister, P., Acuna, G. P., Grohmann, D. & Tinnefeld, P. Breaking the concentration limit of optical single-molecule detection. Chem. Soc. Rev. 43, 1014–1028 (2014).
Jain, A. et al. Probing cellular protein complexes using single-molecule pull-down. Nature 473, 484–488 (2011).
Mao, C. P. et al. Protein detection in blood with single-molecule imaging. Sci Adv 7, eabg6522 (2021).
Hartmann, A. et al. An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics. Nat. Commun. 14, 6511 (2023).
Severins, I., Joo, C. & van Noort, J. Exploring molecular biology in sequence space: the road to next-generation single-molecule biophysics. Mol. Cell 82, 1788–1805 (2022).
Pati, A. K. et al. Tuning the Baird aromatic triplet-state energy of cyclooctatetraene to maximize the self-healing mechanism in organic fluorophores. Proc. Natl Acad. Sci. USA 117, 24305–24315 (2020).
Isselstein, M. et al. Self-healing dyes—keeping the promise? J. Phys. Chem. Lett. 11, 4462–4480 (2020).
Zhang, Y. et al. General strategy to improve the photon budget of thiol-conjugated cyanine dyes. J. Am. Chem. Soc. https://doi.org/10.1021/jacs.2c12635 (2023).
Sow, M. et al. High-throughput nitrogen-vacancy center imaging for nanodiamond photophysical characterization and pH nanosensing. Nanoscale 12, 21821–21831 (2020).
Glushkov, E. et al. Engineering optically active defects in hexagonal boron nitride using focused ion beam and water. ACS Nano 16, 3695–3703 (2022).
Vermeer, B. & Schmid, S. Can DyeCycling break the photobleaching limit in single-molecule FRET? Nano Res. 15, 9818–9830 (2022).
Kummerlin, M., Mazumder, A. & Kapanidis, A. N. Bleaching-resistant, near-continuous single-molecule fluorescence and FRET based on fluorogenic and transient DNA binding. Chemphyschem 24, e202300175 (2023).
Sharonov, A. & Hochstrasser, R. M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc. Natl Acad. Sci. USA 103, 18911–18916 (2006).
Albitz, E. et al. Bioorthogonal ligation-activated fluorogenic FRET dyads. Angew. Chem. Int. Ed. 61, e202111855 (2022).
Ploetz, E. et al. Forster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers. Sci. Rep. 6, 33257 (2016).
Qiu, Y. et al. Srs2 prevents Rad51 filament formation by repetitive motion on DNA. Nat. Commun. 4, 2281 (2013).
Schubert, J., Schulze, A., Prodromou, C. & Neuweiler, H. Two-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics. Nat. Commun. 12, 6964 (2021).
Schmid, S., Stommer, P., Dietz, H. & Dekker, C. Nanopore electro-osmotic trap for the label-free study of single proteins and their conformations. Nat. Nanotechnol. 16, 1244–1250 (2021).
Tang, L. et al. Combined quantum tunnelling and dielectrophoretic trapping for molecular analysis at ultra-low analyte concentrations. Nat. Commun. 12, 913 (2021).
Gordon, R. Future prospects for biomolecular trapping with nanostructured metals. ACS Photonics 9, 1127–1135 (2022).
Hou, S., Exell, J. & Welsher, K. Real-time 3D single molecule tracking. Nat. Commun. 11, 3607 (2020).
Dyla, M. et al. Dynamics of P-type ATPase transport revealed by single-molecule FRET. Nature 551, 346–351 (2017).
Zhang, J. et al. Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand. eLife 7, e39518 (2018).
Hildebrandt, L. L., Preus, S. & Birkedal, V. Quantitative single molecule FRET efficiencies using TIRF microscopy. Faraday Discuss. 184, 131–142 (2015).
Acknowledgements
The authors thank the National Institutes of Health (NIH) for funding (R35 GM 122569 to T.H.) and the National Research Foundation of Korea (NRF-2023R1A2C2006606 to N.K.L.). T.H. is an investigator with the Howard Hughes Medical Institute.
Author information
Authors and Affiliations
Contributions
Introduction (T.H.); Experimentation (T.H., N.K.L. and S.Y.); Results (T.H., J.F. and S.P.); Applications (T.H. and R.L.G.); Reproducibility and data deposition (T.H. and S.S.); Limitations and optimizations (T.H.); Outlook (T.H. and S.S.); Overview of the Primer (T.H.).
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Reviews Methods Primers thanks the anonymous reviewer(s) for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Related links
µManager: https://micro-manager.org
Glossary
- Dwell times
-
Durations of time that single molecules spend in a specific fluorescence resonance energy transfer (FRET) state before transitioning to a different FRET state or photobleaching.
- Excitation volume
-
The spatial region of the excitation laser around an imaged object in which impurities and background fluorophores may also be excited.
- FRET states
-
Compositional or conformational states of a biomolecule that are distinguished by their fluorescence resonance energy transfer (FRET) efficiencies.
- Multi-colour smFRET
-
Single-molecule fluorescence resonance energy transfer (smFRET) using three or more fluorophores with distinct emission colours, allowing the simultaneous monitoring of multiple energy transfer events.
- Photobleaching
-
A photochemical reaction between the excited electronic state of a fluorophore and molecular oxygen (O2) that destroys the fluorophore and manifests as a single-step decrease in the emission signal of a single fluorophore to the background level.
- Photon budgets
-
The number of photons detected from a single fluorophore until photobleaching.
- Triplet-state blinking
-
Turning off and on of a single fluorophore caused by transitions to and from a triplet electronic state, respectively.
- Two-colour smFRET
-
The most common implementation of single-molecule fluorescence resonance energy transfer (smFRET), involving a single donor and a single acceptor fluorophore of distinct emission colours.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Ha, T., Fei, J., Schmid, S. et al. Fluorescence resonance energy transfer at the single-molecule level. Nat Rev Methods Primers 4, 21 (2024). https://doi.org/10.1038/s43586-024-00298-3
Accepted:
Published:
DOI: https://doi.org/10.1038/s43586-024-00298-3
