ABSTRACT Argonaute (Ago) proteins are programmable nuclease found in both eukaryotes and prokaryotes. Prokaryotic Argonaute proteins (pAgos) share a high degree of structural homology with eukaryotic Argonaute proteins (eAgos) and eAgos are considered to evolve from pAgos. However, the majority of studied pAgos prefer to cleave DNA targets, and eAgos exclusively cleave RNA targets. Here, we characterize a novel pAgo, Mbp Ago, from psychrotolerant bacteria Mucilaginibacter paludis that can be programmed with DNA guides and prefers to cleave RNA targets rather than DNA targets. Mbp Ago can be active at a wide range of temperatures (4-65°C). In comparison with previously studied pAgos, Mbp Ago is able to utilize 16-nt long 5’phosphorylated and 5’hydroxylated DNA guides for efficient and precise cleavage and displays no obvious preference for the 5’end nucleotide of a guide. Furthermore, the cleavage efficiency can be regulated by mismatches in the central and 3’supplementary regions of the guide. Mbp Ago can efficiently cleave highly-structured RNA targets using both 5’phosphorylated and 5’hydroxylated DNA guides in the presence of Mg 2+ or Mn 2+ . In conclusion, we have demonstrated that Mbp Ago is a unique programmable nuclease that has a strong preference for RNA targets, with great potential applications in the field of nucleic acid biotechnology.

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