RNA-Cleaving Deoxyribozymes Differentiate Methylated Cytidine Isomers in RNA.

Deoxyribozymes are emerging as modification-specific endonucleases for the analysis of epigenetic RNA modifications. Here, we report RNA-cleaving deoxyribozymes that differentially respond to the presence of natural methylated cytidines, 3-methylcytidine (m3 C), N4 -methylcytidine (m4 C), and 5-methylcytidine (m5 C), respectively. Using in vitro selection, we found several DNA catalysts, which are selectively activated by only one of the three cytidine isomers, and display 10- to 30-fold accelerated cleavage of their target m3 C-, m4 C- or m5 C-modified RNA. An additional deoxyribozyme is strongly inhibited by any of the three methylcytidines, but effectively cleaves unmodified RNA. The mX C-detecting deoxyribozymes are programmable for the interrogation of natural RNAs of interest, as demonstrated for human mitochondrial tRNAs containing known m3 C and m5 C sites. The results underline the potential of synthetic functional DNA to shape highly selective active sites.