Enzymatic Ligation Strategies for the Preparation of Purine Riboswitches with Site-Specific Chemical Modifications

  • Renate Rieder
  • Claudia Höbartner
  • Ronald MicuraEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 540)


One of the most versatile riboswitch classes refers to purine nucleoside metabolism. In the cell, purine riboswitches of the respective mRNAs either act at the transcriptional or translational level and off- or on-regulate genes upon binding to their dedicated ligands. Biophysical studies on ligand-induced folding of these RNA domains in vitro contribute to understanding their regulation mechanisms in vivo. For such studies, in particular, for approaches using fluorescence spectroscopy, the preparation of large RNAs with site-specific chemical modifications is required. Here, we describe a strategy for the preparation of riboswitch aptamers and aptamers adjoined to their expression platforms by chemical synthesis and enzymatic ligation. The modular design enables fast access to a large number of purine riboswitch derivatives with the modification of interest at any strand position. We exemplarily provide a detailed protocol for the preparation of adenosine deaminase (add) A-riboswitch variants with 2-aminopurine (AP) modifications at the 40-nmol scale.

Key words

Ligation Riboswitch Aminopurine RNA modification 



We thank the Austrian Science Fund FWF (P17864) and the BMWF (Gen-AU program; projects “Non-coding RNAs” No. P7260-012-011 and No. P7260-012-012) for funding.


  1. 1.
    Mandal, M., Boese, B., Barrick, J. E., Winkler, W. C., and Breaker, R. R. (2003). Riboswitches control fundamental biochemical pathways in Bacillus subtilis and other bacteria. Cell 113, 577–586.PubMedCrossRefGoogle Scholar
  2. 2.
    Mandal, M. and Breaker, R. R. (2004). Adenine riboswitches and gene activation by disruption of a transcription terminator. Nat. Struct. Mol. Biol. 11, 29–35.PubMedCrossRefGoogle Scholar
  3. 3.
    Batey, R. T., Gilbert, S. D., and Montange, R. K. (2004). Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine. Nature 432,411–415.PubMedCrossRefGoogle Scholar
  4. 4.
    Serganov, A., Yuan, Y. R., Pikovskaya, O., Polonskaia, A., Malinina, L., Phan, A. T., Höbartner, C., Micura, R., Breaker, R. R., and Patel, D. J. (2004). Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs. Chem. Biol. 11, 1729–1741.PubMedCrossRefGoogle Scholar
  5. 5.
    Gilbert, S. D., Stoddard, C. D., Wise, S. J., and Batey, R. T. (2006). Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain. J. Mol. Biol. 359, 754–768.PubMedCrossRefGoogle Scholar
  6. 6.
    Lemay, J.-F., Penedo, J. C., Tremblay, R., Lilley, D. M. J., and Lafontaine, D. A. (2006). Folding of the adenine riboswitch. Chem. Biol. 13, 857–868.PubMedCrossRefGoogle Scholar
  7. 7.
    Wickiser, J. K., Cheah, M. T., Breaker, R. R., and Crothers, D. M. (2005). The Kinetics of ligand binding by an adenine-sensing riboswitch. Biochemistry 44, 13404–13414.PubMedCrossRefGoogle Scholar
  8. 8.
    Lemay, J. F. and Lafontaine, D. A. (2007). Core requirements of the adenine riboswitch aptamer for ligand binding. RNA 13, 339–350.PubMedCrossRefGoogle Scholar
  9. 9.
    Mulhbacher, J. and Lafontaine, D. A. (2007). Ligand recognition determinants of guanine riboswitches. Nucl. Acids Res. 35, 5568–5580.PubMedCrossRefGoogle Scholar
  10. 10.
    Noeske, J. Buck, J. Fürtig, B. Nasiri, H. R., Schwalbe, H., and Wöhnert, J. (2007). Interplay of ‘induced fit’ and preorganization in the ligand induced folding of the aptamer domain of the guanine binding riboswitch. Nucl. Acids Res 35, 572–583.PubMedCrossRefGoogle Scholar
  11. 11.
    Noeske, J., Schwalbe, H., and Wöhnert, J. (2007). Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain. Nucl. Acids Res. 35, 5262–5273.PubMedCrossRefGoogle Scholar
  12. 12.
    Rieder, R., Lang, K., Graber, D., and Micura, R. (2007). Ligand-induced folding of the adenosine deaminase A-riboswitch and implications on riboswitch translational control. ChemBioChem 8, 896–902.PubMedCrossRefGoogle Scholar
  13. 13.
    Micura, R. (2002). Small interfering RNAs and their chemical synthesis. Angew. Chem. Int. Ed. 41, 2265–2269.CrossRefGoogle Scholar
  14. 14.
    Höbartner, C. and Micura, R. (2004). 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.PubMedCrossRefGoogle Scholar
  15. 15.
    Höbartner, C., Rieder, R., Puffer, B., Lang, K., Polonskaia, A., Serganov, A., and Micura, R. (2005). 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, 12035–45.PubMedCrossRefGoogle Scholar
  16. 16.
    Persson, T., Willkomm, D. K., and Hartmann, R. K. (2005). T4 RNA ligase, in Handbook of RNA Biochemistry (Hartmann, R. K., Bindereif, A., Schön, A., and Westhof, E., eds.), Wiley-VCH, Weinheim, Germany, pp. 53–74.CrossRefGoogle Scholar
  17. 17.
    Frilander, J. M. and Turunen, J. J. (2005). RNA ligation using T4 DNA ligase, in Handbook of RNA Biochemistry (Hartmann, R. K., Bindereif, A., Schön, A., and Westhof, E., eds.), Wiley-VCH, Weinheim, Germany, pp. 36–52.Google Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Renate Rieder
    • 1
  • Claudia Höbartner
    • 1
  • Ronald Micura
    • 1
    Email author
  1. 1.Institute of Organic ChemistryLeopold Franzens UniversityAustria

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