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 Mg²⁺ 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.

核酸催化剂（核酶、DNA 酶和 XNA 酶）以高特异性切割靶 (m)RNA，但在临床应用中的功效有限。在此，我们报告了由 2′-脱氧-2′-氟-β- d-阿拉伯核酸 (FANA) 组成的高度特异性模块化 RNA 核酸内切酶 XNAzyme FR6_1 的体外进化和工程设计。 FR6_1 克服了之前 DNA 酶和 XNAzyme 的活性限制，可重新定位以在生理 Mg ²⁺浓度下切割高度结构化的全长 (>5 kb) BRAF和KRAS mRNA，并具有肿瘤相关的等位基因选择性（ BRAF V600E 和KRAS G12D） ）突变。硫代磷酸酯-FANA 修饰可增强 FR6_1 生物稳定性，并通过体内 XNAzyme 裂解活性提供的 G12D 等位基因特异性成分，能够在培养的人腺癌细胞中快速敲低KRAS mRNA。这些结果为开发基于 FANA 或其他 XNA 化学物质的改进基因沉默剂提供了起点。