Studies On Methods For Nucleic Acid Detection Based On Pyrococcus Furiosus Argonaute Protein

As one of the main methods of molecular diagnosis,nucleic acid detection has played an important role in many different fields such as health care,environmental monitoring,biosecurity and food analysis.Therefore,it is important to develop nucleic acid diagnostic methods combining the sensitivity,specificity with the ease and cost effectiveness.In recent years,a series of highly sensitive and specific nucleic acid detection technologies based on CRISPR/Cas(Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system)have made remarkable progress in clinical diagnosis.However,the application of CRISPR/Cas is restricted due to use RNA as guide and the reliance on specific sequence,such as protospacer-adjacent motif(PAM)or Protospacer flanking site(PFS).Analogous to CRISPR/Cas,Argonaute proteins(Agos)are also a kind of nucleic acid-guided endonuclease.PfAgo(Pyrococcus furiosus Argonaute)protein,as one of the prokaryotic Argonaute,uses short phosphorylated DNA guides(>15 nt)to cut DNA target between the 10^th and 11^thnucleotides of the duplex,counting from the 5'end of the guide DNA.This study found that the cleaved ss DNA could be used as guide trigger the new cleavage.Based on this principle,we constructed a nucleic acid detection method with high sensitivity,high specificity,and multiplexed.The feasibility and application potential of this method was proved by the detection of mocked nucleic acid samples and clinical nucleic acid samples.Then,it was improved for detecting SARS-CoV-2.Meanwhile,we proposed ultralshort PCR and combined with PfAgo to develop a simple and rapid nucleic acid detection technology based on the effect of g DNA length on PfAgo digestion.Overview,this study established nucleic acid detection platform with high sensitivity,high specificity and high accuracy by exploring the enzymatic properties of PfAgo,provides a new method choice for molecular diagnosis as well as new idea for the application of Argonaute proteins.Details works are as follows:1.Expression,purification,and characterization of PfAgo protein.High purity PfAgo protein was obtained by heat treatment and Ni-NTA based on the high thermostability of PfAgo protein.It was found that the mismatch of single base between guide DNA(g DNA)and target DNA(t DNA)would significantly affect the cleavage efficiency of target DNA by PfAgo.Besides,the 3'-end protrusion of g DNA did not affect the cleavage activity of PfAgo,and 5'-end protrusion affected the cleavage efficiency and cleavage site of PfAgo.At the same time,we found that PfAgo protein could use the 5'-end ss DNA produced by cutting single-stranded DNA as a new g DNA to mediate the targeted cleavage of complementary ss DNA by PfAgo,indicating that the enzymatic cleavage reaction of PfAgo could be transmissible.2.Combining the transferable of PfAgo cleavage reaction and PCR technology to develop Pyrococcus furiosus Argonaute-mediated nucleic acid detection(PAND).Three g DNAs(input g DNA)targeting different positions of double-stranded DNA were designed to mediate the cleavage of PfAgo.A single-stranded DNA released from the double-stranded DNA target can be used as a new g DNA(generated g DNA)to bind to PfAgo protein,and cleave the corresponding molecular beacon thereby producing significant fluorescence signals.This method could reach attomolar sensitivity,single-base specificity,and five-channel multiplexed detection.The detection of simulated and clinical samples proved the potential application of this method in the detection of 16s r DNA,drug resistance mutations of Mycobacterium tuberculosis and hepatitis B virus,single nucleotide polymorphism of BRAC1 gene,circulating tumor DNA,and multi-subtype typing of human papillomavirus(HPV).3.Developing PfAgo-based detection of SARS-CoV-2(SARS-CoV-2 PAND)combined with reverse transcription PCR and PfAgo cleavage reaction.By screening the best PfAgo/g DNA complex,established a molecular diagnosis platform with high sensitivity and specificity for detecting SARS-CoV-2 by single input g DNA,the limit of detection was 1 copy per reaction.The using time of the Real-time fluorescence quantitative PCR instrument was shortened,hence the shortage of the expensive Real-time PCR instrument is alleviated.The diagnostic results of nucleic acid extracts from throat swabs of 44 suspected patients infected with the novel coronavirus were consistent with quantitative reverse transcription PCR(RT-q PCR).At the same time,D614G mutation of 36 positive samples was also tested,which proved that SARS-CoV-2 PAND could be used to detect the mutation of SARS-CoV-2.4.Nucleic acid detection via combination of ultrashort PCR and Pyrococcus furiosus Argonaute(USPCRP).On the basis that the cleavage activity of PfAgo could only be facilitated by g DNA longer than 15 nt,the amplification product of ultrashort PCR initiated by phosphorylated short primers was used as g DNA to guide PfAgo to cleave molecular beacon for DNA detection.It was proved that this method had 100 a M detection sensitivity and sequence specificity.The HPV16E6 gene and SARS-CoV-2 ORF1ab gene of clinical samples were successfully detected by this method,which proved that this method could be applied to clinical diagnosis.