A universal strategy for enhancing TtAgo-Mediated nucleic acid detection via 2'-O-methyl guide DNA modification: Ultrasensitive and specific identification of African swine fever virus.

Thermus thermophilus Argonaute (TtAgo) is a DNA-guided nuclease that has been widely applied in molecular diagnostics. However, its limited cleavage activity on long double-stranded DNA (dsDNA) remains a significant challenge, restricting its broader application. To address this issue, we propose a universal strategy that utilizes 2'-O-methyl (2'-OMe) modified guide DNA (2'-OMe-gDNA) to enhance TtAgo activity. The results demonstrated that 2'-OMe-gDNA significantly improves the cleavage efficiency of TtAgo on long dsDNA, while retaining its ability to discriminate single-nucleotide variations. Kinetic and binding analyses further reveal that the 2'-OMe modification accelerates the reaction turnover rate, enhances the binding affinity between gDNA and TtAgo, and facilitates gDNA strand invasion into dsDNA, thereby enhancing the cleavage activity of TtAgo. To demonstrate the potential application of this strategy, we integrated the enhanced TtAgo system with recombinase polymerase amplification (RPA) to develop an RPA-ETtAgo platform for African swine fever virus (ASFV) detection and genotyping. Our results show that 2'-OMe-gDNA improves the fluorescence signal by at least 2.8-fold compared to unmodified gDNA. Additionally, leveraging its single-nucleotide resolution, the platform is capable of precise genotypic discrimination of ASFV, with detectable fluorescence signals observed at target concentrations as low as 2 copies/μL. The analysis of 119 tissue and environmental samples further validated the platform's diagnostic accuracy, with results showing complete concordance with qPCR results. This study provides a simple and universal gDNA design strategy for developing highly sensitive TtAgo-based systems with broad application potential.