Pyrococcus furiosus Argonaute-Based FluorometricBiosensor for One-Tube Detection ofCancer-Associated Single Nucleotide Polymorphisms in MicroRNAs

MicroRNA-related single nucleotide polymorphisms (miR-SNPs) are promising biomarkers for cancer diagnostics, yet accurate detection methods remain limited. Here, we introduce a ligation-triggered Pyrococcus furiosus Argonaute (<i>Pf</i>Ago) cleavage (LTAC) strategy for the sensitive detection of miR-SNPs, demonstrated using the rs11614913 SNP in miR-196a2, which is associated with nonsmall cell lung cancer (NSCLC). The mutant miR-196a2T serves as a scaffold for the formation of guide DNA (gDNA) catalyzed by the SplintR ligase, leading to <i>Pf</i>Ago activation and enhanced fluorescence. In contrast, wild-type miR-196a2C cannot facilitate gDNA formation and thus fails to activate <i>Pf</i>Ago. This method exhibits a linear relationship with the logarithm of the miR-196a2T concentration over a range of 0.2 pM to 100 nM, achieving a low detection limit of 0.15 pM. Analysis of NSCLC patient samples using LTAC reveals elevated levels of the rs11614913 SNP in miR-196a2 compared to healthy controls, underscoring the diagnostic potential of LTAC.