Chemical evolution of an autonomous DNAzyme with allele-specific gene silencing activity

• Published in: Nature communications Vol. 14; no. 1; p. 2413
• Main Authors: Nguyen, Kim, Malik, Turnee N, Chaput, John C
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 27.04.2023; Nature Publishing Group UK; Nature Portfolio
• Subjects: Alleles; Biological evolution; Catalysis; Catalytic activity; Chemical evolution; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA, Catalytic - metabolism; Enzymes; Evolution; Evolution, Chemical; Gene Silencing; Magnesium; mRNA; Physicochemical properties; Physiology; Ribonuclease H1; Therapeutic applications
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-023-38100-9

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 Mg 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.