Biophysical Reviews

, 1:161 | Cite as

Single-molecule Förster resonance energy transfer studies of RNA structure, dynamics and function

  • Mark HelmEmail author
  • Andrei Yu Kobitski
  • G. Ulrich Nienhaus


Single-molecule fluorescence microscopy experiments on RNA molecules brought to light the highly complex dynamics of key biological processes, including RNA folding, catalysis of ribozymes, ligand sensing of riboswitches and aptamers, and protein synthesis in the ribosome. By using highly advanced biophysical spectroscopy techniques in combination with sophisticated biochemical synthesis approaches, molecular dynamics of individual RNA molecules can be observed in real time and under physiological conditions in unprecedented detail that cannot be achieved with bulk experiments. Here, we review recent advances in RNA folding and functional studies of RNA and RNA-protein complexes addressed by using single-molecule Förster (fluorescence) resonance energy transfer (smFRET) technique.


RNA RNA folding RNA-protein complexes Single-molecule Förster resonance energy transfer technique 



The authors acknowledge funding by the Volkswagen Foundation and the Deutsche Forschungsgemeinschaft (DFG). We thank Martin Hengesbach for technical assistance.


  1. Abbondanzieri EA, Bokinsky G, Rausch JW, Zhang JX, Le Grice SF, Zhuang X (2008) Dynamic binding orientations direct activity of HIV reverse transcriptase. Nature 453:184–189PubMedGoogle Scholar
  2. Aitken CE, Marshall RA, Puglisi JD (2008) An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys J 94:1826–1835PubMedGoogle Scholar
  3. Al-Hashimi HM, Walter NG (2008) RNA dynamics: it is about time. Curr Opin Struct Biol 18:321–329PubMedGoogle Scholar
  4. Altman S (2007) A view of RNase P. Mol Biosyst 3:604–607PubMedGoogle Scholar
  5. Amirgoulova E, Groll J, Heyes CD, Ameringer T, Röcker C, Möller M, Nienhaus GU (2004) Biofunctionalized polymer surfaces exhibiting minimal interaction towards immobilized proteins. Chem Phys Chem 5:552–555PubMedGoogle Scholar
  6. Bartley LE, Zhuang X, Das R, Chu S, Herschlag D (2003) Exploration of the transition state for tertiary structure formation between an RNA helix and a large structured RNA. J Mol Biol 328:1011–1026PubMedGoogle Scholar
  7. Bates M, Blosser TR, Zhuang X (2005) Short-range spectroscopic ruler based on a single-molecule optical switch. Phys Rev Lett 94:108101PubMedGoogle Scholar
  8. Blanchard SC (2009) Single-molecule observations of ribosome function. Curr Opin Struct Biol 19:103–109PubMedGoogle Scholar
  9. Blanchard SC, Gonzalez RL, Kim HD, Chu S, Puglisi JD (2004a) tRNA selection and kinetic proofreading in translation. Nat Struct Mol Biol 11:1008–1014PubMedGoogle Scholar
  10. Blanchard SC, Kim HD, Gonzalez RL Jr, Puglisi JD, Chu S (2004b) tRNA dynamics on the ribosome during translation. Proc Natl Acad Sci USA 101:12893–12898PubMedGoogle Scholar
  11. Blount KF, Breaker RR (2006) Riboswitches as antibacterial drug targets. Nat Biotechnol 24:1558–1564PubMedGoogle Scholar
  12. Bokinsky G, Zhuang X (2005) Single-molecule RNA folding. Acc Chem Res 38:566–573PubMedGoogle Scholar
  13. Bokinsky G, Rueda D, Misra VK, Rhodes MM, Gordus A, Babcock HP, Walter NG, Zhuang X (2003) Single-molecule transition-state analysis of RNA folding. Proc Natl Acad Sci USA 100:9302–9307PubMedGoogle Scholar
  14. Bokinsky G, Nivon LG, Liu S, Chai G, Hong M, Weeks KM, Zhuang X (2006) Two distinct binding modes of a protein cofactor with its target RNA. J Mol Biol 361:771–784PubMedGoogle Scholar
  15. Boukobza E, Sonnenfeld A, Haran G (2001) Immobilization in surface-tethered lipid vesicles as a new tool for single biomolecule spectroscopy. J Phys Chem B 105:12165–12170Google Scholar
  16. Breaker RR (2002) Engineered allosteric ribozymes as biosensor components. Curr Opin Biotechnol 13:31–39PubMedGoogle Scholar
  17. Brion P, Westhof E (1997) Hierarchy and dynamics of RNA folding. Annu Rev Biophys Biomol Struct 26:113–137PubMedGoogle Scholar
  18. Cate JH, Gooding AR, Podell E, Zhou K, Golden BL, Kundrot CE, Cech TR, Doudna JA (1996) Crystal structure of a group I ribozyme domain: principles of RNA packing. Science 273:1678–1685PubMedGoogle Scholar
  19. Cech T (1995) Group I introns: new molecular mechanisms for mRNA repair. Biotechnology (NY) 13:323–326Google Scholar
  20. Cech TR (2000) Structural biology. The ribosome is a ribozyme. Science 289:878–879Google Scholar
  21. Chan B, Weidemaier K, Yip WT, Barbara PF, Musier-Forsyth K (1999) Intra-tRNA distance measurements for nucleocapsid proteindependent tRNA unwinding during priming of HIV reverse transcription. Proc Natl Acad Sci USA 96:459–464PubMedGoogle Scholar
  22. Christensen SM, Stamou D (2007) Surface-based lipid vesicle reactor systems: fabrication and applications. Soft Matter 3:828–836Google Scholar
  23. Cisse I, Okumus B, Joo C, Ha T (2007) Fueling protein DNA interactions inside porous nanocontainers. Proc Natl Acad Sci USA 104:12646–12650PubMedGoogle Scholar
  24. Cornish PV, Ermolenko DN, Noller HF, Ha T (2008) Spontaneous intersubunit rotation in single ribosomes. Mol Cell 30:578–588PubMedGoogle Scholar
  25. Cornish PV, Ermolenko DN, Staple DW, Hoang L, Hickerson RP, Noller HF, Ha T (2009) Following movement of the L1 stalk between three functional states in single ribosomes. Proc Natl Acad Sci USA 106:2571–2576PubMedGoogle Scholar
  26. de Silva C, Walter NG (2009) Leakage and slow allostery limit performance of single drug-sensing aptazyme molecules based on the hammerhead ribozyme. RNA 15:76–84PubMedGoogle Scholar
  27. Dittrich PS, Muller B, Schwille P (2004) Studying reaction kinetics by simultaneous FRET and cross-correlation analysis in a miniaturized continuous flow reactor. Phys Chem Chem Phys 6:4416–4420Google Scholar
  28. Ditzler MA, Aleman EA, Rueda D, Walter NG (2007) Focus on function: single molecule RNA enzymology. Biopolymers 87:302–316PubMedGoogle Scholar
  29. Ditzler MA, Rueda D, Mo J, Hakansson K, Walter NG (2008) A rugged free energy landscape separates multiple functional RNA folds throughout denaturation. Nucleic Acids Res 36:7088–7099PubMedGoogle Scholar
  30. Dorywalska M, Blanchard SC, Gonzalez RL, Kim HD, Chu S, Puglisi JD (2005) Site-specific labeling of the ribosome for single-molecule spectroscopy. Nucleic Acids Res 33:182–189PubMedGoogle Scholar
  31. Eggeling C, Widengren J, Brand L, Schaffer J, Felekyan S, Seidel CA (2006) Analysis of photobleaching in single-molecule multicolor excitation and Forster resonance energy transfer measurements. J Phys Chem A 110:2979–2995PubMedGoogle Scholar
  32. Ehrenschwender T, Wanninger-Weiss C, Wagenknecht HA (2008) BODIPY-modified uridines as potential fluorescent probes for nucleic acids that are recognized by DNA-polymerases. Nucleic Acids Symp Ser 52:349–350Google Scholar
  33. Elenko MP, Szostak JW, van Oijen AM (2009) Single-Molecule Imaging of an in Vitro-Evolved RNA Aptamer Reveals Homogeneous Ligand Binding Kinetics. J Am Chem Soc 131:9866–9867PubMedGoogle Scholar
  34. Famulok M, Mayer G (1999) Aptamers as tools in molecular biology and immunology. Curr Top Microbiol Immunol 243:123–136PubMedGoogle Scholar
  35. Fang XW, Pan T, Sosnick TR (1999) Mg2+-dependent folding of a large ribozyme without kinetic traps. Nat Struct Biol 6:1091–1095PubMedGoogle Scholar
  36. Fei J, Kosuri P, MacDougall DD, Gonzalez RL Jr (2008) Coupling of ribosomal L1 stalk and tRNA dynamics during translation elongation. Mol Cell 30:348–359PubMedGoogle Scholar
  37. Fersht AR, Matouschek A, Serrano L (1992) The folding of an enzyme. I. Theory of protein engineering analysis of stability and pathway of protein folding. J Mol Biol 224:771–782PubMedGoogle Scholar
  38. Frank J (1997) The ribosome at higher resolution - the donut takes shape. Curr Opin Struct Biol 7:266–272PubMedGoogle Scholar
  39. Frank J, Agrawal RK (2000) A ratchet-like inter-subunit reorganization of the ribosome during translocation. Nature 406:318–322PubMedGoogle Scholar
  40. Frank J, Gao H, Sengupta J, Gao N, Taylor DJ (2007) The process of mRNA-tRNA translocation. Proc Natl Acad Sci USA 104:19671–19678PubMedGoogle Scholar
  41. Gesteland RF, Atkins JF (1993) The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  42. Gold L, Polisky B, Uhlenbeck O, Yarus M (1995) Diversity of oligonucleotide functions. Annu Rev Biochem 64:763–797PubMedGoogle Scholar
  43. Gonzalez RL Jr, Chu S, Puglisi JD (2007) Thiostrepton inhibition of tRNA delivery to the ribosome. RNA 13:2091–2097PubMedGoogle Scholar
  44. Gopich IV, Szabo A (2009) Decoding the Pattern of Photon Colors in Single-Molecule FRET. J Phys Chem B (in press)Google Scholar
  45. Greenleaf WJ, Frieda KL, Foster DA, Woodside MT, Block SM (2008) Direct observation of hierarchical folding in single riboswitch aptamers. Science 319:630–633PubMedGoogle Scholar
  46. Groll J, Amirgoulova EV, Ameringer T, Heyes CD, Röcker C, Nienhaus GU, Möller M (2004) Biofunctionalized, ultrathin coatings of cross-linked star-shaped poly(ethylene oxide) allow reversible folding of immobilized proteins. J Am Chem Soc 126:4234–4239PubMedGoogle Scholar
  47. Grundy FJ, Henkin TM (2006) From ribosome to riboswitch: control of gene expression in bacteria by RNA structural rearrangements. Crit Rev Biochem Mol Biol 41:329–338PubMedGoogle Scholar
  48. Guerrier-Takada C, Gardiner K, Marsh T, Pace N, Altman S (1983) The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35:849–857PubMedGoogle Scholar
  49. Guo F, Gooding AR, Cech TR (2004) Structure of the Tetrahymena ribozyme: base triple sandwich and metal ion at the active site. Mol Cell 16:351–362PubMedGoogle Scholar
  50. Ha T, Zhuang X, Kim HD, Orr JW, Williamson JR, Chu S (1999) Ligand-induced conformational changes observed in single RNA molecules. Proc Natl Acad Sci USA 96:9077–9082PubMedGoogle Scholar
  51. Haustein E, Schwille P (2007) Trends in fluorescence imaging and related techniques to unravel biological information. HFSP J 1:169–180PubMedGoogle Scholar
  52. Heilemann M, Margeat E, Kasper R, Sauer M, Tinnefeld P (2005) Carbocyanine dyes as efficient reversible single-molecule optical switch. J Am Chem Soc 127:3801–3806PubMedGoogle Scholar
  53. Hengesbach M, Kobitski A, Voigts-Hoffmann F, Frauer C, Nienhaus GU, Helm M (2008) RNA intramolecular dynamics by single-molecule FRET. Curr Protoc Nucleic Acid Chem Chapter 11:Unit 11 12Google Scholar
  54. Heyes CD, Kobitski AY, Amirgoulova EV, Nienhaus GU (2004) Biocompatible surfaces for specific tethering of individual protein molecules. J Phys Chem B 108:13387–13394Google Scholar
  55. Heyes CD, Groll J, Moller M, Nienhaus GU (2007) Synthesis, patterning and applications of star-shaped poly(ethylene glycol) biofunctionalized surfaces. Mol Biosyst 3:419–430PubMedGoogle Scholar
  56. Hohng S, Joo C, Ha T (2004a) Single-molecule three-color FRET. Biophys J 87:1328–1337PubMedGoogle Scholar
  57. Hohng S, Wilson TJ, Tan E, Clegg RM, Lilley DM, Ha T (2004b) Conformational flexibility of four-way junctions in RNA. J Mol Biol 336:69–79PubMedGoogle Scholar
  58. Horenko I, Dittmer E, Fischer A, Christof S (2007) Automated model reduction for complex systems exhibiting metastability. Multiscale Model Simul 5:802–827Google Scholar
  59. Iqbal A, Arslan S, Okumus B, Wilson TJ, Giraud G, Norman DG, Ha T, Lilley DM (2008a) Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids. Proc Natl Acad Sci USA 105:11176–11181PubMedGoogle Scholar
  60. Iqbal A, Wang L, Thompson KC, Lilley DM, Norman DG (2008b) The structure of cyanine 5 terminally attached to double-stranded DNA: implications for FRET studies. Biochemistry 47:7857–7862PubMedGoogle Scholar
  61. Jia Y, Sytnik A, Li L, Vladimirov S, Cooperman BS, Hochstrasser RM (1997) Nonexponential kinetics of a single tRNAPhe molecule under physiological conditions. Proc Natl Acad Sci USA 94:7932–7936PubMedGoogle Scholar
  62. Johansen LE, Nygaard P, Lassen C, Agerso Y, Saxild HH (2003) Definition of a second Bacillus subtilis pur regulon comprising the pur and xpt-pbuX operons plus pbuG, nupG (yxjA), and pbuE (ydhL). J Bacteriol 185:5200–5209PubMedGoogle Scholar
  63. Julian P, Konevega AL, Scheres SH, Lazaro M, Gil D, Wintermeyer W, Rodnina MV, Valle M (2008) Structure of ratcheted ribosomes with tRNAs in hybrid states. Proc Natl Acad Sci USA 105:16924–16927PubMedGoogle Scholar
  64. Kalinin S, Felekyan S, Antonik M, Seidel CA (2007) Probability distribution analysis of single-molecule fluorescence anisotropy and resonance energy transfer. J Phys Chem B 111:10253–10262PubMedGoogle Scholar
  65. Kapanidis A, Weiss S (2002) Fluorescent probes and bioconjugation chemistries for single-molecule fluorescence analysis of biomolecules. J Chem Phys 117:10953–10964Google Scholar
  66. Kapanidis AN, Laurence TA, Lee NK, Margeat E, Kong X, Weiss S (2005) Alternating-laser excitation of single molecules. Acc Chem Res 38:523–533PubMedGoogle Scholar
  67. Kauffmann E, Darnton NC, Austin RH, Batt C, Gerwert K (2001) Lifetimes of intermediates in the beta -sheet to alpha -helix transition of beta -lactoglobulin by using a diffusional IR mixer. Proc Natl Acad Sci USA 98:6646–6649PubMedGoogle Scholar
  68. Kim HD, Nienhaus GU, Ha T, Orr JW, Williamson JR, Chu S (2002) Mg2+-dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules. Proc Natl Acad Sci USA 99:4284–4289PubMedGoogle Scholar
  69. Klein DJ, Ferre-D’Amare AR (2006) Structural basis of glmS ribozyme activation by glucosamine-6-phosphate. Science 313:1752–1756PubMedGoogle Scholar
  70. Klein DJ, Been MD, Ferre-D’Amare AR (2007) Essential role of an active-site guanine in glmS ribozyme catalysis. J Am Chem Soc 129:14858–14859PubMedGoogle Scholar
  71. Kobitski AY, Nierth A, Helm M, Jaschke A, Nienhaus GU (2007) Mg2+-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis. Nucleic Acids Res 35:2047–2059PubMedGoogle Scholar
  72. Kobitski AY, Hengesbach M, Helm M, Nienhaus GU (2008) Sculpting an RNA conformational energy landscape by a methyl group modification–a single-molecule FRET study. Angew Chem Int Ed Engl 47:4326–4230PubMedGoogle Scholar
  73. Koopmans WJ, Schmidt T, van Noort J (2008) Nucleosome immobilization strategies for single-pair FRET microscopy. ChemPhysChem 9:2002–2009PubMedGoogle Scholar
  74. Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE, Cech TR (1982) Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell 31:147–157PubMedGoogle Scholar
  75. Kuzmenkina EV, Heyes CD, Nienhaus GU (2006) Single-molecule FRET study of denaturant induced unfolding of RNase H. J Mol Biol 357:313–324PubMedGoogle Scholar
  76. Landes CF, Zeng Y, Liu HW, Musier-Forsyth K, Barbara PF (2007) Single-molecule study of the inhibition of HIV-1 transactivation response region DNA/DNA annealing by argininamide. J Am Chem Soc 129:10181–10188PubMedGoogle Scholar
  77. Lee JY, Okumus B, Kim DS, Ha T (2005) Extreme conformational diversity in human telomeric DNA. Proc Natl Acad Sci USA 102:18938–18943PubMedGoogle Scholar
  78. Lee NK, Kapanidis AN, Koh HR, Korlann Y, Ho SO, Kim Y, Gassman N, Kim SK, Weiss S (2007a) Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances. Biophys J 92:303–312PubMedGoogle Scholar
  79. Lee TH, Blanchard SC, Kim HD, Puglisi JD, Chu S (2007b) The role of fluctuations in tRNA selection by the ribosome. Proc Natl Acad Sci USA 104:13661–13665PubMedGoogle Scholar
  80. Lee TH, Lapidus LJ, Zhao W, Travers KJ, Herschlag D, Chu S (2007c) Measuring the folding transition time of single RNA molecules. Biophys J 92:3275–3283PubMedGoogle Scholar
  81. Lemay JF, Penedo JC, Tremblay R, Lilley DM, Lafontaine DA (2006) Folding of the adenine riboswitch. Chem Biol 13:857–368PubMedGoogle Scholar
  82. Lipman EA, Schuler B, Bakajin O, Eaton WA (2003) Single-molecule measurement of protein folding kinetics. Science 301:1233–1235PubMedGoogle Scholar
  83. Liu S, Bokinsky G, Walter NG, Zhuang X (2007) Dissecting the multistep reaction pathway of an RNA enzyme by single-molecule kinetic "fingerprinting". Proc Natl Acad Sci USA 104:12634–12639PubMedGoogle Scholar
  84. Liu S, Abbondanzieri EA, Rausch JW, Le Grice SF, Zhuang X (2008) Slide into action: dynamic shuttling of HIV reverse transcriptase on nucleic acid substrates. Science 322:1092–1097PubMedGoogle Scholar
  85. Marshall RA, Aitken CE, Dorywalska M, Puglisi JD (2008a) Translation at the single-molecule level. Annu Rev Biochem 77:177–203PubMedGoogle Scholar
  86. Marshall RA, Dorywalska M, Puglisi JD (2008b) Irreversible chemical steps control intersubunit dynamics during translation. Proc Natl Acad Sci USA 105:15364–15369PubMedGoogle Scholar
  87. McKinney SA, Joo C, Ha T (2006) Analysis of single-molecule FRET trajectories using hidden Markov modeling. Biophys J 91:1941–1951PubMedGoogle Scholar
  88. Moerner WE, Fromm D (2003) Methods of single-molecule fluorescence spectroscopy and microscopy. Rev Sci Instrum 74:3597–3619Google Scholar
  89. Montange RK, Batey RT (2008) Riboswitches: emerging themes in RNA structure and function. Annu Rev Biophys 37:117–133PubMedGoogle Scholar
  90. Muller BK, Zaychikov E, Brauchle C, Lamb DC (2005) Pulsed interleaved excitation. Biophys J 89:3508–3522PubMedGoogle Scholar
  91. Munro JB, Altman RB, O’Connor N, Blanchard SC (2007) Identification of two distinct hybrid state intermediates on the ribosome. Mol Cell 25:505–517PubMedGoogle Scholar
  92. Munro JB, Vaiana A, Sanbonmatsu KY, Blanchard SC (2008) A new view of protein synthesis: mapping the free energy landscape of the ribosome using single-molecule FRET. Biopolymers 89:565–577PubMedGoogle Scholar
  93. Nahas MK, Wilson TJ, Hohng S, Jarvie K, Lilley DM, Ha T (2004) Observation of internal cleavage and ligation reactions of a ribozyme. Nat Struct Mol Biol 11:1107–1113PubMedGoogle Scholar
  94. Noe F, Fischer S (2008) Transition networks for modeling the kinetics of conformational change in macromolecules. Curr Opin Struct Biol 18:154–162PubMedGoogle Scholar
  95. Nygaard P, Saxild HH (2005) The purine efflux pump PbuE in Bacillus subtilis modulates expression of the PurR and G-box (XptR) regulons by adjusting the purine base pool size. J Bacteriol 187:791–794PubMedGoogle Scholar
  96. Okumus B, Wilson TJ, Lilley DM, Ha T (2004) Vesicle encapsulation studies reveal that single molecule ribozyme heterogeneities are intrinsic. Biophys J 87:2798–2806PubMedGoogle Scholar
  97. Pereira MJ, Nikolova EN, Hiley SL, Jaikaran D, Collins RA, Walter NG (2008) Single VS ribozyme molecules reveal dynamic and hierarchical folding toward catalysis. J Mol Biol 382:496–509PubMedGoogle Scholar
  98. Pfeil SH, Wickersham CE, Hoffmann A, Lipman EA (2009) A microfluidic mixing system for single-molecule measurements. Rev Sci Instrum 80:5105Google Scholar
  99. Pljevaljcic G, Millar DP, Deniz AA (2004) Freely diffusing single hairpin ribozymes provide insights into the role of secondary structure and partially folded states in RNA folding. Biophys J 87:457–467PubMedGoogle Scholar
  100. Qu X, Smith GJ, Lee KT, Sosnick TR, Pan T, Scherer NF (2008) Single-molecule nonequilibrium periodic Mg2+-concentration jump experiments reveal details of the early folding pathways of a large RNA. Proc Natl Acad Sci U S A 105:6602–6607PubMedGoogle Scholar
  101. Ramakrishnan V (2002) Ribosome structure and the mechanism of translation. Cell 108:557–572PubMedGoogle Scholar
  102. Rasnik I, Myong S, Cheng W, Lohman TM, Ha T (2004) 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 336:395–408PubMedGoogle Scholar
  103. Rasnik I, McKinney SA, Ha T (2005) Surfaces and orientations: much to FRET about? Acc Chem Res 38:542–548PubMedGoogle Scholar
  104. Rasnik I, McKinney SA, Ha T (2006) Nonblinking and long-lasting single-molecule fluorescence imaging. Nat Methods 3:891–893PubMedGoogle Scholar
  105. Rhoades E, Gussakovsky E, Haran G (2003) Watching proteins fold one molecule at a time. Proc Natl Acad Sci USA 100:3197–3202PubMedGoogle Scholar
  106. Rieger R, Rocker C, Nienhaus GU (2005) Fluctuation correlation spectroscopy for the advanced physics laboratory. Am J Phys 73:1129–1134Google Scholar
  107. Roy R, Hohng S, Ha T (2008) A practical guide to single-molecule FRET. Nat Methods 5:507–516PubMedGoogle Scholar
  108. Rueda D, Bokinsky G, Rhodes MM, Rust MJ, Zhuang X, Walter NG (2004) Single-molecule enzymology of RNA: essential functional groups impact catalysis from a distance. Proc Natl Acad Sci USA 101:10066–10071PubMedGoogle Scholar
  109. Russell R, Zhuang X, Babcock HP, Millett IS, Doniach S, Chu S, Herschlag D (2002) Exploring the folding landscape of a structured RNA. Proc Natl Acad Sci USA 99:155–160PubMedGoogle Scholar
  110. Sattin BD, Zhao W, Travers K, Chu S, Herschlag D (2008) Direct measurement of tertiary contact cooperativity in RNA folding. J Am Chem Soc 130:6085–6087PubMedGoogle Scholar
  111. Schuler B, Lipman EA, Steinbach PJ, Kumke M, Eaton WA (2005) Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence. Proc Natl Acad Sci USA 102:2754–2759PubMedGoogle Scholar
  112. Seelig B, Jaschke A (1999) A small catalytic RNA motif with Diels-Alderase activity. Chem Biol 6:167–176PubMedGoogle Scholar
  113. Seelig B, Keiper S, Stuhlmann F, Jaschke A (2000) Enantioselective ribozyme catalysis of a bimolecular cycloaddition reaction. Angew Chem Int Ed Engl 39:4576–4579PubMedGoogle Scholar
  114. Silverman SK, Deras ML, Woodson SA, Scaringe SA, Cech TR (2000) Multiple folding pathways for the P4–P6 RNA domain. Biochemistry 39:12465–12475PubMedGoogle Scholar
  115. Smith GJ, Lee KT, Qu X, Xie Z, Pesic J, Sosnick TR, Pan T, Scherer NF (2008) A large collapsed-state RNA can exhibit simple exponential single-molecule dynamics. J Mol Biol 378:943–953PubMedGoogle Scholar
  116. Soukup GA, Breaker RR (1999) Engineering precision RNA molecular switches. Proc Natl Acad Sci USA 96:3584–3589PubMedGoogle Scholar
  117. Soukup GA, Emilsson GA, Breaker RR (2000) Altering molecular recognition of RNA aptamers by allosteric selection. J Mol Biol 298:623–632PubMedGoogle Scholar
  118. Stone MD, Mihalusova M, O’Connor CM, Prathapam R, Collins K, Zhuang X (2007) Stepwise protein-mediated RNA folding directs assembly of telomerase ribonucleoprotein. Nature 446:458–461PubMedGoogle Scholar
  119. Sudarsan N, Lee ER, Weinberg Z, Moy RH, Kim JN, Link KH, Breaker RR (2008) Riboswitches in eubacteria sense the second messenger cyclic di-GMP. Science 321:411–413PubMedGoogle Scholar
  120. Sytnik A, Vladimirov S, Jia Y, Li L, Cooperman BS, Hochstrasser RM (1999) Peptidyl transferase center activity observed in single ribosomes. J Mol Biol 285:49–54PubMedGoogle Scholar
  121. Tan E, Wilson TJ, Nahas MK, Clegg RM, Lilley DM, Ha T (2003) A four-way junction accelerates hairpin ribozyme folding via a discrete intermediate. Proc Natl Acad Sci USA 100:9308–9313PubMedGoogle Scholar
  122. Thirumalai D, Hyeon C (2005RNA and protein folding: common themes and variations. Biochemistry 44:4957–4970PubMedGoogle Scholar
  123. Tinnefeld P, Sauer M (2005) Branching out of single-molecule fluorescence spectroscopy: challenges for chemistry and influence on biology. Angew Chem Int Ed Engl 44:2642–2671PubMedGoogle Scholar
  124. Uemura S, Dorywalska M, Lee TH, Kim HD, Puglisi JD, Chu S (2007) Peptide bond formation destabilizes Shine-Dalgarno interaction on the ribosome. Nature 446:454–457PubMedGoogle Scholar
  125. Vogelsang J, Kasper R, Steinhauer C, Person B, Heilemann M, Sauer M, Tinnefeld P (2008) A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew Chem Int Ed Engl 47:5465–5469PubMedGoogle Scholar
  126. Voigts-Hoffmann F, Hengesbach M, Kobitski AY, van Aerschot A, Herdewijn P, Nienhaus GU, Helm M (2007) A methyl group controls conformational equilibrium in human mitochondrial tRNA(Lys). J Am Chem Soc 129:13382–11383PubMedGoogle Scholar
  127. Walter NG (2003) Probing RNA structural dynamics and function by fluorescence resonance energy transfer (FRET). Curr Protoc Nucleic Acid Chem Chapter 11:Unit 11 10Google Scholar
  128. Walter NG, Huang CY, Manzo AJ, Sobhy MA (2008) Do-it-yourself guide: how to use the modern single-molecule toolkit. Nat Methods 5:475–489PubMedGoogle Scholar
  129. Wen JD, Lancaster L, Hodges C, Zeri AC, Yoshimura SH, Noller HF, Bustamante C, Tinoco I (2008) Following translation by single ribosomes one codon at a time. Nature 452:598–603PubMedGoogle Scholar
  130. Widengren J, Kudryavtsev V, Antonik M, Berger S, Gerken M, Seidel CA (2006) Single-molecule detection and identification of multiple species by multiparameter fluorescence detection. Anal Chem 78:2039–2050PubMedGoogle Scholar
  131. Widengren J, Chmyrov A, Eggeling C, Lofdahl PA, Seidel CA (2007) Strategies to improve photostabilities in ultrasensitive fluorescence spectroscopy. J Phys Chem A 111:429–440PubMedGoogle Scholar
  132. Wilson DS, Szostak JW (1999) In vitro selection of functional nucleic acids. Annu Rev Biochem 68:611–647PubMedGoogle Scholar
  133. Wozniak AK, Nottrott S, Kuhn-Holsken E, Schroder GF, Grubmuller H, Luhrmann R, Seidel CA, Oesterhelt F (2005) Detecting protein-induced folding of the U4 snRNA kink-turn by single-molecule multiparameter FRET measurements. RNA 11:1545–1554PubMedGoogle Scholar
  134. Xie Z, Srividya N, Sosnick TR, Pan T, Scherer NF (2004) Single-molecule studies highlight conformational heterogeneity in the early folding steps of a large ribozyme. Proc Natl Acad Sci USA 101:534–539PubMedGoogle Scholar
  135. Zeng Y, Liu HW, Landes CF, Kim YJ, Ma X, Zhu Y, Musier-Forsyth K, Barbara PF (2007) Probing nucleation, reverse annealing, and chaperone function along the reaction path of HIV-1 single-strand transfer. Proc Natl Acad Sci USA 104:12651–12656PubMedGoogle Scholar
  136. Zhuang X (2005) Single-molecule RNA science. Annu Rev Biophys Biomol Struct 34:399–414PubMedGoogle Scholar
  137. Zhuang X, Rief M (2003) Single-molecule folding. Curr Opin Struct Biol 13:88–97PubMedGoogle Scholar
  138. Zhuang X, Bartley LE, Babcock HP, Russell R, Ha T, Herschlag D, Chu S (2000) A single-molecule study of RNA catalysis and folding. Science 288:2048–2051PubMedGoogle Scholar
  139. Zhuang X, Kim H, Pereira MJ, Babcock HP, Walter NG, Chu S (2002) Correlating structural dynamics and function in single ribozyme molecules. Science 296:1473–1476PubMedGoogle Scholar

Copyright information

© International Union for Pure and Applied Biophysics (IUPAB) and Springer 2009

Authors and Affiliations

  • Mark Helm
    • 1
    • 2
    Email author
  • Andrei Yu Kobitski
    • 3
  • G. Ulrich Nienhaus
    • 3
    • 4
  1. 1.Institute of PharmacyUniversity of MainzMainzGermany
  2. 2.Institute of Pharmacy and Molecular BiotechnologyHeidelberg UniversityHeidelbergGermany
  3. 3.Institute of Applied Physics and Center for Functional Nanostructures (CFN)Karlsruhe Institute of Technology (KIT)KarlsruheGermany
  4. 4.Department of PhysicsUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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