A practical guide to single-molecule FRET

Feynman, R.P. There's plenty of room at the bottom. J. Microelectromech. Syst. 1, 60–66 (1992).

Bustamante, C., Bryant, Z. & Smith, S.B. Ten years of tension: single-molecule DNA mechanics. Nature 421, 423–427 (2003).

Moerner, W.E. & Fromm, D.P. Methods of single-molecule fluorescence spectroscopy and microscopy. Rev. Sci. Instrum. 74, 3597–3619 (2003).An extensive review of single-molecule fluorescence methods.

Förster, T. Experimental and theoretical investigation of the intermolecular transfer of electronic excitation energy. Z. Naturforsch. A 4, 321–327 (1949).

Ha, T. Single-molecule fluorescence resonance energy transfer. Methods 25, 78–86 (2001).

Weiss, S. Fluorescence spectroscopy of single biomolecules. Science 283, 1676–1683 (1999).

Joo, C. & Ha, T. Single-molecule FRET with total internal reflection microscopy. in Single Molecule Techniques: a Laboratory Manual. (eds. P. Selvin & T. Ha) 3–36 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2007).A step-by-step 'how-to' manual for single-molecule FRET with TIR microscopy.

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).First detection of single-molecule FRET.

Kapanidis, A.N. et al. Alternating-laser excitation of single molecules. Acc. Chem. Res. 38, 523–533 (2005).A review of the alternating laser excitation (ALEX) methods for probing FRET in diffusing single-molecules in solution.

Michalet, X., Weiss, S. & Jager, M. Single-molecule fluorescence studies of protein folding and conformational dynamics. Chem. Rev. 106, 1785–1813 (2006).

Seidel, R. & Dekker, C. Single-molecule studies of nucleic acid motors. Curr. Opin. Struct. Biol. 17, 80–86 (2007).

Smiley, R.D. & Hammes, G.G. Single molecule studies of enzyme mechanisms. Chem. Rev. 106, 3080–3094 (2006).

Zhuang, X. Single-molecule RNA science. Annu. Rev. Biophys. Biomol. Struct. 34, 399–414 (2005).

Förster, T. Delocalized excitation and excitation transfer. in Modern Quantum Chemistry (ed., O. Shinanoglu) 93–137 (Academic Press, New York, 1967).

Stryer, L. & Haugland, R.P. Energy transfer: a spectroscopic ruler. Proc. Natl. Acad. Sci. USA 58, 719–726 (1967).

Deniz, A.A. et al. Single-pair fluorescence resonance energy transfer on freely diffusing molecules: observation of Förster distance dependence and subpopulations. Proc. Natl. Acad. Sci. USA 96, 3670–3675 (1999).

Best, R.B. et al. Effect of flexibility and cis residues in single-molecule FRET studies of polyproline. Proc. Natl. Acad. Sci. USA 104, 18964–18969 (2007).

Merchant, K.A., Best, R.B., Louis, J.M., Gopich, I.V. & Eaton, W.A. Characterizing the unfolded states of proteins using single-molecule FRET spectroscopy and molecular simulations. Proc. Natl. Acad. Sci. USA 104, 1528–1533 (2007).

Schuler, B. & Eaton, W.A. Protein folding studied by single-molecule FRET. Curr. Opin. Struct. Biol. 18, 16–26 (2008).A review of single-molecule FRET studies applied to extract quantitative distance information during protein folding.

Ha, T., Chemla, D.S., Enderle, T. & Weiss, S. Single molecule spectroscopy with automated positioning. Appl. Phys. Lett. 70, 782–784 (1997).

Sabanayagam, C.R., Eid, J.S. & Meller, A. High-throughput scanning confocal microscope for single molecule analysis. Appl. Phys. Lett. 84, 1216–1218 (2004).

Zhuang, X. et al. A single-molecule study of RNA catalysis and folding. Science 288, 2048–2051 (2000).

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).

Hohng, S. & Ha, T. Near-complete suppression of quantum dot blinking in ambient conditions. J. Am. Chem. Soc. 126, 1324–1325 (2004).

Kapanidis, A.N. & Weiss, S. Fluorescent probes and bioconjugation chemistries for single-molecule fluorescence analysis of biomolecules. J. Chem. Phys. 117, 10953–10964 (2002).A review of fluorescent dyes and conjugation chemistries for single-molecule fluorescence experiments.

Hubner, C.G., Renn, A., Renge, I. & Wild, U.P. Direct observation of the triplet lifetime quenching of single dye molecules by molecular oxygen. J. Chem. Phys. 115, 9619–9622 (2001).

Rasnik, I., McKinney, S.A. & Ha, T. Nonblinking and long-lasting single-molecule fluorescence imaging. Nat. Methods 3, 891–893 (2006).

Rust, M.J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

Widengren, J., Chmyrov, A., Eggeling, C., Lofdahl, P.A. & Seidel, C. Strategies to improve photostabilities in ultrasensitive fluorescence spectroscopy. J. Phys. Chem. A 111, 429–440 (2007).

Benesch, R.E. & Benesch, R. Enzymatic removal of oxygen for polarography and related methods. Science 118, 447–448 (1953).

Aitken, C.E., Marshall, R.A. & Puglisi, J. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys. J. 94, 1826–1835 (2008).

Wu, P.G. & Brand, L. Resonance energy transfer: methods and applications. Anal. Biochem. 218, 1–13 (1994).

Clegg, R.M. Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol. 211, 353–388 (1992).

Murphy, M.C., Rasnik, I., Cheng, W., Lohman, T.M. & Ha, T. Probing single-stranded DNA conformational flexibility using fluorescence spectroscopy. Biophys. J. 86, 2530–2537 (2004).

Ryu, Y.H. & Schultz, P.G. Efficient incorporation of unnatural amino acids into proteins in Escherichia coli. Nat. Methods 3, 263–265 (2006).

Higuchi, R., Krummel, B. & Saiki, R.K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16, 7351–7367 (1988).

Braman, J. In vitro mutagenesis protocols, vol. 182,. 2nd edn. (Humana Press, Totowa, New Jersey, 2001).

Pennington, M.W. Site-specific chemical modification procedures. Methods Mol. Biol. 35, 171–185 (1994).

Axelrod, D. Total internal reflection fluorescence at biological surfaces. in Noninvasive Techniques in Cell Biology. (eds. J.K. Foskett & S. Grinstein) 93–127 (Wiley-Liss, New York, 1990).

Axelrod, D. Total internal reflection fluorescence microscopy in cell biology. Methods Enzymol. 361, 1–33 (2003).

Michalet, X. et al. Detectors for single-molecule fluorescence imaging and spectroscopy. J. Mod. Opt. 54, 239–281 (2007).

Hohng, S., Joo, C. & Ha, T. Single-molecule three-color FRET. Biophys. J. 87, 1328–1337 (2004).Design and validation of three-color FRET at the single-molecule level.

Ha, T. et al. Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419, 638–641 (2002).

Sofia, S.J., Premnath, V.V. & Merrill, E.W. Poly(ethylene oxide) grafted to silicon surfaces: grafting density and protein adsorption. Macromolecules 31, 5059–5070 (1998).

Schroeder, C.M., Blainey, P.C., Kim, S. & Xie, X.S. Hydrodynamic flow-stretching assay for single-molecule studies of nucleic acid–protein interactions. in Single Molecule Techniques: A Laboratory Manual. (eds. P. Selvin & T. Ha) 461–492 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; 2007).

Heyes, C.D., Kobitski, A.Y., Amirgoulova, E.V. & Nienhaus, G.U. Biocompatible surfaces for specific tethering of individual protein molecules. J. Phys. Chem. B 108, 13387–13394 (2004).

Heyes, C.D., Groll, J., Moller, M. & Nienhaus, G.U. Synthesis, patterning and applications of star-shaped poly(ethylene glycol) biofunctionalized surfaces. Mol. Biosyst. 3, 419–430 (2007).

Cha, T., Guo, A. & Zhu, X.Y. Enzymatic activity on a chip: the critical role of protein orientation. Proteomics 5, 416–419 (2005).

Rhoades, E., Gussakovsky, E. & Haran, G. Watching proteins fold one molecule at a time. Proc. Natl. Acad. Sci. USA 100, 3197–3202 (2003).

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).

Benitez, J.J. et al. Probing transient copper chaperone-Wilson disease protein interactions at the single-molecule level with nanovesicle trapping. J. Am. Chem. Soc. 130, 2446–2447 (2008).

Cisse, I., Okumus, B., Joo, C. & Ha, T. Fueling protein-DNA interactions inside porous nanocontainers. Proc. Natl. Acad. Sci. USA 104, 12646–12650 (2007).

Myong, S., Rasnik, I., Joo, C., Lohman, T.M. & Ha, T. Repetitive shuttling of a motor protein on DNA. Nature 437, 1321–1325 (2005).

Van der Meer, B.W. Resonance energy transfer. (Wiley, Chichester, UK, 1999).

Ha, T. et al. Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism. Proc. Natl. Acad. Sci. USA 96, 893–898 (1999).

Joo, C. et al. Real-time observation of RecA filament dynamics with single monomer resolution. Cell 126, 515–527 (2006).

Luo, G., Wang, M., Konigsberg, W.H. & Xie, X.S. Single-molecule and ensemble fluorescence assays for a functionally important conformational change in T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 104, 12610–12615 (2007).Use of intensity fluctuations of a single fluorophore to report on the biochemical reactions of a DNA-enzyme complex.

Clegg, R.M., Murchie, A.I., Zechel, A. & Lilley, D.M. Observing the helical geometry of double-stranded DNA in solution by fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 90, 2994–2998 (1993).

Cooper, J.P. & Hagerman, P.J. Analysis of fluorescence energy transfer in duplex and branched DNA molecules. Biochemistry 29, 9261–9268 (1990).

Lee, S.P., Porter, D., Chirikjian, J.G., Knutson, J.R. & Han, M.K. A fluorometric assay for DNA cleavage reactions characterized with BamHI restriction endonuclease. Anal. Biochem. 220, 377–383 (1994).

Ha, T.J. et al. Temporal fluctuations of fluorescence resonance energy transfer between two dyes conjugated to a single protein. Chem. Phys. 247, 107–118 (1999).

Colquhoun, D. & Hawkes, A.G. The principles of the stochastic interpretation of ion-channel mechanism. in Single Channel Recording. (eds. B. Sakmann & E. Neher) 397–482 (Plenum Press, New York, 1995).This chapter explains determination of kinetic parameters from stochastic fluctuations of single ion-channel time trajectories. The same concepts are equally applicable to single-molecule FRET trajectories, and the chapter is highly recommended to the beginners in the field.

Kim, H.D. et al. Mg2+-dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules. Proc. Natl. Acad. Sci. USA 99, 4284–4289 (2002).

McKinney, S.A., Joo, C. & Ha, T. Analysis of single-molecule FRET trajectories using hidden Markov modeling. Biophys. J. 91, 1941–1951 (2006).Hidden Markov model–based analysis of single-molecule FRET time trajectories.

Munro, J.B., Altman, R.B., O'Connor, N. & Blanchard, S.C. Identification of two distinct hybrid state intermediates on the ribosome. Mol. Cell 25, 505–517 (2007).

Myong, S., Bruno, M.M., Pyle, A.M. & Ha, T. Spring-loaded mechanism of DNA unwinding by hepatitis C virus NS3 helicase. Science 317, 513–516 (2007).

Yang, H. & Xie, X.S. Probing single-molecule dynamics photon by photon. J. Chem. Phys. 117, 10965–10979 (2002).

Andrec, M., Levy, R.M. & Talaga, D.S. Direct determination of kinetic rates from single-molecule photon arrival trajectories using hidden Markov models. J. Phys. Chem. A 107, 7454–7464 (2003).

Schroder, G.F. & Grubmuller, H. Maximum likelihood trajectories from single molecule fluorescence resonance energy transfer experiments. J. Chem. Phys. 119, 9920–9924 (2003).

Blanchard, S.C., Gonzalez, R.L., Kim, H.D., Chu, S. & Puglisi, J.D. tRNA selection and kinetic proofreading in translation. Nat. Struct. Mol. Biol. 11, 1008–1014 (2004).

Smith, G.J., Sosnick, T.R., Scherer, N.F. & Pan, T. Efficient fluorescence labeling of a large RNA through oligonucleotide hybridization. RNA 11, 234–239 (2005).

Dorywalska, M. et al. Site-specific labeling of the ribosome for single-molecule spectroscopy. Nucleic Acids Res. 33, 182–189 (2005).

Deniz, A.A. et al. Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2. Proc. Natl. Acad. Sci. USA 97, 5179–5184 (2000).

Jager, M., Nir, E. & Weiss, S. Site-specific labeling of proteins for single-molecule FRET by combining chemical and enzymatic modification. Protein Sci. 15, 640–646 (2006).

Dale, R.E., Eisinger, J. & Blumberg, W.E. Orientational freedom of molecular probes: orientation factor in intra-molecular energy transfer. Biophys. J. 26, 161–193 (1979).

Schuler, B., Lipman, E.A., Steinbach, P.J., Kumke, M. & Eaton, W.A. Polyproline and the “spectroscopic ruler” revisited with single-molecule fluorescence. Proc. Natl. Acad. Sci. USA 102, 2754–2759 (2005).

Rothwell, P.J. et al. Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes. Proc. Natl. Acad. Sci. USA 100, 1655–1660 (2003).

Rasnik, I., Myong, S., Cheng, W., Lohman, T.M. & Ha, T. DNA-binding orientation and domain conformation of the E. coli rep helicase monomer bound to a partial duplex junction: single-molecule studies of fluorescently labeled enzymes. J. Mol. Biol. 336, 395–408 (2004).

Andrecka, J. et al. Single-molecule tracking of mRNA exiting from RNA polymerase II. Proc. Natl. Acad. Sci. USA 105, 135–140 (2008).

Clamme, J.P. & Deniz, A.A. Three-color single-molecule fluorescence resonance energy transfer. ChemPhysChem 6, 74–77 (2005).

Heinze, K.G., Jahnz, M. & Schwille, P. Triple-color coincidence analysis: one step further in following higher order molecular complex formation. Biophys. J. 86, 506–516 (2004).

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).

Muller, B.K., Zaychikov, E., Brauchle, C. & Lamb, D.C. Pulsed interleaved excitation. Biophys. J. 89, 3508–3522 (2005).

Lee, N.K. et al. Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances. Biophys. J. 92, 303–312 (2007).

Heilemann, M. et al. Multistep energy transfer in single molecular photonic wires. J. Am. Chem. Soc. 126, 6514–6515 (2004).

Lang, M., Fordyce, P. & Block, S. Combined optical trapping and single-molecule fluorescence. J. Biol. 2, 6 (2003).

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 nm(3). Nano Lett. 5, 1509–1514 (2005).

Hohng, S. et al. Fluorescence-force spectroscopy maps two-dimensional reaction landscape of the holliday junction. Science 318, 279–283 (2007).

Borisenko, V. et al. Simultaneous optical and electrical recording of single gramicidin channels. Biophys. J. 84, 612–622 (2003).

Harms, G.S. et al. Probing conformational changes of gramicidin ion channels by single-molecule patch-clamp fluorescence microscopy. Biophys. J. 85, 1826–1838 (2003).

Tan, E. et al. A four-way junction accelerates hairpin ribozyme folding via a discrete intermediate. Proc. Natl. Acad. Sci. USA 100, 9308–9313 (2003).

Jager, M., Michalet, X. & Weiss, S. Protein-protein interactions as a tool for site-specific labeling of proteins. Protein Sci. 14, 2059–2068 (2005).

Ratner, V., Kahana, E., Eichler, M. & Haas, E. A general strategy for site-specific double labeling of globular proteins for kinetic FRET studies. Bioconjug. Chem. 13, 1163–1170 (2002).