Use of SHAPE to Select 2AP Substitution Sites for RNA–Ligand Interactions and Dynamics Studies

  • Marie F. Soulière
  • Ronald Micura
Part of the Methods in Molecular Biology book series (MIMB, volume 1103)


Most regulatory RNA molecules must adopt a precise secondary fold and tertiary structure to allow their function in cells. A number of experimental approaches, such as the 2-Aminopurine-Based RNA Folding Analysis (2ApFold), have therefore been developed to offer insights into the folding and folding dynamics of RNA. A crucial requirement for this method is the selection of proper 2AP labeling positions. In that regard, we recently discovered that Selective 2′-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) offers a reliable path to identify appropriate nucleotides for 2AP substitution on a target RNA. This chapter describes the straightforward procedure to select 2AP substitution sites in RNA molecules using SHAPE probing. The protocols detail the preparation of the target RNA by transcription, and the SHAPE steps including (1) probing of the RNA, (2) reverse transcription with a radiolabeled primer, (3) sequencing gel, and (4) analysis of the obtained band pattern.

Key words

SHAPE RNA probing 2-Aminopurine 2ApFold preQ1cII riboswitch SAFA 


  1. 1.
    Strobel SA, Cochrane JC (2007) RNA catalysis: ribozymes, ribosomes, and riboswitches. Curr Opin Chem Biol 11:636–643PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Serganov A, Patel DJ (2007) Ribozymes, riboswitches and beyond: regulation of gene expression without proteins. Nat Rev Genet 8:776–790PubMedCrossRefGoogle Scholar
  3. 3.
    Inui M, Martello G, Piccolo S (2010) MicroRNA control of signal transduction. Nat Rev Mol Cell Biol 11:252–263PubMedCrossRefGoogle Scholar
  4. 4.
    Batey RT, Rambo RP, Doudna JA (1999) Tertiary motifs in RNA structure and folding. Angew Chem Int Ed 38:2326–2343CrossRefGoogle Scholar
  5. 5.
    Roth A, Breaker RR (2009) The structural and functional diversity of metabolite-binding riboswitches. Annu Rev Biochem 78:305–334PubMedCrossRefGoogle Scholar
  6. 6.
    Schwalbe H, Buck J, Furtig B, Noeske J, Wohnert J (2007) Structures of RNA switches: insight into molecular recognition and tertiary structure. Angew Chem Int Ed 46:1212–1219CrossRefGoogle Scholar
  7. 7.
    Haller A, Souliere MF, Micura R (2011) The dynamic nature of RNA as key to understanding riboswitch mechanisms. Acc Chem Res 44:1339–1348PubMedCrossRefGoogle Scholar
  8. 8.
    Haller A, Rieder U, Aigner M, Blanchard SC, Micura R (2011) Conformational capture of the SAM-II riboswitch. Nat Chem Biol 7:393–400PubMedCrossRefGoogle Scholar
  9. 9.
    Rieder U, Kreutz C, Micura R (2010) Folding of a transcriptionally acting preQ1 riboswitch. Proc Natl Acad Sci U S A 2107:10804–10809CrossRefGoogle Scholar
  10. 10.
    Rieder R, Lang K, Graber D, Micura R (2007) Ligand-induced folding of the adenosine deaminase A-riboswitch and implications on riboswitch translational control. Chembiochem 8:896–902PubMedCrossRefGoogle Scholar
  11. 11.
    Lang K, Rieder R, Micura R (2007) Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach. Nucleic Acids Res 35:5370–5378PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Jean JM, Hall KB (2001) 2-Aminopurine fluorescence quenching and lifetimes: role of base stacking. Proc Natl Acad Sci U S A 98:37–41PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Sinkeldam RW, Greco N, Tor Y (2010) Fluorescent analogs of biomolecular building blocks: design properties and applications. Chem Rev 110:2579–2619PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Souliere MF, Haller A, Rieder R, Micura R (2011) A powerful approach for the selection of 2-aminopurine substitution sites to investigate RNA folding. J Am Chem Soc 133:16161–16167PubMedCrossRefGoogle Scholar
  15. 15.
    Merino EJ, Wilkinson KA, Coughlan JL, Weeks KM (2005) RNA structure analysis at single nucleotide resolution by selective 2′-hydroxyl acylation and primer extension (SHAPE). J Am Chem Soc 127:4223–4231PubMedCrossRefGoogle Scholar
  16. 16.
    Wilkinson KA, Merino EJ, Weeks KM (2006) Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution. Nat Protoc 1:1610–1616PubMedCrossRefGoogle Scholar
  17. 17.
    Wilkinson KA, Vasa SM, Deigan KE, Mortimer SA, Giddings MC, Weeks KM (2009) Influence of nucleotide identity on ribose 29-hydroxylreactivity in RNA. RNA 15:1314–1321PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Gherghe CM, Shajani Z, Wilkinson KA, Varani G, Weeks KM (2008) Strong correlation between SHAPE chemistry and the generalized NMR order parameter (S2) in RNA. J Am Chem Soc 130:12244–12245PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Laederach A, Das R, Vicens Q, Pearlman SM, Brenowitz M, Herschlag D, Altman RB (2008) Semi-automated and rapid quantification of nucleic acid footprinting and structure mapping experiment. Nat Protoc 3(9):1395–1401PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Marie F. Soulière
    • 1
  • Ronald Micura
    • 1
  1. 1.Institute of Organic Chemistry, Center for Chemistry and BiomedicineLeopold Franzens UniversityInnsbruckAustria

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