A programmable pAgo nuclease with RNA target preference from the psychrotolerant bacterium Mucilaginibacter paludis

Argonaute (Ago) proteins are programmable nucleases found in eukaryotes and prokaryotes. Prokaryotic Agos (pAgos) share a high degree of structural homology with eukaryotic Agos (eAgos), and eAgos originate from pAgos. Although eAgos exclusively cleave RNA targets, most characterized pAgos cleave DNA targets. This study characterized a novel pAgo, MbpAgo, from the psychrotolerant bacterium Mucilaginibacter paludis which prefers to cleave RNA targets rather than DNA targets. Compared to previously studied Agos, MbpAgo can utilize both 5 ' phosphorylated(5 ' P) and 5 ' hydroxylated(5 ' OH) DNA guides (gDNAs) to efficiently cleave RNA targets at the canonical cleavage site if the guide is between 15 and 17 nt long. Furthermore, MbpAgo is active at a wide range of temperatures (4-65 degrees C) and displays no obvious preference for the 5 '-nucleotide of a guide. Single-nucleotide and most dinucleotide mismatches have no or little effects on cleavage efficiency, except for dinucleotide mismatches at positions 11-13 that dramatically reduce target cleavage. MbpAgo can efficiently cleave highly structured RNA targets using both 5 ' P and 5 ' OH gDNAs in the presence of Mg2+ or Mn2+. The biochemical characterization of MbpAgo paves the way for its use in RNA manipulations such as nucleic acid detection and clearance of RNA viruses.

Automated summary

This study characterized a novel prokaryotic Ago protein, MbpAgo, from the psychrotolerant bacterium Mucilaginibacter paludis, which prefers cleaving RNA targets over DNA targets. MbpAgo is active at a wide range of temperatures and shows no obvious preference for the 5'-nucleotide of a guide.