Study On Catalytic Properties Of Argonaute Nuclease From Verrucomicrobia Bacterium

Argonaute（Ago）proteins are conserved programmable nucleic acid enzymes that exist in eukaryotes and prokaryotes.Eukaryotic argonautes（e Agos）use guide RNA（g RNA）to cleave RNA targets and are key players in the RNA interference（RNAi）pathway.Most reported prokaryotic argonautes（p Agos）use guide DNA（g DNA）to cleave DNA targets and play a role in protecting hosts from foreign nucleic acid and cell replication.Recently,several RNA-targeting p Agos have been reported.Although the studies on these p Agos are beginning to be explored,the great application prospects in RNA targeting have been shown.To search for novel RNA-targeting p Agos,we selected candidate p Agos genes from the mesophilic bacteria by the web interface of the BLASTp program with the PIWI domain sequence of Km Ago as a query and tested the cleavage activity of the candidate p Agos.The experiment results show that VbAgo from Verrucomicrobia utilizes 5′-phosphorylated guide DNA（5′P-g DNA）and 5’-hydroxylated guide DNA（5′OH-g DNA）to specifically cleave RNA targets.This study explores the characteristics and catalytic properties of VbAgo.The research results are as follows:1.VbAgo utilizes 5′P-g DNA and 5′OH-g DNA to cleave RNA targets from 25℃ to 65℃,specifically cleaves RNA targets but not DNA targets at 37℃ and shows weak DNA targets cleavage activity above 45℃;2.VbAgo is sensitive to salt concentration,and the cleavage activity reduces when the concentration of Na Cl is≥100 m M;3.VbAgo cleaves RNA targets loaded with 12-44 nt 5′P-g DNA and 13-44 nt 5′OH-g DNA and the cleavage activity reaches the maximum with 15-16 nt g DNA;4.VbAgo requires Mn²⁺and Mg²⁺to cleave targets,and Mg²⁺is better than Mn²⁺;5.VbAgo presents a weak tolerance for mismatches between g DNAs and RNA targets.The single-nucleotide mismatch at positions 6、9、11-12 and dinucleotide mismatches at positions 3-15 remarkably reduce the efficiency of RNA targets cleavage;6.VbAgo shows no obvious preference to the first nucleotide of the guide and functions as a multiple-turnover enzyme;7.VbAgo uses 5′P-g DNA to cleave highly-structured RNA targets at 37℃ and 50℃;In summary,VbAgo specifically cleaves RNA targets at 37℃,shows no obvious preference to the first nucleotide of the guide and cleaves highly-structured RNA targets.These characteristics broaden our understanding of Ago protein.VbAgo may be applied to the RNA editing,and expends the p Agos-based RNA manipulation toolbox.