Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Dissecting RNA folding by nucleotide analog interference mapping (NAIM)

Nature Protocols volume 3, pages 811–823 (2008)Cite this article

Abstract

Nucleotide analog interference mapping (NAIM) is a powerful chemogenetic approach that allows RNA structure and function to be characterized at the atomic level. Random modifications of base or backbone moieties are incorporated into the RNA transcript as nucleotide analog phosphorothioates. The resulting RNA pool is then subjected to a stringent selection step, in which the RNA has to accomplish a specific task, for example, folding. RNA functional groups important for this process can be identified by physical isolation of the functional and the nonfunctional RNA molecules and subsequent mapping of the modified nucleotide positions in both RNA populations by iodine cleavage of the susceptible phosphorothioate linkage. This approach has been used to analyze a variety of aspects of RNA biochemistry, including RNA structure, catalysis and ligand interaction. Here, I describe how to set up a NAIM assay for studying RNA folding. This protocol can be readily adapted to study any RNAs and their properties. The time required to complete the experiment is dependent on the length of the RNA and the number of atomic modifications tested. In general, a single NAIM experiment can be completed in 1–2 weeks, but expect a time frame of several weeks to obtain reliable and statistically meaningful results.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Commercially available analogs.
Figure 2: Experimental design.
Figure 3: Functional groups critical for the tertiary collapse of the D135 ribozyme.

Similar content being viewed by others

References

  1. Brunel, C. & Romby, P. Probing RNA structure and RNA-ligand complexes with chemical probes. Methods Enzymol. 318, 3–21 (2000).

    Article  CAS  PubMed  Google Scholar 

  2. Shcherbakova, I., Mitra, S., Beer, R.H. & Brenowitz, M. Fast Fenton footprinting: a laboratory-based method for the time-resolved analysis of DNA, RNA and proteins. Nucleic Acids Res. 34, e48 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Hampel, K.J. & Burke, J.M. Time-resolved hydroxyl-radical footprinting of RNA using Fe(II)-EDTA. Methods 23, 233–239 (2001).

    Article  CAS  PubMed  Google Scholar 

  4. Urlaub, H., Hartmuth, K. & Luhrmann, R. A two-tracked approach to analyze RNA-protein crosslinking sites in native, nonlabeled small nuclear ribonucleoprotein particles. Methods 26, 170–181 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Reed, R. & Chiara, M.D. Identification of RNA-protein contacts within functional ribonucleoprotein complexes by RNA site-specific labeling and UV crosslinking. Methods 18, 3–12 (1999).

    Article  CAS  PubMed  Google Scholar 

  6. Noah, J.W. & Lambowitz, A.M. Effects of maturase binding and Mg2+ concentration on group II intron RNA folding investigated by UV cross-linking. Biochemistry 42, 12466–12480 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Sigel, R.K. & Pyle, A.M. Lanthanide ions as probes for metal ions in the structure and catalytic mechanism of ribozymes. Met. Ions Biol. Syst. 40, 477–512 (2003).

    CAS  PubMed  Google Scholar 

  8. Streicher, B., Westhof, E. & Schroeder, R. The environment of two metals ions surrounding the splice site of a group I intron. EMBO J. 15, 2556–2564 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Polacek, N. & Barta, A. Metal ion probing of rRNAs: evidence for evolutionarily conserved divalent cation binding pockets. RNA 4, 1282–1294 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Fedorova, O., Boudvillain, M., Kawaoka, J. & Pyle, A.M. Nucleotide analog interference mapping and suppression: specific applications in studies of RNA tertiary structure, dynamic helicase mechanism and RNA–protein interactions. in Handbook of RNA Biochemistry (eds. Hartmann, R.K., Bindereif, A., Schön, A. & Westhof, E.) pp. 259–293 (Wiley-VCH, Weinheim, 2005).

    Chapter  Google Scholar 

  11. Ryder, S.P., Ortoleva-Donnelly, L., Kosek, A.B. & Strobel, S.A. Chemical probing of RNA by nucleotide analog interference mapping. Methods Enzymol. 317, 92–109 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Vortler, L.C. & Eckstein, F. Phosphorothioate modification of RNA for stereochemical and interference analyses. Methods Enzymol. 317, 74–91 (2000).

    Article  CAS  PubMed  Google Scholar 

  13. Christian, E.L. & Yarus, M. Metal coordination sites that contribute to structure and catalysis in the group I intron from Tetrahymena. Biochemistry 32, 4475–4480 (1993).

    Article  CAS  PubMed  Google Scholar 

  14. Kazantsev, A.V. & Pace, N.R. Identification by modification-interference of purine N-7 and ribose 2′- OH groups critical for catalysis by bacterial ribonuclease P. RNA 4, 937–947 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Rox, C., Feltens, R., Pfeiffer, T. & Hartmann, R.K. Potential contact sites between the protein and RNA subunit in the Bacillus subtilis RNase P holoenzyme. J. Mol. Biol. 315, 551–560 (2002).

    Article  CAS  PubMed  Google Scholar 

  16. Siew, D., Zahler, N.H., Cassano, A.G., Strobel, S.A. & Harris, M.E. Identification of adenosine functional groups involved in substrate binding by the ribonuclease P ribozyme. Biochemistry 38, 1873–1883 (1999).

    Article  CAS  PubMed  Google Scholar 

  17. Ortoleva-Donnelly, L., Szewczak, A.A., Gutell, R.R. & Strobel, S.A. The chemical basis of adenosine conservation throughout the Tetrahymena ribozyme. RNA 4, 498–519 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Strauss-Soukup, J.K. & Strobel, S.A. A chemical phylogeny of group I introns based upon interference mapping of a bacterial ribozyme. J. Mol. Biol. 302, 339–358 (2000).

    Article  CAS  PubMed  Google Scholar 

  19. Strobel, S.A. & Shetty, K. Defining the chemical groups essential for Tetrahymena group I intron function by nucleotide analog interference mapping. Proc. Natl. Acad. Sci. USA 94, 2903–2908 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Boudvillain, M. & Pyle, A.M. Defining functional groups, core structural features and inter-domain tertiary contacts essential for group II intron self-splicing: a NAIM analysis. EMBO J. 17, 7091–7104 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Fedorova, O. & Pyle, A.M. Linking the group II intron catalytic domains: tertiary contacts and structural features of domain 3. EMBO J. 24, 3906–3916 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Waldsich, C. & Pyle, A.M. A folding control element for tertiary collapse of a group II intron ribozyme. Nat. Struct. Mol. Biol. 14, 37–44 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Waldsich, C. & Pyle, A.M. A kinetic intermediate that regulates proper folding of a group II intron RNA. J. Mol. Biol. 375, 572–580 (2008).

    Article  CAS  PubMed  Google Scholar 

  24. Oyelere, A.K., Kardon, J.R. & Strobel, S.A. pK(a) perturbation in genomic Hepatitis Delta Virus ribozyme catalysis evidenced by nucleotide analogue interference mapping. Biochemistry 41, 3667–3675 (2002).

    Article  CAS  PubMed  Google Scholar 

  25. Tuschl, T., Ng, M.M., Pieken, W., Benseler, F. & Eckstein, F. Importance of exocyclic base functional groups of central core guanosines for hammerhead ribozyme activity (published erratum appears in Biochemistry 33, 848 (1994)). Biochemistry 32, 11658–11668 (1993).

    Article  CAS  PubMed  Google Scholar 

  26. Williams, D.M., Pieken, W.A. & Eckstein, F. Function of specific 2′-hydroxyl groups of guanosines in a hammerhead ribozyme probed by 2′ modifications. Proc. Natl. Acad. Sci. USA 89, 918–921 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Chowrira, B.M. & Burke, J.M. Extensive phosphorothioate substitution yields highly active and nuclease-resistant hairpin ribozymes. Nucleic Acids Res. 20, 2835–2840 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ryder, S.P. et al. Investigation of adenosine base ionization in the hairpin ribozyme by nucleotide analog interference mapping. RNA 7, 1454–1463 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Ryder, S.P. & Strobel, S.A. Nucleotide analog interference mapping of the hairpin ribozyme: implications for secondary and tertiary structure formation. J. Mol. Biol. 291, 295–311 (1999).

    Article  CAS  PubMed  Google Scholar 

  30. Jones, F.D. & Strobel, S.A. Ionization of a critical adenosine residue in the Neurospora Varkud Satellite ribozyme active site. Biochemistry 42, 4265–4276 (2003).

    Article  CAS  PubMed  Google Scholar 

  31. Ruffner, D.E. & Uhlenbeck, O.C. Thiophosphate interference experiments locate phosphates important for the hammerhead RNA self-cleavage reaction. Nucleic Acids Res. 18, 6025–6029 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Vortler, C.S., Fedorova, O., Persson, T., Kutzke, U. & Eckstein, F. Determination of 2′-hydroxyl and phosphate groups important for aminoacylation of Escherichia coli tRNAAsp: a nucleotide analogue interference study. RNA 4, 1444–1454 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Jansen, J.A., McCarthy, T.J., Soukup, G.A. & Soukup, J.K. Backbone and nucleobase contacts to glucosamine-6-phosphate in the glmS ribozyme. Nat. Struct. Mol. Biol. 13, 517–523 (2006).

    Article  CAS  PubMed  Google Scholar 

  34. Cochrane, J.C., Batey, R.T. & Strobel, S.A. Quantitation of free energy profiles in RNA-ligand interactions by nucleotide analog interference mapping. RNA 9, 1282–1289 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Basu, S. et al. A specific monovalent metal ion integral to the AA platform of the RNA tetraloop receptor. Nat. Struct. Biol. 5, 986–992 (1998).

    Article  CAS  PubMed  Google Scholar 

  36. Luptak, A. & Doudna, J.A. Distinct sites of phosphorothioate substitution interfere with folding and splicing of the Anabaena group I intron. Nucleic Acids Res. 32, 2272–2280 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Eckstein, F. Nucleoside phosphorothioates. Annu. Rev. Biochem. 54, 367–402 (1985).

    Article  CAS  PubMed  Google Scholar 

  38. Eckstein, F. & Gish, G. Phosphorothioates in molecular biology. Trends Biochem. Sci. 14, 97–100 (1989).

    Article  CAS  PubMed  Google Scholar 

  39. Gish, G. & Eckstein, F. DNA and RNA sequence determination based on phosphorothioate chemistry. Science 240, 1520–1522 (1988).

    Article  CAS  PubMed  Google Scholar 

  40. Ryder, S.P. & Strobel, S.A. Nucleotide analog interference mapping. Methods 18, 38–50 (1999).

    Article  CAS  PubMed  Google Scholar 

  41. Strobel, S.A. A chemogenetic approach to RNA function/structure analysis. Curr. Opin. Struct. Biol. 9, 346–352 (1999).

    Article  CAS  PubMed  Google Scholar 

  42. Cochrane, J.C. & Strobel, S.A. Probing RNA structure and function by nucleotide analog interference mapping. in Current Protocols in Nucleic Acid Chemistry (eds. Beaucage, S.L., Bergstrom, D.E., Glick, G.D. & Jones, R.A.) 6.9.1–6.9.21 (John Wiley & Sons, Hoboken, New Jersey, 2004).

    Chapter  Google Scholar 

  43. Ryder, S.P. & Strobel, S.A. Comparative analysis of hairpin ribozyme structures and interference data. Nucleic Acids Res. 30, 1287–1291 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Basu, S. & Strobel, S.A. Biochemical detection of monovalent metal ion binding sites within RNA. Methods 23, 264–275 (2001).

    Article  CAS  PubMed  Google Scholar 

  45. Soukup, J.K., Minakawa, N., Matsuda, A. & Strobel, S.A. Identification of A-minor tertiary interactions within a bacterial group I intron active site by 3-deazaadenosine interference mapping. Biochemistry 41, 10426–10438 (2002).

    Article  CAS  PubMed  Google Scholar 

  46. Boudvillain, M., Delencastre, A. & Pyle, A.M. A new RNA tertiary interaction that links active-site domains of a group II intron and anchors them at the site of catalysis. Nature 406, 315–318 (2000).

    Article  CAS  PubMed  Google Scholar 

  47. Szewczak, A.A., Ortoleva-Donnelly, L., Ryder, S.P., Moncoeur, E. & Strobel, S.A. A minor groove RNA triple helix within the catalytic core of a group I intron. Nat. Struct. Biol. 5, 1037–1042 (1998).

    Article  CAS  PubMed  Google Scholar 

  48. Arabshahi, A. & Frey, P.A. A simplified procedure for synthesizing nucleoside 1-thiotriphosphates: dATP alpha S, dGTP alpha S, UTP alpha S, and dTTP alpha S. Biochem. Biophys. Res. Commun. 204, 150–155 (1994).

    Article  CAS  PubMed  Google Scholar 

  49. Oyelere, A.K. & Strobel, S.A. Biochemical detection of cytidine protonation within RNA. J. Am. Chem. Soc. 122, 10259–10267 (2000).

    Article  CAS  Google Scholar 

  50. Ortoleva-Donnelly, L., Kronman, M. & Strobel, S.A. Identifying RNA minor groove tertiary contacts by nucleotide analogue interference mapping with N2-methylguanosine. Biochemistry 37, 12933–12942 (1998).

    Article  CAS  PubMed  Google Scholar 

  51. Doherty, E.A., Batey, R.T., Masquida, B. & Doudna, J.A. A universal mode of helix packing in RNA. Nat. Struct. Biol. 8, 339–343 (2001).

    Article  CAS  PubMed  Google Scholar 

  52. Pyle, A.M., Fedorova, O. & Waldsich, C. Folding of group II introns: a model system for large, multidomain RNAs? Trends Biochem. Sci. 32, 138–145 (2007).

    Article  CAS  PubMed  Google Scholar 

  53. Su, L.J., Waldsich, C. & Pyle, A.M. An obligate intermediate along the slow folding pathway of a group II intron ribozyme. Nucleic Acids Res. 33, 6674–6687 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Gutell, R.R., Cannone, J.J., Shang, Z., Du, Y. & Serra, M.J. A story: unpaired adenosine bases in ribosomal RNAs. J. Mol. Biol. 304, 335–354 (2000).

    Article  CAS  PubMed  Google Scholar 

  55. Basu, S., Pazsint, C. & Chowdhury, G. Analysis of ribozyme structure and function by nucleotide analog interference mapping. Methods Mol. Biol. 252, 57–75 (2004).

    CAS  PubMed  Google Scholar 

  56. Sousa, R. & Padilla, R. A mutant T7 RNA polymerase as a DNA polymerase. EMBO J. 14, 4609–4621 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Swisher, J., Su, L., Brenowitz, M., Anderson, V. & Pyle, A. Productive folding to the native state by a group II intron ribozyme. J. Mol. Biol. 315, 297–310 (2002).

    Article  CAS  PubMed  Google Scholar 

  58. Huang, Z. & Szostak, J.W. A simple method for 3′-labeling of RNA. Nucleic Acids Res. 24, 4360–4361 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Das, R., Laederach, A., Pearlman, S.M., Herschlag, D. & Altman, R.B. SAFA: semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments. RNA 11, 344–354 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Shcherbakova, I. & Brenowitz, M. Perturbation of the hierarchical folding of a large RNA by the destabilization of its Scaffold's tertiary structure. J. Mol. Biol. 354, 483–496 (2005).

    Article  CAS  PubMed  Google Scholar 

  61. Christian, E.L. & Yarus, M. Analysis of the role of phosphate oxygens in the group I intron from Tetrahymena. J. Mol. Biol. 228, 743–758 (1992).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Anna Marie Pyle is acknowledged for her support and helpful suggestions. I thank Olga Fedorova for many invaluable discussions and for critically reading the manuscript. Funding was in part provided by National Institutes of Health (NIH) grant GM50313 to A.M. Pyle when I was a postdoctoral associate in her lab and by the Austrian Science Foundation (FWF; J2332) to C.W.

Author information

Authors and Affiliations

  1. Department of Biochemistry, Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9/5, Vienna, 1030, Austria

    Christina Waldsich

Authors
  1. Christina Waldsich
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christina Waldsich.

Supplementary information

Supplementary Table 1

Sample NAIM data (XLS 28 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Waldsich, C. Dissecting RNA folding by nucleotide analog interference mapping (NAIM). Nat Protoc 3, 811–823 (2008). https://doi.org/10.1038/nprot.2008.45

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2008.45

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing