Abstract
AbstractDeoxyribozymes 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 (m3C), N4‐methylcytidine (m4C), and 5‐methylcytidine (m5C), 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 m3C‐, m4C‐ or m5C‐modified RNA. An additional deoxyribozyme is strongly inhibited by any of the three methylcytidines, but effectively cleaves unmodified RNA. The mXC‐detecting deoxyribozymes are programmable for the interrogation of natural RNAs of interest, as demonstrated for human mitochondrial tRNAs containing known m3C and m5C sites. The results underline the potential of synthetic functional DNA to shape highly selective active sites.
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