Study On The Activity Of PfAgo Cutting DsDNA In Vitro

The Argonaute of the archaeon Pyrococcus furiosus（PfAgo）can utilizes small5’-phosphorylated DNA guides to recognize and cleave DNA targets specifically.PfAgo is one of the most widely studied and applied prokaryotic Agos due to its excellent thermal stability.It was reported that PfAgo functions as a DNA-guided DNA endonuclease that requires divalent cations such as Mn²⁺or Co²⁺,but some p Agos with high homology to PfAgo can be active with Mg²⁺.Because of the closely relationship and similar catalytic site,it is assumed that PfAgo should also be active under a specific salt concentration and p H with Mg²⁺.In addition,Mg²⁺is the divalent cation that most polymerases depend on,so if we can find a buffer for PfAgo to be active with Mg²⁺and study the related properties,so that it can work with polymerases,we can expand its application to some extent.At the same time,Mn²⁺and Co²⁺are heavy metals and have serious toxicity to organisms,so if it can be active with Mg²⁺,PfAgo will have the potential to become a tool for gene editing.Therefore,we constructed PfAgo expression vector,expressed and purified PfAgo,obtained about 0.4 mg PfAgo for subsequent experiments and then tried various Mg²⁺-containing polymerase buffers,and identified Pfu Buffer that could make PfAgo active,and studied the properties of PfAgo cutting ds DNA under Pfu Buffer.We find that PfAgo can perform cutting activity with g DNA in the length range of 18-50nt,and the longer the guide,the lower the cutting efficiency;PfAgo needs phosphorylated g DNA to cut ds DNA;PfAgo can cut ss DNA above 65℃,lower temperature were not tested,while complete cutting of ds DNA could only be above 90℃due to ds DNA unstranding;different sites would slightly affect the cutting efficiency of PfAgo.In addition,PfAgo has the sensitivity to recognize single nucleotide mutations under both Mg²⁺and Mn²⁺conditions,the sensitivity is higher under Mn²⁺conditions.We further used PfAgo in conjunction with Pfu,the two can work together under Pfu Buffer to recognize templates of 300copies,which improves the PCR sensitivity,and expand the application of PfAgo for its better role in more aspects such as in vitro diagnosis and clone construction.