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:

The preparation of site-specifically modified riboswitch domains as an example for enzymatic ligation of chemically synthesized RNA fragments

Nature Protocols volume 3, pages 1457–1466 (2008)Cite this article

Abstract

This protocol describes an efficient method for the preparation of riboswitch domains comprising up to ∼200 nt containing site-specific nucleoside modifications. The strategy is based on enzymatic ligation of chemically synthesized RNA fragments. The design of ligation sites strictly follows the criterion that all fragments comprise less than ∼50 nt. This allows the researcher to rely on custom synthesis services and to utilize the large pool of commercially available, functionalized nucleoside phosphoramidites for solid-phase RNA synthesis. Importantly, this design renders utmost flexibility to position a chemical modification (e.g., a fluorescence label) within the RNA. Selection of the appropriate ligation type (using T4 RNA or T4 DNA ligase) is subordinate to the criteria above and is detailed in the protocol. The whole concept is demonstrated for 2-aminopurine containing thiamine pyrophosphate responsive riboswitch domains that are applied in fluorescence spectroscopic folding studies. Labeled samples in 5–35 nmol quantities are obtained within 3–4 d, not including the time for fragment synthesis.

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: Basic requirements for enzymatic ligation of RNA strands.
Figure 2: The thiM Escherichia coli thiamine pyrophosphate (TPP) responsive riboswitch.
Figure 3: Preparation of a 2-aminopurine (AP) modified thiamine pyrophosphate riboswitch by successive ligation of four strands.
Figure 4: Characterization of ligation products by mass spectrometry.
Figure 5: Modular concept for the construction of chemically modified thiamine pyrophosphate riboswitch domains.
Figure 6: Fluorescence spectroscopy of a 2-aminopurine (AP) labeled thiamine pyrophosphate (TPP) riboswitch domain (U62AP 151 nt).

Similar content being viewed by others

References

  1. Moore, M.J. & Sharp, P.A. Site-specific modification of pre-mRNA: the 2′-hydroxyl groups at the splice sites. Science 256, 992–997 (1992).

    Article  CAS  Google Scholar 

  2. Persson, T., Willkomm, D.K. & Hartmann, R.K. T4 RNA ligase. In Handbook of RNA Biochemistry (eds. Hartmann, R.K., Bindereif, A., Schön, A. & Westhof, E.) 53–74 (Wiley-VCH, Weinheim, Germany, 2005).

    Chapter  Google Scholar 

  3. Frilander, J.M. & Turunen, J.J. RNA ligation using T4 DNA ligase. In Handbook of RNA Biochemistry (eds. Hartmann, R.K., Bindereif, A., Schön, A. & Westhof, E.) 36–52 (Wiley-VCH, Weinheim, Germany, 2005).

    Chapter  Google Scholar 

  4. Winkler, W., Nahvi, A. & Breaker, R.R. Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419, 952–966 (2002).

    Article  CAS  Google Scholar 

  5. Nahvi, A. et al. Genetic control by a metabolite binding mRNA. Chem. Biol. 9, 1043 (2002).

    Article  CAS  Google Scholar 

  6. Miranda-Ríos, J., Navarro, M. & Soberón, M. A conserved RNA structure (thi box) is involved in regulation of thiamin biosynthetic gene expression in bacteria. Proc. Natl. Acad. Sci. USA 98, 9736–9741 (2001).

    Article  Google Scholar 

  7. Mironov, A.S. et al. Sensing small molecules by nascent RNA: a mechanism to control transcription in bacteria. Cell 111, 747–756 (2002).

    Article  CAS  Google Scholar 

  8. Barrick, J.E. & Breaker, R.R. The distributions, mechanisms, and structures of metabolite-binding riboswitches. Genome Biol. 8, R239 (2007).

    Article  Google Scholar 

  9. Serganov, A. & Patel, D.J. Ribozymes, riboswitches and beyond: regulation of gene expression without proteins. Nat. Rev. Genet. 8, 776–790 (2007).

    Article  CAS  Google Scholar 

  10. Edwards, T.E., Klein, D.J. & Ferré-D'Amaré, A.R. Riboswitches: small-molecule recognition by gene regulatory RNAs. Curr. Opin. Struct. Biol. 17, 273–279 (2007).

    Article  CAS  Google Scholar 

  11. Bocobza, S. et al. Riboswitch-dependent gene regulation and its evolution in the plant kingdom. Genes Dev. 21, 2874–2879 (2007).

    Article  CAS  Google Scholar 

  12. Grundy, F.J. & Henkin, T.M. From ribosome to riboswitch: control of gene expression in bacteria by RNA structural rearrangements. Crit. Rev. Biochem. Mol. Biol. 41, 329–338 (2006).

    Article  CAS  Google Scholar 

  13. Cheah, M.T., Wachter, A., Sudarsan, N. & Breaker, R.R. Control of alternative RNA splicing and gene expression by eukaryotic riboswitches. Nature 447, 497–500 (2007).

    Article  CAS  Google Scholar 

  14. Wachter, A. et al. Riboswitch control of gene expression in plants by splicing and alternative 3′ end processing of mRNAs. Plant Cell 19, 3437–3450 (2007).

    Article  CAS  Google Scholar 

  15. Schwalbe, H., Buck, J., Fürtig, B., Noeske, J. & Wöhnert, J. Structures of RNA switches: insight into molecular recognition and tertiary structure. Angew. Chem. Int. Ed. Engl. 46, 1212–1219 (2007).

    Article  CAS  Google Scholar 

  16. Mandal, M. & Breaker, R.R. Gene regulation by riboswitches. Nat. Rev. Mol. Cell Biol. 5, 451–463 (2004).

    Article  CAS  Google Scholar 

  17. Nudler, E. & Mironov, A.S. The riboswitch control of bacterial metabolism. Trends Biochem. Sci. 29, 11–17 (2004).

    Article  CAS  Google Scholar 

  18. Wickiser, J.K., Winkler, W.C., Breaker, R.R. & Crothers, D.M. The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch. Mol. Cell 18, 49–60 (2005).

    Article  CAS  Google Scholar 

  19. Rieder, R., Lang, K., Graber, D. & Micura, R. Folding of the adenosine deaminase A-riboswitch and implications on translational control. Chembiochem 8, 896–902 (2007).

    Article  CAS  Google Scholar 

  20. Lang, K., Rieder, R. & Micura, R. Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach. Nucleic Acids Res. 35, 5370–5378 (2007).

    Article  CAS  Google Scholar 

  21. Höbartner, C. et al. Syntheses of RNAs with up to 100 nucleotides containing site-specific 2′-methylseleno labels for use in X-ray crystallography. J. Am. Chem. Soc. 127, 12035–12045 (2005).

    Article  Google Scholar 

  22. Lemay, J.F., Penedo, J.C., Tremblay, R., Lilley, D.M. & Lafontaine, D.A. Folding of the adenine riboswitch. Chem. Biol. 13, 857–868 (2006).

    Article  CAS  Google Scholar 

  23. Edwards, T.E. & Sigurdsson, S.T. Site-specific incorporation of nitroxide spin-labels into 2′-position of nucleic acids. Nat. Protoc. 2, 1954–1962 (2007).

    Article  CAS  Google Scholar 

  24. Tzakos, A.G., Easton, L.E. & Lukavsky, P.J. Preparation of large oligoribonucleotides with complementary isotope-labeled segments for NMR structural studies. Nat. Protoc. 2, 2139–2147 (2007).

    Article  CAS  Google Scholar 

  25. Zhang, Q. & Al-Hashimi, H.M. Extending the NMR spatial resolution limit for RNA by motional couplings. Nat. Methods 5, 243–245 (2008).

    Article  CAS  Google Scholar 

  26. Bailor, M.H. et al. Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings. Nat. Protoc. 2, 1536–1546 (2007).

    Article  CAS  Google Scholar 

  27. Schiemann, O., Piton, N., Plackmeyer, J., Bode, B.E., Prisner, T.F. & Engels, J.W. Spin labeling of oligonucleotides with the nitroxide TPA and the use of PELDOR, a pulse EPR method, to measure intramolecular distances. Nat. Protoc. 2, 904–922 (2007).

    Article  CAS  Google Scholar 

  28. Srivatsan, S.G. & Tor, Y. Synthesis and enzymatic incorporation of a fluorescent pyrimidine ribonucleotide. Nat. Protoc. 2, 1547–1555 (2007).

    Article  CAS  Google Scholar 

  29. Chaulk, S.G. & MacMillan, A.M. Synthesis of oligo-RNAs with photocaged adenosine 2′-hydroxyls. Nat. Protoc. 2, 1052–1058 (2007).

    Article  CAS  Google Scholar 

  30. Puffer, B., Moroder, H., Aigner, M. & Micura, R. 2′-Methylseleno modified oligoribonucleotides for X-ray crystallography synthesized by the ACE RNA solid-phase approach. Nucleic Acids Res. 36, 970–983 (2008).

    Article  CAS  Google Scholar 

  31. Moroder, H., Kreutz, C., Lang, K., Serganov, A. & Micura, R. Synthesis, oxidation behavior, crystallization and structure of 2′-methylseleno guanosine containing RNAs. J. Am. Chem. Soc. 128, 9909–9918 (2006).

    Article  CAS  Google Scholar 

  32. Höbartner, C. & Micura, R. Chemical synthesis of selenium-modified oligoribonucleotides and their enzymatic ligation leading to an U6 snRNA stem-loop segment. J. Am. Chem. Soc. 126, 1141–1149 (2004).

    Article  Google Scholar 

  33. Pallan, P.S. & Egli, M. Selenium modification of nucleic acids: preparation of phosphoroselenoate derivatives for crystallographic phasing of nucleic acid structures. Nat. Protoc. 2, 640–646 (2007).

    Article  CAS  Google Scholar 

  34. Pallan, P.S. & Egli, M. Selenium modification of nucleic acids: preparation of oligonucleotides with incorporated 2′-SeMe-uridine for crystallographic phasing of nucleic acid structures. Nat. Protoc. 2, 647–651 (2007).

    Article  CAS  Google Scholar 

  35. Arn, E.A. & Abelson, J. RNA ligases: function, mechanism, and sequence conservation. In RNA Structure and Function (eds. Simons, R.W. & Grunberg-Manago, M.) 695–726 (CSHL Press, New York, 1998).

    Google Scholar 

  36. Stark, M.R., Pleiss, J.A., Deras, M., Scaringe, S.A. & Rader, S.D. An RNA ligase-mediated method for the efficient creation of large synthetic RNAs. RNA 12, 2014–2019 (2006).

    Article  CAS  Google Scholar 

  37. Sherlin, L.D. et al. Chemical and enzymatic synthesis of tRNAs for high-throughput crystallization. RNA 7, 1671–1678 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Kurschat, W., Müller, J., Wombacher, R. & Helm, M. Optimizing splinted ligation of highly structured small RNAs. RNA 11, 1909–1914 (2005).

    Article  CAS  Google Scholar 

  39. Stutz, A.Ph.D. Thesis ETH Nr. 15141. Methoden zur Ligation von Oligoribonukleotiden und ihre Anwendung bei der Totalsynthese aminoacylierter tRNAs. ETH Zurich (2003).

  40. Sudarsan, N., Barrick, J.E. & Breaker, R.R. Metabolite-binding RNA domains are present in the genes of eukaryotes. RNA 9, 644–647 (2003).

    Article  CAS  Google Scholar 

  41. Galagan, J.E., Henn, M.R., Ma, L.J., Cuomo, C.A. & Birren, B. Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature 438, 1105–1115 (2005).

    Article  CAS  Google Scholar 

  42. Kubodera, T. et al. Thiamine-regulated gene expression of Aspergillus oryzae thiA requires splicing of the intron containing a riboswitch-like domain in the 5′-UTR. FEBS Lett. 555, 516–520 (2003).

    Article  CAS  Google Scholar 

  43. Yamauchi, T. et al. Roles of Mg2+ in TPP-dependent riboswitch. FEBS Lett. 579, 2583–2588 (2005).

    Article  CAS  Google Scholar 

  44. Sudarsan, N. et al. Tandem riboswitch architectures exhibit complex gene control functions. Science 314, 300–304 (2006).

    Article  CAS  Google Scholar 

  45. Welz, R. & Breaker, R.R. Ligand binding and genetic control characteristics of tandem riboswitches in Bacillus anthracis. RNA 13, 308–317 (2007).

    Article  Google Scholar 

  46. Serganov, A., Polonskaia, A., Phan, A.T., Breaker, R.R. & Patel, D.J. Structural basis for gene regulation by thiamine pyrophosphate-sensing riboswitch. Nature 441, 1167–1171 (2006).

    Article  CAS  Google Scholar 

  47. Thore, S., Leibundgut, M. & Ban, N. Structure of the eukaryotic thiamine pyrophosphate riboswitch with its regulatory ligand. Science 312, 1208–1211 (2006).

    Article  CAS  Google Scholar 

  48. Edwards, T.E. & Ferré-D'Amaré, A.R. Crystal structure of the thi-box riboswitch bound to thiamine pyrophosphate analogs reveal adaptive RNA-small molecule recognition. Structure 14, 1459–1468 (2006).

    Article  CAS  Google Scholar 

  49. Micura, R. Small interfering RNAs and their chemical synthesis. Angew. Chem. Int. Ed. Engl. 41, 2265–2269 (2002).

    Article  CAS  Google Scholar 

  50. Pitsch, S., Weiss, P.A., Jenny, L., Stutz, A. & Wu, X.L. Reliable chemical synthesis of oligoribonucleotides (RNA) with 2′-O-[(triisopropylsilyl)oxy]methyl(2′-O-tom)-protected phosphoramidites. Helv. Chim. Acta. 84, 3773–3795 (2001).

    Article  CAS  Google Scholar 

  51. Höbartner, C., Ebert, M.O., Jaun, B. & Micura, R. RNA two-state conformation equilibria and the effect of nucleobase methylation. Angew. Chem. Int. Ed. Engl. 41, 605–609 (2002).

    Article  Google Scholar 

  52. Höbartner, C., Mittendorfer, H., Breuker, K. & Micura, R. Triggering of RNA secondary structures by a functionalized nucleobase. Angew. Chem. Int. Ed. Engl. 43, 3922–3925 (2004).

    Article  Google Scholar 

  53. Micura, R. et al. Preparation of 2′-deoxy-2′-methylseleno-modified RNA. Curr. Protoc. Nucleic Acid Chem. 1, C1.15.1–C1.15.34 (2007).

    Google Scholar 

  54. Stephenson, F. Calculations for Molecular Biology and Biotechnology: A Guide to Mathematics in the Laboratory (Academic Press, Amsterdam, 2003).

Download references

Acknowledgements

The work is supported by the Austrian Science Fund FWF (P17864) and the bm:wf (Gen-AU programme; project 'Non-coding RNAs' no. P7260-012-011) for funding.

Author information

Authors and Affiliations

  1. Institute of Organic Chemistry, Center for Molecular Biosciences Innsbruck (CMBI), Leopold Franzens University, Innrain 52a, Innsbruck, A-6020, Austria

    Kathrin Lang & Ronald Micura

Authors
  1. Kathrin Lang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ronald Micura
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ronald Micura.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lang, K., Micura, R. The preparation of site-specifically modified riboswitch domains as an example for enzymatic ligation of chemically synthesized RNA fragments. Nat Protoc 3, 1457–1466 (2008). https://doi.org/10.1038/nprot.2008.135

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2008.135

This article is cited by

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