A mesophilic Argonaute from Clostridium formicaceticum with efficient DNA cleavage activity guided by small DNA

Abstract

We characterized a new Argonaute protein (pAgo), CfAgo, from the mesophilic bacterium Clostridium formicaceticum. CfAgo possesses DNA-guided DNA endonuclease activity and cleaves DNA targets at the canonical site. It is active from 28°C to 75°C and prefers DNA guides with a 5′-phosphate group and thymidine as the first nucleotide. Cleavage activity is reduced by single-nucleotide mismatches in the seed, central, and 3′-supplementary regions of guides, with stronger mismatch discrimination observed for 5′hydroxylated (5′OH) guides compared to 5′phosphorylated (5′P) guides. Moreover, structural analysis suggests that the MID domain of CfAgo is crucial for recognizing the 5′ guide and it influences the binding specificity. CfAgo catalyzes programmable cleavage of double-stranded DNA in AT-rich regions in the presence of Mn²⁺ and Mg²⁺ ions at appropriate salt concentrations. These properties could make CfAgo a promising tool for DNA manipulation such as nucleic acid detection and cleavage.