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A Post-Labeling Approach for the Characterization and Quantification of RNA Modifications Based on Site-Directed Cleavage by DNAzymes

  • Madeleine Meusburger
  • Martin Hengesbach
  • Mark HelmEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 718)

Abstract

Deoxyribozymes or DNAzymes are small DNA molecules with catalytic activity originating from in vitro selection experiments. Variants of the two most popular DNAzymes with RNase activity, the 10–23 DNAzyme and the 8–17 DNAzyme, promote efficient in vitro cleavage of the phosphodiester bond in at least 11 out of 16 possible dinucleotide permutations. Judicious choice of the sequences flanking the active core of the DNAzymes permits to direct cleavage activity with high sequence specificity. Here, the harnessing of these features for the analysis of RNA nucleotide modifications by a post-labeling approach is described in detail. DNAzymes are designed such that RNase cleavage is directed precisely to the 5′ end of the nucleotide to be analyzed. Iterative complex formation of DNAzyme and RNA substrate and subsequent cleavage are performed by temperature cycling. The DNAzyme activity liberates the analyte nucleotide on the very 5′-end of an RNA fragment, whose hydroxyl group can be conveniently phosphorylated with 32P. The labeled RNA is digested to mononucleotides, and analyzed by thin layer chromatography.

Key words

DNAzyme Modified nucleotides Catalytic DNA RNA cleavage Post-labeling Thin-layer chromatography 

Notes

Acknowledgments

Mark Helm for the DFG funding (HE 3397/4). Martin Hengesbach acknowledges funding by the Landesgradu­iertenförderung Baden-Württemberg. We thank Andres Jäschke for generous support.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Madeleine Meusburger
    • 1
  • Martin Hengesbach
    • 1
  • Mark Helm
    • 2
    • 3
    Email author
  1. 1.Institute of Pharmacy and Molecular BiotechnologyUniversity of HeidelbergHeidelbergGermany
  2. 2.Department of Chemistry, Institute of Pharmacy and Molecular BiotechnologyRuprecht-Karls Universität HeidelbergHeidelbergGermany
  3. 3.Institute of Pharmacy and BiochemistryJohannes Gutenberg-University MainzMainzGermany

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