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 N1-methyladenosine (m1A), one of the most prevalent RNA modifications that plays important regulatory roles in gene expression and human cancers. A cis-acting deoxyribozyme, RCD1-S2m1A, exhibits an observed rate constant (kobs) of 5.3 × 10−2 min−1, resulting in up to 105-fold faster cleavage of the m1A-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.
This article is Open Access
Please wait while we load your content…
Something went wrong. Try again?