A programmable pAgo DNase from Hydrogenophilus thermoluteolus guided by small DNA and RNA

Argonaute (Ago) proteins are programmable nucleases found in both eukaryotes and prokaryotes that use small nucleic acid guides to cleave complementary DNA or RNA targets. However, the limited catalytic efficiency and substrate versatility of currently characterized prokaryotic Argonautes (pAgos) have restricted their broader applications. Here, we characterized HthAgo, a pAgo from the thermophilic bacterium Hydrogenophilus thermoluteolus, and show that it possesses broad and precise nucleic acid cleavage activities. HthAgo efficiently cleaves DNA targets when loaded with 5'-phosphorylated DNA guides (5'P-gDNA), 5'-hydroxylated DNA guides (5'OH-gDNA), or 5'-phosphorylated RNA guides (5'P-gRNA), and also cleaves RNA targets when guided by 5'P-gDNA or 5'OH-gDNA. Remarkably, HthAgo maintains precise cleavage with both 5'P- and 5'OH-gDNA, with 5'P-guides directing canonical cleavage between guide positions 10 and 11, whereas 5'OH-guides shift cleavage exclusively to positions 11 and 12. Biochemical analyses showed that HthAgo remains active over a broad range of reaction conditions, displays optimal activity at 60-70 °C in the presence of Mn²⁺, and exhibits no apparent preference for the 5'-terminal nucleotide of the guide. Binding and thermal stability analyses further suggest that HthAgo preferentially utilizes DNA guides and DNA targets owing to their stronger binding affinity and higher complex stability. In addition, HthAgo enables efficient and accurate cleavage of double-stranded DNA at elevated temperature using paired DNA guides. Together, these findings expand the functional landscape of thermophilic pAgos and establish HthAgo as a promising programmable nuclease platform for versatile nucleic acid manipulation.