Chemically Evolved XNAzyme Platforms for Effective In Vivo Gene Regulation.

DNAzymes are programmable nucleic acid catalysts for RNA cleavage, but their application in biological systems is limited by low catalytic efficiency and poor stability under physiological conditions. Here we report the chemical evolution of the 8-17 DNAzyme to generate xeno-nucleic acid enzymes (XNAzymes) with enhanced functionality. Through rational modifications of sugar and backbone components within the catalytic core, the optimized XNAzyme exhibits up to a 14-fold increase in catalytic activity under near-physiological conditions while fully preserving sequence programmability and structural modularity. Additional terminal modifications improve nuclease resistance and reduce RNase H-mediated degradation, favoring efficient catalytic RNA cleavage. Functionally, the XNAzyme platform enables sequence-specific silencing of both exogenous and endogenous mRNAs in mammalian cells, and can be programmed to cleave oncogenic miRNA, resulting in upregulation of downstream genes. In zebrafish embryos, XNAzyme-mediated RNA targeting induces gene-specific developmental alterations. This study establishes chemical evolution of the catalytic core as a general strategy to enhance the performance of nucleic acid enzymes and highlights XNAzyme as a versatile platform for programmable RNA regulation in complex biological environments.