Low activity has been the primary obstacle impeding the use of DNA enzymes (DNAzymes) as gene silencing agents in clinical applications. Here we describe the chemical evolution of a DNAzyme with strong catalytic activity under near physiological conditions. The enzyme achieves ~65 turnovers in 30 minutes, a feat only previously witnessed by the unmodified parent sequence under forcing conditions of elevated Mg²⁺ and pH. Structural constraints imposed by the chemical modifications drive catalysis toward a highly preferred UGUD motif (cut site underlined) that was validated by positive and negative predictions. Biochemical assays support an autonomous RNA cleavage mechanism independent of RNase H1 engagement. Consistent with its strong catalytic activity, the enzyme exhibits persistent allele-specific knock-down of an endogenous mRNA encoding an undruggable oncogenic KRAS target. Together, these results demonstrate that chemical evolution offers a powerful approach for discovering new chemotype combinations that can imbue DNAzymes with the physicochemical properties necessary to support therapeutic applications.

The hydrolytic deamination of adenosine-to-inosine (A-to-I) by RNA editing is a widespread post-transcriptional modification catalyzed by the adenosine deaminase acting on RNA (ADAR) family of proteins. ADAR-mediated RNA editing modulates cellular pathways involved in innate immunity, RNA splicing, RNA interference, and protein recoding, and has been investigated as a strategy for therapeutic intervention of genetic disorders. Despite advances in basic and translational research, the mechanisms regulating RNA editing are poorly understood. Though several trans-acting regulators of editing have been shown to modulate ADAR protein expression, previous studies have not identified factors that modulate ADAR catalytic activity. Here, we show that RNA editing increases upon intracellular acidification, and that these effects are predominantly explained by both enhanced ADAR base-flipping and deamination rate at acidic pH. We also show that the extent of RNA editing increases with the reduction in pH associated with conditions of cellular hypoxia.