A bacterial Argonaute with efficient DNA and RNA cleavage activity guided by small DNA and RNA

Argonaute proteins are widespread in prokaryotes and eukaryotes with diversified catalytic activities. Here, we describe an Argonaute from Marinitoga hydrogenitolerans (MhAgo) with all eight cleavage activities. Utilization of all four types of guides and efficient cleavage of single-stranded DNA (ssDNA) and RNA targets are revealed. The preference for the 5'-terminus nucleotides of 5'P guides, but no obvious preferences for that in 5'OH guides, is further uncovered. Moreover, the cleavage efficiency is heavily impaired by mis-matches in the central and 3'-supplementary regions of guides, and the affinity between guides or guides/ target duplex and MhAgo is proved as one of the factors affecting cleavage efficiency. Structural and muta-tional analyses imply some unknown distinctive structural features behind the cleavage activity of MhAgo. Meanwhile, 5'OH-guide RNA (gRNA)-mediated plasmid cleavage activity is unveiled. Conclusively, MhAgo is versatile, and its biochemical characteristics improve our understanding of pAgos and the pAgo-based techniques.

Automated summary

This study describes an Argonaute protein (MhAgo) from Marinitoga hydrogenitolerans that exhibits all eight cleavage activities. MhAgo shows preference for specific nucleotides and mismatches in the guideRNA can significantly impair its cleavage efficiency. The study also unveils the plasmid cleavage activity mediated by 5'OH-guide RNA (gRNA). These findings enhance our understanding of pAgos and pAgo-based techniques.