In vitro selection of N ¹-methyladenosine-sensitive RNA-cleaving deoxyribozymes with 10⁵-fold selectivity over unmethylated RNA.

ABSTRACT: 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 (m¹A), 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 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.

SUBMITTER: Shi J 

PROVIDER: S-EPMC11339963 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature