selection of -methyladenosine-sensitive RNA-cleaving deoxyribozymes with 10-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 selection that cleave RNA/DNA chimera containing -methyladenosine (mA), one of the most prevalent RNA modifications that plays important regulatory roles in gene expression and human cancers. A -acting deoxyribozyme, RCD1-S2, exhibits an observed rate constant ( ) of 5.3 × 10 min, resulting in up to 10-fold faster cleavage of the mA-modified unmethylated RNA. Furthermore, a -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.