A modular XNAzyme cleaves long, structured RNAs under physiological conditions and enables allele-specific gene silencing

Nucleic-acid catalysts (ribozymes, DNA- and XNAzymes) cleave target (m)RNAs with high specificity but have shown limited efficacy in clinical applications. Here we report on the in vitro evolution and engineering of a highly specific modular RNA endonuclease XNAzyme, FR6_1, composed of 2′-deoxy-2′-fluoro-β- d -arabino nucleic acid (FANA). FR6_1 overcomes the activity limitations of previous DNA- and XNAzymes and can be retargeted to cleave highly structured full-length (>5 kb) BRAF and KRAS mRNAs at physiological Mg 2+ concentrations with allelic selectivity for tumour-associated ( BRAF V600E and KRAS G12D) mutations. Phosphorothioate-FANA modification enhances FR6_1 biostability and enables rapid KRAS mRNA knockdown in cultured human adenocarcinoma cells with a G12D-allele-specific component provided by in vivo XNAzyme cleavage activity. These results provide a starting point for the development of improved gene-silencing agents based on FANA or other XNA chemistries. Oligonucleotide catalysts such as ribozymes and DNAzymes can cleave RNA efficiently and specifically but are typically dependent on high concentrations of divalent cations, limiting their biological applications. A modular XNAzyme catalyst composed of 2′-deoxy-2′-fluoro-β- d -arabino nucleic acid (FANA) has now been developed that can cleave long (>5 kb), highly structured mRNAs under physiological conditions and enables allele-specific catalytic RNA knockdown inside cells.