<i>N</i>⁶‐Isopentenyladenosine in RNA Determines the Cleavage Site of Endonuclease Deoxyribozymes

Abstract

RNA-cleaving deoxyribozymes can serve as selective sensors and catalysts to examine the modification state of RNA. However, site-specific endonuclease deoxyribozymes that selectively cleave post-transcriptionally modified RNA are extremely rare and their specificity over unmodified RNA is low. We report that the native tRNA modification N⁶ -isopentenyladenosine (i⁶ A) strongly enhances the specificity and has the power to reconfigure the active site of an RNA-cleaving deoxyribozyme. Using in vitro selection, we identified a DNA enzyme that cleaves i⁶ A-modified RNA at least 2500-fold faster than unmodified RNA. Another deoxyribozyme shows unique and unprecedented behaviour by shifting its cleavage site in the presence of the i⁶ A RNA modification. Together with deoxyribozymes that are strongly inhibited by i⁶ A, these results highlight that post-transcriptional RNA modifications modulate the catalytic activity of DNA in various intricate ways.