In vitro selection of N 1 -methyladenosine-sensitive RNA-cleaving deoxyribozymes with 10 5 -fold selectivity over unmethylated RNA.

RNA-cleaving DNAzymes (RCDs) are catalytically active DNA molecules that cleave a wide range of RNA targets with extremely high sequence-selectivity, but none is able to faithfully discriminate methylated from unmethylated RNA (typically <30-fold). We report the first efforts to isolate RCDs from a random-sequence DNA pool by in vitro selection that cleave RNA/DNA chimera containing N 1 -methyladenosine (m 1 A), one of the most prevalent RNA modifications that plays important regulatory roles in gene expression and human cancers. A cis -acting deoxyribozyme, RCD1-S2 m1A , exhibits an observed rate constant ( k obs ) of 5.3 × 10 -2 min -1 , resulting in up to 10 5 -fold faster cleavage of the m 1 A-modified versus unmethylated RNA. Furthermore, a trans -acting fluorogenic deoxyribozyme was constructed by labeling a fluorophore and a quencher at the 5' and 3' ends of the chimeric substrate, respectively. It permits the synchronization of RNA-cleaving with real-time fluorescence signaling, thus allowing the selective monitoring of ALKBH3-mediated demethylation and inhibitor screening in living cells.