A modular XNAzyme cleaves long, structured RNAs under physiological conditions and enables allele-specific gene silencing.
Alexander I TaylorChristopher J K WanMaria J DondeSew Peak ChewPhilipp HolligerPublished in: Nature chemistry (2022)
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.
Keyphrases
- nucleic acid
- wild type
- endothelial cells
- induced apoptosis
- squamous cell carcinoma
- dna repair
- circulating tumor
- positron emission tomography
- oxidative stress
- radiation therapy
- cell proliferation
- cell free
- metastatic colorectal cancer
- dna binding
- induced pluripotent stem cells
- locally advanced
- structural basis
- dna damage
- quantum dots