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

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 ³² P. The labeled RNA is digested to mononucleotides, and analyzed by thin layer chromatography.