Allele-specific knockdown by an engineered DNAzyme capable of RNase H1 evasion.

DNA enzymes (DNAzymes) offer an attractive therapeutic approach for targeting disease-associated mutations in mRNA transcripts, but face limitations in development due to unintended engagement by RNase H1. Although chemical optimization has led to designs with improved catalytic activity, strategies to mitigate RNase H1 recognition remain underexplored. Here, we report the incorporation of threose nucleic acid (TNA) into the backbone architecture of the 10-23 DNAzyme variant known as Dz46. Substitution of the dC3 position in the catalytic loop with TNA increases activity, whereas installation of two TNA residues in the binding arm abrogates competition by RNase H1. The resulting enzyme enables allele-specific knockdown of an oncogenic KRAS mutation in mammalian cells and facilitates general knockdown of PCSK9 and GATA3 targets. Together, these results demonstrate the utility of TNA as a chemical tool for enhancing DNAzyme performance and evading RNase H1 activity in cells.