Exploiting inosine as a distinctive cleavage site for deriving a highly specific RNA-cleaving DNAzyme.

Most RNA-cleaving DNAzymes are limited to cleaving between combinations of the four canonical ribonucleotides. Through in vitro selection, we isolated ICD11-1T, a DNAzyme that selectively cleaves at inosine, a non-canonical RNA modification generated by adenosine deamination. ICD11-1T cleaves inosine-containing substrates with a rate constant of 0.1 min-1 and achieves >103-fold selectivity over adenosine, discriminating between nucleosides that differ at the C6 and N1 positions of the purine ring. Molecular dynamics simulations revealed that the inosine positions the cleavage site near the catalytic core, whereas the adenosine complex sequesters it away, accounting for the stringent specificity. Using ICD11-1T, we constructed a fluorescent biosensor that enabled imaging of endogenous ADAR (adenosine deaminase acting on RNA) editing activity in fixed and permeabilized cells. This work provides a proof-of-concept tool for studying adenosine-to-inosine (A-to-I) RNA editing and establishes a strategy for expanding the functionality of DNAzymes.