N-6-Methyladenosine-Sensitive RNA-Cleaving Deoxyribozymes

Deoxyribozymes are synthetic enzymes made of DNA that can catalyze the cleavage or formation of phosphodiester bonds and are useful tools for RNA biochemistry. Herein, we report new RNA-cleaving deoxyribozymes to interrogate the methylation status of target RNAs, thereby providing an alternative method for the biochemical validation of RNA methylation sites containing N-6-methyladenosine, which is the most wide-spread and extensively investigated natural RNA modification. The developed deoxyribozymes are sensitive to the presence of N-6-methyladenosine in RNA near the cleavage site. One class of these DNA enzymes shows faster cleavage of methylated RNA, while others are strongly inhibited by the modified nucleotide. The general applicability of the new deoxyribozymes is demonstrated for several examples of natural RNA sequences, including a lncRNA and a set of C/D box snoRNAs, which have been suggested to contain m(6)A as a regulatory element that influences RNA folding and protein binding.