Abstract
RNA folding pathways can be complex and even include kinetic traps or misfolded intermediates that can be slow to resolve. Characterizing these pathways is critical to understanding how RNA molecules acquire their biological function. We have previously developed a novel approach to help characterize such misfolded intermediates. Laser-assisted single-molecule refolding (LASR) is a powerful technique that combines temperature-jump (T-jump) kinetics with single-molecule detection. In a typical LASR experiment, the temperature is rapidly increased and conformational dynamics are characterized, in real-time, at the single-molecule level using single-molecule fluorescence resonance energy transfer (smFRET). Here, we provide detailed protocols for performing LASR experiments including sample preparation, temperature calibration, and data analysis.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Burge CB, Tuschl TA, Sharp PA (1999) Splicing of precursors to mRNAs by the spliceosomes. In: Gesteland RF, Cech TR, Atkins JF (eds) The RNA world, 2nd edn. Cold Spring Harbor Press, Cold Spring Harbor, NY, pp 525–560
Eddy SR (2001) Non-coding RNA genes and the modern RNA world. Nat Rev Genet 2:919–929
Tinoco I Jr, Bustamante C (1999) How RNA folds. J Mol Biol 293:271–281
Brion P, Westhof E (1997) Hierarchy and dynamics of RNA folding. Annu Rev Biophys Biomol Struct 26:113–137
Pyle AM (2002) Metal ions in the structure and function of RNA. J Biol Inorg Chem 7:679–690
Woodson SA (2005) Metal ions and RNA folding: a highly charged topic with a dynamic future. Curr Opin Chem Biol 9:104–109
Greenleaf WJ, Frieda KL, Foster DA et al (2008) Direct observation of hierarchical folding in single riboswitch aptamers. Science 319:630–633
Dill KA, Chan HS (1997) From levinthal to pathways to funnels. Nat Struct Biol 4:10–19
Zhuang X (2005) Single-molecule RNA science. Annu Rev Biophys Biomol Struct 34:399–414
Chen SJ (2008) RNA folding: conformational statistics, folding kinetics, and ion electrostatics. Annu Rev Biophys 37:197–214
Treiber DK, Williamson JR (2001) Beyond kinetic traps in RNA folding. Curr Opin Struct Biol 11:309–314
Zhuang X, Kim H, Pereira MJ et al (2002) Correlating structural dynamics and function in single ribozyme molecules. Science 296:1473–1476
Ditzler MA, Rueda D, Mo J et al (2008) A rugged free energy landscape separates multiple functional RNA folds throughout denaturation. Nucleic Acids Res 36:7088–7099
Solomatin SV, Greenfeld M, Chu S et al (2010) Multiple native states reveal persistent ruggedness of an RNA folding landscape. Nature 463:681–684
Aleman EA, Lamichhane R, Rueda D (2008) Exploring RNA folding one molecule at a time. Curr Opin Chem Biol 12:647–654
Karunatilaka KS, Rueda D (2009) Single-molecule fluorescence studies of RNA: a decade’s progress. Chem Phys Lett 476:1–10
Fiore JL, Kraemer B, Koberling F et al (2009) Enthalpy-driven RNA folding: single-molecule thermodynamics of tetraloop-receptor tertiary interaction. Biochemistry 48:2550–2558
Szewczak AA, Podell ER, Bevilacqua PC et al (1998) Thermodynamic stability of the P4-P6 domain RNA tertiary structure measured by temperature gradient gel electrophoresis. Biochemistry 37:11162–11170
Nixon PL, Giedroc DP (2000) Energetics of a strongly pH dependent RNA tertiary structure in a frameshifting pseudoknot. J Mol Biol 296:659–671
Hammann C, Cooper A, Lilley DM (2001) Thermodynamics of ion-induced RNA folding in the hammerhead ribozyme: an isothermal titration calorimetric study. Biochemistry 40:1423–1429
Feig AL (2007) Applications of isothermal titration calorimetry in RNA biochemistry and biophysics. Biopolymers 87:293–301
Rueda D, Walter NG (2006) Fluorescent energy transfer readout of an aptazyme-based biosensor. Methods Mol Biol 335:289–310
Reymond C, Bisaillon M, Perreault JP (2009) Monitoring of an RNA multistep folding pathway by isothermal titration calorimetry. Biophys J 96:132–140
Phillips CM, Mizutani Y, Hochstrasser RM (1995) Ultrafast thermally induced unfolding of RNase A. Proc Natl Acad Sci USA 92:7292–7296
Ballew RM, Sabelko J, Gruebele M (1996) Observation of distinct nanosecond and microsecond protein folding events. Nat Struct Biol 3:923–926
Proctor DJ, Ma H, Kierzek E et al (2004) Folding thermodynamics and kinetics of YNMG RNA hairpins: specific incorporation of 8-bromoguanosine leads to stabilization by enhancement of the folding rate. Biochemistry 43:14004–14014
Dyer RB, Brauns EB (2009) Laser-induced temperature jump infrared measurements of RNA folding. Methods Enzymol 469:353–372
Kirschner K, Eigen M, Bittman R et al (1966) The binding of nicotinamide-adenine dinucleotide to yeast d-glyceraldehyde-3-phosphate dehydrogenase: temperature-jump relaxation studies on the mechanism of an allosteric enzyme. Proc Natl Acad Sci USA 56:1661–1667
Zhao R, Marshall M, Aleman EA et al (2010) Laser-assisted single-molecule refolding (LASR). Biophys J 99:1925–1931
Thirumalai D, Woodson SA (1996) Kinetics of folding of proteins and RNA. Acc Chem Res 29:433–439
Stephens DJ, Allan VJ (2003) Light microscopy techniques for live cell imaging. Science 300:82–86
Walter NG, Huang CY, Manzo AJ et al (2008) Do-it-yourself guide: how to use the modern single-molecule toolkit. Nat Methods 5:475–489
Mattheyses AL, Simon SM, Rappoport JZ (2010) Imaging with total internal reflection fluorescence microscopy for the cell biologist. J Cell Sci 123:3621–3628
Axelrod D (2001) Total internal reflection fluorescence microscopy in cell biology. Traffic 2:764–774
Ha T (2001) Single-molecule fluorescence resonance energy transfer. Methods 25:78–86
Roy R, Hohng S, Ha T (2008) A practical guide to single-molecule FRET. Nat Methods 5:507–516
Pljevaljcic G, Millar DP (2008) Single-molecule fluorescence methods for the analysis of RNA folding and ribonucleoprotein assembly. Methods Enzymol 450:233–252
Zhao R, Rueda D (2009) RNA folding dynamics by single-molecule fluorescence resonance energy transfer. Methods 49:112–117
Haustein E, Schwille P (2004) Single-molecule spectroscopic methods. Curr Opin Struct Biol 14:531–540
Rueda D, Walter NG (2005) Single molecule fluorescence control for nanotechnology. J Nanosci Nanotechnol 5:1990–2000
Bassi GS, Murchie AI, Walter F et al (1997) Ion-induced folding of the hammerhead ribozyme: a fluorescence resonance energy transfer study. EMBO J 16:7481–7489
Ha T (2001) Single-molecule fluorescence methods for the study of nucleic acids. Curr Opin Struct Biol 11:287–292
Zhuang X, Rief M (2003) Single-molecule folding. Curr Opin Struct Biol 13:88–97
Gregorian RS Jr, Crothers DM (1995) Determinants of RNA hairpin loop–loop complex stability. J Mol Biol 248:968–984
Wood S, Ferre-D’Amare AR, Rueda D (2012) Allosteric tertiary interactions preorganize the c-di-GMP riboswitch and accelerate ligand binding. ACS Chem Biol 7:920–927
Steiner M, Karunatilaka KS, Sigel RK et al (2008) Single-molecule studies of group II intron ribozymes. Proc Natl Acad Sci USA 105:13853–13858
Holmstrom ED, Nesbitt DJ (2010) Real-time infrared overtone laser control of temperature in picoliter H(2)O samples: “Nanobathtubs” for single molecule microscopy. J Phys Chem Lett 1:2264–2268
Santalucia J Jr (2000) The use of spectroscopic techniques in the study of DNA stability. In: Gore GM (ed) Spectrometry and spectrofluorimetry. Oxford University Press, New York, pp 329–354
Lamichhane R, Solem A, Black W et al (2010) Single-molecule FRET of protein-nucleic acid and protein–protein complexes: surface passivation and immobilization. Methods 52:192–200
Walter NG, Burke JM (2000) Fluorescence assays to study structure, dynamics, and function of RNA and RNA-ligand complexes. Methods Enzymol 317:409–440
Crawford DJ, Hoskins AA, Friedman LJ et al (2008) Visualizing the splicing of single pre-mRNA molecules in whole cell extract. RNA 14:170–179
Aitken CE, Marshall RA, Puglisi JD (2008) An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys J 94:1826–1835
Salim N, Lamichhane R, Zhao R et al (2012) Thermodynamic and kinetic analysis of an RNA kissing interaction and its resolution into an extended duplex. Biophys J 102:1097–1107
Acknowledgements
This is supported by the National Science Foundation (NSF CAREER award 0747285 to D.R.).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Paudel, B., Rueda, D. (2014). RNA Folding Dynamics Using Laser-Assisted Single-Molecule Refolding. In: Waldsich, C. (eds) RNA Folding. Methods in Molecular Biology, vol 1086. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-667-2_17
Download citation
DOI: https://doi.org/10.1007/978-1-62703-667-2_17
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-666-5
Online ISBN: 978-1-62703-667-2
eBook Packages: Springer Protocols