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

ABSTRACT Argonaute proteins are widespread in prokaryotes and eukaryotes. Most prokaryotic Argonaute proteins (pAgos) use 5’P-gDNA to target complementary DNA. However, more and more studies on the properties of pAgos make their functions more diversified. Previously reported pAgos only possess several forms of high activity in all eight cleavage patterns, which limits their practical applications. Here, we described a unique pAgo from Marinitoga hydrogenitolerans (MhAgo) with eight cleavage activities. MhAgo can utilize all four types of guides (5’OH-gDNA, 5’P-gDNA, 5’OH-gRNA, and 5’P-gRNA) for ssDNA and RNA cleavage. Further studies demonstrated that MhAgo had high activities with 16-21 nt guides and no obvious preferences for the 5’-end nucleotides of 5’OH-guides. Unexpectedly, MhAgo had different preferences for the 5’-end nucleotides of 5’P-guides depending on the types of targets. Although the specificity of MhAgo was related to the types of guides, single mismatches in the central and 3’-supplementary regions of guides greatly reduced the cleavage efficiency. Additionally, the electrophoretic mobility shift assay (EMSA) demonstrated MhAgo had the weakest affinity for 5’P-gRNA:tRNA duplex, which was consistent with its cleavage efficiency. In conclusion, MhAgo is highly active under a wide range of conditions and can be used for programmable endonucleolytic cleavage of both ssDNA and RNA substrates. The abundant biochemical characteristics of MhAgo broaden our understanding of pAgos and expand the potential application in nucleic acids manipulations.