Site‐Specific Recognition of Inosine‐Modified RNA by Deoxyribozyme with Distant Cleavage Site

Abstract We report an RNA‐cleaving DNAzyme (RCD), RCD‐I4T3, discovered through in vitro selection, that can specifically recognize an inosine‐modified site in RNA and cleave at a remote site (eight nucleotides downstream of the inosine). RCD‐I4T3 represents the first DNAzyme with spatially separated domains for target recognition and catalytic cleavage, functionally analogous to Type IIS restriction endonucleases and endonuclease V, which is not previously observed in DNA catalysts. The RCD‐I4T3 serves as a potent RNA‐cleaving tool, exhibiting an observed rate constant ( k obs ) of 1.9 × 10 −2 min −1 for inosine‐modified RNA, and successfully targeting natural inosine‐containing mRNA fragments with high specificity. This modular architecture minimizes the off‐target activity through stringent two‐step validation (i.e., recognition and cleavage) and enables independent functional optimization. The unique structural and mechanistic features of RCD‐I4T3 expand the functional repertoire of DNAzymes for biological applications.