Study On The Properties Of An Argonaute Protein From Monosporascus Cannonballus

As a programmable nuclease,Argonaute（Ago）protein widely distributed in eukaryotes and prokaryotes.The eukaryoticAgo protein plays a key role in the RNA interference pathway,and can recognize and even cleave the complementary RNA under the guidance of small RNA or small DNA.ProkaryoticAgo protein may be involved in host defense and DNA replication,and prefer to cut complementary DNA under the guidance of small DNA.TheAgo protein has been applied in nucleic acid detection and genetic manipulation.In order to further expand the nucleic acid manipulation tools based on theAgo protein,we have retrieved a kind ofAgo protein（McAgo）from the fungus Monosporascus cannonballus through bioinformatics methods.The amino acid sequence of McAgo is less than 25%identical to the reported sequence ofAgo proteins,but it has a conservative catalytic tetrad DEDD.It is speculated that McAgo may be a new activeAgo protein.The McAgo gene was optimized by E.coli codons and cloned into the p ET28a vector.In order to further purify the McAgo protein,a CL7 tag was fused to the N-terminus of the McAgo gene and a high-purity McAgo protein was obtained by the IM7-CL7 purification method.Nucleic acid extraction experiments show that McAgo binds 20-33 nt small RNA like mostAgo proteins.Activity assay experiments show that McAgo can use 5’-end phosphorylated or hydroxylated RNA as a guide to cleave complementary RNA,and cleave the complementary target at a site between the10 and 11 nucleotides starting from the 5′end of the guide.McAgo requires divalent metal ions to activate cleavage,and it works best under Mg²⁺conditions.In addition,McAgo can effectively cleave target RNA at a sodium chloride concentration of 50-750 m M and a p H of 6.8-8.8.McAgo has no obvious preference for the first base at the 5’end of the guide RNA（g RNA）;McAgo can cleave RNA in the range of 30-61°C,and the optimum temperature is 42°C;Circular dichroism analysis shows that the thermal stability of the binary complex formed by g RNA and McAgo is improved compared with empty McAgo,and the melting temperature（Tm）is increased from 48.8℃to 58.5℃;McAgo can use 12-30 nt length of g RNA to cleave the target RNA,and the maximum cleave efficiency is when the length of g RNA is 20 nt and 21 nt;McAgo has single-base specificity and is a high-conversion number enzyme.In summary,McAgo has potential applications,such as using McAgo as the core to develop high-sensitivity nucleic acid detection methods,and as a tool for RNA level regulation,etc.The discovery of McAgo protein and the exploration of biochemical properties provide new applications based onAgo protein.