Skip to main content
SpringerLink
Log in

Single Molecule FRET Characterization of Large Ribozyme Folding

  • Protocol
  • First Online:
Ribozymes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 848))

Abstract

A procedure to investigate the folding of group II intron by single molecule Fluorescence Resonance Energy Transfer (smFRET) using total internal reflection fluorescence microscopy (TIRFM) is described in this chapter. Using our previous studies on the folding and dynamics of a large ribozyme in the presence of metal ions (i.e., Mg2+ and Ca2+) and/or the DEAD-box protein Mss116 as an example, we here describe step-by-step procedures to perform experiments. smFRET allows the investigation of individual molecules, thus, providing kinetic and mechanistic information hidden in ensemble averaged experiments.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Hinterdorfer P., Oijen A. v. (2009) Handbook of Single-Molecule Biophysics. Springer, New York.

    Google Scholar 

  2. Cornish P. V., Ha T. (2007) A survey of single-molecule techniques in chemical biology. ACS Chem Biol 2, 53–61.

    Article  PubMed  CAS  Google Scholar 

  3. Greenleaf W. J., Woodside M. T., Block S. M. (2007) High-resolution, single-molecule measurements of biomolecular motion. Annu Rev Biophys Biomol Struct 36, 171–190.

    Article  PubMed  CAS  Google Scholar 

  4. Kulzer F., Orrit M. (2004) Single-molecule optics. Annu Rev Phys Chem 55, 585-611.

    Article  PubMed  CAS  Google Scholar 

  5. Kapanidis A. N., Strick T. (2009) Biology, one molecule at a time. Trends Biochem Sci 34, 234–243.

    Article  PubMed  CAS  Google Scholar 

  6. Moerner W. E. (2007) New directions in single-molecule imaging and analysis Proc Natl Acad Sci U S A 104, 12596–12602.

    Article  PubMed  CAS  Google Scholar 

  7. Karunatilaka K. S., Rueda D. (2009) Single-molecule fluorescence studies of RNA: A decade’s progress. Chem Phys Lett 476, 1–10.

    Article  PubMed  CAS  Google Scholar 

  8. Fedorova O., Solem A., Pyle A. M. (2010) Protein-facilitated folding of group II intron ribozymes. J Mol Biol 397, 799–813.

    Article  PubMed  CAS  Google Scholar 

  9. Lilley D. M. J. (2005) Structure, folding and mechanisms of ribozymes. Curr Opin Struct Biol 15, 313–323.

    Article  PubMed  CAS  Google Scholar 

  10. Thirumalai D., Hyeon C. (2009) Theory of RNA folding: from hairpins to ribozymes. In: Walter N. G., Woodson S. A., Batey R. T. (ed) Non-Protein Coding RNAs. Springer, Heidelberg Germany

    Google Scholar 

  11. Lilley D. M. J., Eckstein F. (2008) Ribozymes and RNA catalysis. RCS Publishing, Cambridge UK.

    Google Scholar 

  12. Lee M. K., Gal M., Frydman L., Varani G. Real-time multidimensional NMR follows RNA folding with second resolution. Proc Natl Acad Sci U S A 107, 9192–9197.

    Google Scholar 

  13. Solomatin S., Herschlag D. (2009) Methods of site-specific labeling of RNA with fluorescent dyes. Methods Enzymol 469, 47–68.

    Article  PubMed  CAS  Google Scholar 

  14. Johannsen S., Korth M. M. T., Schnabl J., Sigel R. K. O. (2009) Exploring metal ion coordination to nucleic acids by NMR. Chimia 63, 146–152.

    Article  CAS  Google Scholar 

  15. Schlatterer J. C., Brenowitz M. (2009) Complementing global measures of RNA folding with local reports of backbone solvent accessibility by time resolved hydroxyl radical footprinting. Methods 49, 142–147.

    Article  PubMed  CAS  Google Scholar 

  16. Woodson S. A., Koculi E. (2009) Analysis of RNA folding by native polyacrylamide gel electrophoresis. Methods Enzymol 469, 189–208.

    Article  PubMed  CAS  Google Scholar 

  17. Roy R., Hohng S., Ha T. (2008) A practical guide to single-molecule FRET. Nat Methods 5, 507-516.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  19. Uphoff S., Holden S. J., Le Reste L., Periz J., van de Linde S., Heilemann M., Kapanidis A. N. Monitoring multiple distances within a single molecule using switchable FRET. Nat Methods 7, 831–U890.

    Google Scholar 

  20. Blanco M., Walter N. G. (2010) Analisis of complex single molecule FRET time trajectories. Methods Enzymol 472, 153–178.

    Article  PubMed  CAS  Google Scholar 

  21. Ditzler M. A., Aleman E. A., Rueda D., Walter N. G. (2007) Focus on function: single molecule RNA enzymology. Biopolymers 87, 302–316.

    Article  PubMed  CAS  Google Scholar 

  22. Greenfeld M., Herschlag D. (2010) Measuring the energetic coupling of tertiary contacts in RNA folding using Single Molecule Fluorescence Resonance Energy Transfer. Methods Enzymol 472, 205–220.

    Article  PubMed  CAS  Google Scholar 

  23. Kapanidis A. N., Weiss S. (2009) Single-molecule FRET analysis of the path from transcription initiation to elongation. In: Buc V. H., Strick T. (ed) RNA polymerases as molecular motors. RCS Publishing, Cambridge UK

    Google Scholar 

  24. Abelson J., Blanco M., Ditzler M. A., Fuller F., Aravamudhan P., Wood M., Villa T., Ryan D. E., Pleiss J. A., Maeder C., Guthrie C., Walter N. G. (2010) Conformational dynamics of single pre-mRNA molecules during in vitro splicing. Nat Struct Mol Biol 17, 504–U156.

    Article  PubMed  CAS  Google Scholar 

  25. Bokinsky G., Zhuang X. W. (2005) Single-molecule RNA folding. Acc Chem Res 38, 566–573.

    Article  PubMed  CAS  Google Scholar 

  26. Rueda D., Bokinsky G., Rhodes M. M., Rust M. J., Zhuang X. W., Walter N. G. (2004) Single-molecule enzymology of RNA: Essential functional groups impact catalysis from a distance. Proc Natl Acad Sci U S A 101, 10066–10071.

    Article  PubMed  CAS  Google Scholar 

  27. Rueda D., Guo Z. J., Karunatilaka K. (2009) Splicing Mechanisms: Lessons from Single-Molecule Spectroscopy. J Biomol Struct Dyn 26, 48.

    Google Scholar 

  28. Weiss S. (2000) Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy. Nature Struct Biol 7, 724–729.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  30. Ha T., Zhuang X. W., Kim H. D., Orr J. W., Williamson J. R., Chu S. (1999) Ligand-induced conformational changes observed in single RNA molecules. Proc Natl Acad Sci U S A 96, 9077–9082.

    Article  PubMed  CAS  Google Scholar 

  31. Zhuang X. W., Kim H., Pereira M. J. B., Babcock H. P., Walter N. G., Chu S. (2002) Correlating structural dynamics and function in single ribozyme molecules. Science 296, 1473–1476.

    Article  PubMed  CAS  Google Scholar 

  32. Aleman E. A., Lamichhane R., Rueda D. (2008) Exploring RNA folding one molecule at a time. Curr Opin Chem Biol 12, 647–654.

    Article  PubMed  CAS  Google Scholar 

  33. Zhao R., Rueda D. (2009) RNA folding dynamics by single-molecule fluorescence resonance energy transfer. Methods 49, 112-117.

    Article  PubMed  CAS  Google Scholar 

  34. Vukojevic V., Heidkamp M., Ming Y., Johansson B., Terenius L., Rigler R. (2008) Quantitative single-molecule imaging by confocal laser scanning microscopy. Proc Natl Acad Sci U S A 105, 18176–18181.

    Article  PubMed  CAS  Google Scholar 

  35. Steiner M., Karunatilaka K. S., Sigel R. K. O., Rueda D. (2008) Single-molecule studies of group II intron ribozymes. Proc Natl Acad Sci U S A 105, 13853–13858.

    Article  PubMed  CAS  Google Scholar 

  36. Steiner M., Rueda D., Sigel R. K. O. (2009) Ca2+ Induces the Formation of Two Distinct Subpopulations of Group II Intron Molecules. Angew Chem Int Ed Engl 48, 9739–9742.

    PubMed  CAS  Google Scholar 

  37. Karunatilaka K. S., Solem A., Pyle A. M., Rueda D. (2010) Single-molecule analysis of Mss116-mediated group II intron folding. Nature 467, 935–939.

    Article  PubMed  CAS  Google Scholar 

  38. Fedorova O., Zingler N. (2007) Group II introns: structure, folding and splicing mechanism. Biol Chem 388, 665–678.

    Article  PubMed  CAS  Google Scholar 

  39. Sigel R. K. O. (2005) Group II intron ribozymes and metal ions - A delicate relationship. Eur J Inorg Chem 2281–2292.

    Google Scholar 

  40. Solem A., Zingler N., Pyle A. M. (2006) A DEAD protein that activates intron self-splicing without unwinding RNA. Mol Cell 24, 611–617.

    Article  PubMed  CAS  Google Scholar 

  41. Sigel R. K. O., Pyle A. M. (2007) Alternative roles for metal ions in enzyme catalysis and the implications for ribozyme chemistry. Chem Rev 107, 97–113.

    Article  PubMed  CAS  Google Scholar 

  42. Lakowicz J. R. (2006) Principles of fluorescence spectroscopy. Springer, New York.

    Book  Google Scholar 

  43. Pljevaljcic G., Millar D. P., Deniz A. A. (2004) Freely diffusing single hairpin ribozymes provide insights into the role of secondary structure and partially folded states in RNA folding. Biophys J 87, 457–467.

    Article  PubMed  CAS  Google Scholar 

  44. Davanloo P., Rosenberg A. H., Dunn J. J., Studier F. W. (1984) Cloning and expression of the gene for bacteriophage-T7 RNA polymerase. Proc Natl Acad Sci U S A. 81, 2035–2039.

    Article  PubMed  CAS  Google Scholar 

  45. Gallo S., Furler M., Sigel R. K. O. (2005) In vitro transcription and purification of RNAs of different size. Chimia 59, 812–816.

    Article  CAS  Google Scholar 

  46. Lamichhane R., Solem A., Black W., Rueda D. (2010) Single-molecule FRET of protein-nucleic acid and protein-protein complexes: Surface passivation and immobilization. Methods 52, 192–200.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  49. SantaLucia J. J. (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA. 95, 1460–1465.

    Article  PubMed  CAS  Google Scholar 

  50. Cavaluzzi M. J., Borer P. N. (2004) Revised UV extinction coefficients for nucleoside-5′-monophosphates and unpaired DNA and RNA. Nucleic Acids Res 32, e13.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

Financial support by the University of Zürich, an ERC Starting Grant 2010 (259092-MIRNA to R.K.O.S.), as well as the National Science Foundation (MCB0747285 to D.R.) and the National Institutes of Health (R01 GM085116 to D.R.) is gratefully acknowledged.

Author information

Authors and Affiliations

  1. Institute of Inorganic Chemistry, University of Zurich, Zurich, Switzerland

    Lucia Cardo & Roland K. O. Sigel

  2. Wayne State University, Detroit, MI, USA

    Krishanthi S. Karunatilaka & David Rueda

Authors
  1. Lucia Cardo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Krishanthi S. Karunatilaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David Rueda
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Roland K. O. Sigel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David Rueda .

Editor information

Editors and Affiliations

  1. Department of Chemistry, Universität Konstanz, Universitätsstr. 10, Konstanz, 78457, Germany

    Jörg S. Hartig

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Cardo, L., Karunatilaka, K.S., Rueda, D., Sigel, R.K.O. (2012). Single Molecule FRET Characterization of Large Ribozyme Folding. In: Hartig, J. (eds) Ribozymes. Methods in Molecular Biology, vol 848. Humana Press. https://doi.org/10.1007/978-1-61779-545-9_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-545-9_15

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-544-2

  • Online ISBN: 978-1-61779-545-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation