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

## TL;DR

Scientists engineered a DNAzyme to avoid a cellular enzyme, enabling targeted gene knockdown of disease-related mutations.

## Contribution

A TNA-modified DNAzyme was developed to evade RNase H1 and achieve allele-specific gene knockdown.

## Key findings

- TNA substitution in the DNAzyme increased catalytic activity and evaded RNase H1.
- The modified DNAzyme achieved allele-specific knockdown of the KRAS oncogenic mutation.
- The enzyme also enabled general knockdown of PCSK9 and GATA3 targets in mammalian cells.

## Abstract

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.

Graphical Abstract