RNA Folding pp 309-319 | Cite as

RNA Refolding Studied by Light-Coupled NMR Spectroscopy

  • Harald Schwalbe
  • Boris Fürtig
Part of the Methods in Molecular Biology book series (MIMB, volume 1086)


Conformational transitions (refolding) between long-lived conformational states constitute the time-limiting step during the folding process of large RNAs. As the dynamics of these reactions dominate the regulatory and other functional behavior of RNA molecules, it is of importance to characterize them with high spatial and temporal resolution. Here, we describe a method for the investigation of RNA refolding reactions based on the photolytic generation of preselected conformations in a non-equilibrium state, followed by the observation of the folding trajectory with real-time NMR spectroscopy.

Key words

Real-time NMR NMR laser coupling RNA refolding Photo-cleavable protecting groups Long-lived conformational states 



The work is supported by the state of Hesse (BMRZ), the DFG collaborative research center: Molecular principles of RNA-based regulation. H.S. is a member of the DFG center of excellence: macromolecular complexes.


  1. 1.
    Kleckner IR, Foster MP (2011) An introduction to NMR-based approaches for measuring protein dynamics. Biochim Biophys Acta 1814:942–968PubMedCrossRefGoogle Scholar
  2. 2.
    Thirumalai D, Woodson SA (2000) Maximizing RNA folding rates: a balancing act. RNA 6:790–794PubMedCrossRefGoogle Scholar
  3. 3.
    Webb AE, Weeks KM (2001) A collapsed state functions to self-chaperone RNA folding into a native ribonucleoprotein complex. Nat Struct Biol 8:135–140PubMedCrossRefGoogle Scholar
  4. 4.
    Porschke D, Hoffman GW, Senear A (1973) Double helical complex formed from a polynucleotide and a complementary monomer. Nat New Biol 242:45–46PubMedCrossRefGoogle Scholar
  5. 5.
    Schwalbe H, Buck J, Furtig B et al (2007) Structures of RNA switches: insight into molecular recognition and tertiary structure. Angew Chem Int Ed Engl 46:1212–1219PubMedCrossRefGoogle Scholar
  6. 6.
    Dethoff EA, Chugh J, Mustoe AM et al (2012) Functional complexity and regulation through RNA dynamics. Nature 482:322–330PubMedCrossRefGoogle Scholar
  7. 7.
    Treiber DK, Rook MS, Zarrinkar PP et al (1998) Kinetic intermediates trapped by native interactions in RNA folding. Science 279:1943–1946PubMedCrossRefGoogle Scholar
  8. 8.
    Zarrinkar PP, Williamson JR (1994) Kinetic intermediates in RNA folding. Science 265:918–924PubMedCrossRefGoogle Scholar
  9. 9.
    Hobartner C, Micura R (2003) Bistable secondary structures of small RNAs and their structural probing by comparative imino proton NMR spectroscopy. J Mol Biol 325:421–431PubMedCrossRefGoogle Scholar
  10. 10.
    Wenter P, Furtig B, Hainard A et al (2005) Kinetics of photoinduced RNA refolding by real-time NMR spectroscopy. Angew Chem Int Ed Engl 44:2600–2603PubMedCrossRefGoogle Scholar
  11. 11.
    Wenter P, Furtig B, Hainard A et al (2006) A caged uridine for the selective preparation of an RNA fold and determination of its refolding kinetics by real-time NMR. Chembiochem 7:417–420PubMedCrossRefGoogle Scholar
  12. 12.
    Kühn T, Schwalbe H (2000) Monitoring the kinetics of ion-dependent protein folding by time-resolved NMR spectroscopy at atomic resolution. J Am Chem Soc 122:6169–6174CrossRefGoogle Scholar
  13. 13.
    Wirmer J, Kühn T, Schwalbe H (2001) Millisecond time resolved photo-CIDNP NMR reveals a non-native folding intermediate on the ion-induced refolding pathway of bovine α-lactalbumin. Angew Chem 113:4378–4381CrossRefGoogle Scholar
  14. 14.
    Manoharan V, Furtig B, Jaschke A et al (2009) Metal-induced folding of Diels-Alderase ribozymes studied by static and time-resolved NMR spectroscopy. J Am Chem Soc 131:6261–6270PubMedCrossRefGoogle Scholar
  15. 15.
    Micura R, Hobartner C (2003) On secondary structure rearrangements and equilibria of small RNAs. Chembiochem 4:984–990PubMedCrossRefGoogle Scholar
  16. 16.
    Micura R, Pils W, Hobartner C et al (2001) Methylation of the nucleobases in RNA oligonucleotides mediates duplex-hairpin conversion. Nucleic Acids Res 29:3997–4005PubMedGoogle Scholar
  17. 17.
    Reining A, Nozinovic S, Schlepckow K, Buhr F, Fürtig B, Schwalbe H (2013) Three–state mechanism couples ligand and temperature sensing in riboswitches. Nature 499: 355–359PubMedCrossRefGoogle Scholar
  18. 18.
    Furtig B, Wenter P, Reymond L et al (2007) Conformational dynamics of bistable RNAs studied by time-resolved NMR spectroscopy. J Am Chem Soc 129:16222–16229PubMedCrossRefGoogle Scholar
  19. 19.
    Gruber AR, Lorenz R, Bernhart SH et al (2008) The Vienna RNA websuite. Nucleic Acids Res 36:W70–W74PubMedCrossRefGoogle Scholar
  20. 20.
    Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415PubMedCrossRefGoogle Scholar
  21. 21.
    Furtig B, Wenter P, Pitsch S et al (2010) Probing mechanism and transition state of RNA refolding. ACS Chem Biol 5:753–765PubMedCrossRefGoogle Scholar
  22. 22.
    Kawashima E, Kamaike K (2004) Synthesis of stable-isotope (C-13 and N-15) labeled nucleosides and their applications. Mini Rev Org Chem 1:309–332CrossRefGoogle Scholar
  23. 23.
    Heckel A (2007) Nucleobase-caged phosphoramidites for oligonucleotide synthesis. Curr Protoc Nucleic Acid Chem Chapter 1, Unit 1 17Google Scholar
  24. 24.
    Mayer G, Heckel A (2006) Biologically active molecules with a “light switch”. Angew Chem Int Ed Engl 45:4900–4921PubMedCrossRefGoogle Scholar
  25. 25.
    Kuprov I, Hore PJ (2004) Uniform illumination of optically dense NMR samples. J Magn Reson 171:171–175PubMedCrossRefGoogle Scholar
  26. 26.
    Otting G, Wüthrich K (1989) Extended heteronuclear editing of 2D 1H NMR spectra of isotope-labeled proteins, using the X(w1, w2)-double-half-filter. J Magn Reson 85:586–594Google Scholar
  27. 27.
    Buck J, Furtig B, Noeske J et al (2007) Time-resolved NMR methods resolving ligand-induced RNA folding at atomic resolution. Proc Natl Acad Sci USA 104:15699–15704PubMedCrossRefGoogle Scholar
  28. 28.
    Schanda P, Kupce E, Brutscher B (2005) SOFAST-HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins within a few seconds. J Biomol NMR 33:199–211PubMedCrossRefGoogle Scholar
  29. 29.
    Furtig B, Richter C, Wohnert J et al (2003) NMR spectroscopy of RNA. Chembiochem 4:936–962PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Harald Schwalbe
    • 1
  • Boris Fürtig
    • 1
  1. 1.Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic ResonanceJohann Wolfgang Goethe-UniversityFrankfurt am MainGermany

Personalised recommendations