A bacterial Argonaute from Tepiditoga spiralis with the ability of RNA guided plasmid cleavage

The eukaryotic Argonaute proteins (eAgos) play an important role in the RNA interference pathway. The function and mechanism of prokaryotic Argonaute proteins (pAgos) in vivo are still unclear although the structure of pAgos and eAgos are highly homologous. Most of the reported pAgos have a preference for 5'P-gDNA, but MpAgo originated from bacteria Marinitoga piezophila preferentially uses 5'OH-gRNA to target DNA and RNA. To enrich our knowledge of this type of Argonaute proteins, here we report an Argonaute protein derived from Tepiditoga spiralis (TsAgo). Like MpAgo, TsAgo has a preference for 5'OH-gRNA. Meanwhile, TsAgo has DNA and RNA cleavage activity in presence of Mn2+ and Mg2+, and TsAgo has catalytic activity at 37-70 degrees C. In addition, TsAgo can tolerate mismatches in the 5'-end and 3' -tail regions of guides but is sensitive to mismatches in the 5'-seed and central regions of guides, espe-cially the central region. Furthermore, the EMSA assay reveals that TsAgo exhibits a stronger binding affinity for 5'OH-gRNA than 5'P-gRNA which is consistent with its cleavage activity. Moreover, the structural modeling analysis demonstrates that like MpAgo, TsAgo has an ordered a5 at the C terminus of the PIWI domain which may hinder to binding of 5' phosphate. Importantly, we find that TsAgo can target and cut plasmid DNA in vitro at 60 degrees C under the direction of RNA guides. These studies broaden our understanding of pAgos, and demonstrate that TsAgo can be regarded as an RNA-guided programmable nuclease for cleaving plasmids.(c) 2022 Elsevier Inc. All rights reserved.

Automated summary

In this study, a new Argonaute protein called TsAgo was identified from Tepiditoga spiralis. TsAgo, similar to other reported proteins, prefers 5'OH-gRNA. It has been shown that TsAgo has DNA and RNA cleavage activity, can tolerate mismatches in certain regions of guides, and exhibits a stronger binding affinity for 5'OH-gRNA. Importantly, TsAgo can function as an RNA-guided programmable nuclease to cut plasmid DNA at high temperatures.