CN114561374A - Novel thermophilic endonuclease mutant and preparation method and application thereof - Google Patents

Abstract

The invention provides a mutant of a thermophilic nuclease Argonaute (PfAgo) protein, and a preparation method and application thereof. Compared with a wild type, the enzymatic activity of the Ago mutant is obviously improved, the Ago mutant has better high-temperature thermal stability, and the mutant has better distinguishing capability on the selectivity of a target DNA substrate for wild and mutant bases, so that the application potential of the Ago mutant in the technical field of nucleic acid detection is improved, and a foundation is laid for developing a new gene operation tool.

Description

Novel thermophilic endonuclease mutant and preparation method and application thereof

Technical Field

The invention belongs to the technical field of protein engineering, and particularly relates to a mutant of Pyrococcus furiosus PfAgo, in particular to a mutant with improved enzyme activity, improved thermal stability and improved substrate specificity, and a preparation method and application thereof.

Background

The argonaute (ago) protein, an important component of the RNA-induced silencing complex (RISC), plays a very crucial role in the cleavage and recruitment of small RNA molecules, and is a protein family that is of great interest following the discovery of RNA interference (RNAi). At present, the Argonaute protein is widely present in eukaryote and prokaryote, has different functions in the eukaryote and the prokaryote, and plays an important role in forming a RISC complex as the Ago protein mainly participates in the RNAi process in the eukaryote; in prokaryotes, Ago protein mainly plays a host defense function, participates in a defense mechanism of bacteria for invasion of foreign genes and in replication and damage repair of hosts.

In vitro cleavage experiments by TtAgo demonstrated that prokaryotes pAgos can use ssDNA as guide to target ssDNA or RNA (target strand) for targeted cleavage. The Ago protein is firstly combined with guide-DNA to form a binary complex, wherein an MID domain of the Ago protein serves as a5 'end binding site of the guide, a PAZ domain serves as a 3' end binding site, then the guide and the target are subjected to complementary base pairing to target the target DNA to form a guide-Ago-target ternary complex, then a PIWI domain cuts between 10 th and 11 th bases of the guide, a target chain is broken, and finally the guide and the cut target are released through an N domain. Pfago is also a DNA-guided endonuclease that can directionally cleave single-stranded DNA targets under ultra-high temperature conditions.

Since the event of 2016 Han spring rain, the application of Ago proteins was gradually explored by numerous biologists. Thereafter, in 2017-2021, several Ago proteins were characterized in succession, such as Cpago/Ibago in the normal temperature Ago protein, Cbago/LrAgo extracted from intestinal microorganisms. As various Ago proteins were characterized, several applications were followed. The von anser professor group (the group of subjects) in 2018 to 2019 developed sequentially using PfAgo, radar (renewed gda Assisted DNA clean with Argonaute) and MULAN (multiple array-based Nucleic acid detection) technologies for multiple pathogen Nucleic acid detection, and a-STAR (Ago-directed Specific Target expression and detection) technologies for tumor-associated snvs (single nucleotide variants) enrichment detection, and the nano-rod research group developed PAND Nucleic acid detection technology in 2019. In 2020, the Oost team invented NAVIGATER nucleic acid specificity enrichment technology by using TtAgo, and in the same year, the subject group invented LAMP-RADAR virus nucleic acid detection technology, one-tube device design and reaction tube design. In 2021, Huimin Zhao et al developed a SPOT (scalable and Portable) technology based on Pfago that detects COVID-19 quickly, accurately, and portably.

Similar to the CRISPR-Cas9 and CRISPR-Cas12a/13a enzymes now commonly used, pAgos has also been suggested for use as a next generation genome editing tool. However, well-studied TtAgo, PfAgo and MjAgo are not well suited for gene editing due to their thermophilicity (optimum activity temperature ≧ 65 ℃) and low levels of endonuclease activity at corresponding temperatures from 20 to 37 ℃.

Compared with the normal-temperature NgAgo, CbAgo and the like, the application prospects of TtAgo and PfAgo at high temperature are wider at present, and the biggest defect of Ago protein is that the Ago protein cannot open a DNA double chain and does not have a unwinding function compared with a CRISPR/Cas system, which is the most important reason why Ago protein cannot be applied to gene editing. However, the DNA can open double strands by itself under high temperature, which allows the Ago protein to act as a cleavage function of nuclease, and thus, the nucleic acid detection technology is available.

However, the current nucleic acid detection technologies developed by using Pfago protein all find that the sensitivity and time of detection are limited due to the low catalytic activity of the enzyme. Therefore, there is an urgent need in the art to develop a method that can obtain PfAgo mutants with high catalytic activity. And the modification of catalytic activity of Pfago enzyme will further reveal the relationship between the structure and the function.

Disclosure of Invention

The present invention aims to provide mutants of Pyrococcus furiosus PfAgo nuclease, particularly mutants having high enzymatic activity and mutants having improved base discrimination specificity for wild-type and mutant genes.

In a first aspect of the invention there is provided a thermophilic nuclease argonaute (Ago) mutein characterized in that the Ago mutein has a core amino acid mutation at one or more positions selected from the group consisting of:

(Z1)I656Y/S/C

(Z2) Y743L/M/F; and/or

(Z3)I569S/T/C/Y；

Wherein the numbering of the mutation sites is based on the sequence shown in SEQ ID NO. 1;

and the Ago mutein has activity of directed cleavage of a single-stranded DNA target.

In another preferred embodiment, the Ago mutein further has a core amino acid mutation at one or more positions selected from the group consisting of:

(Z4)A573K/H/Q

(Z5)F769Y/R

(Z6) F747P/I/W; and/or

(Z7)L626P/N/V，

Wherein the numbering of the mutation sites is based on the sequence shown in SEQ ID NO. 1.

In another preferred example, the Ago mutein has an activity of directionally cleaving a single-stranded DNA target under hyperthermophilic conditions under guidance of guide DNA.

In another preferred example, the Ago mutein has an activity of directionally cleaving a single-stranded DNA target under ultra-high temperature conditions.

In another preferred embodiment, the ultra-high temperature condition is 80-99.9 ℃, preferably 90-99.9 ℃, more preferably 94-96 ℃, more preferably 95 ℃.

In another preferred embodiment, said Ago mutein is derived from the wild-type thermophilic nuclease Argonaute.

In another preferred embodiment, the protein is derived from the endonuclease Argonaute from any one of the species selected from the group consisting of: the organisms of the genus Thermococcus (Thermococcus eurythermalis), Methanococcus jannaschii (Methanococcus luteus), Methanococcus jannaschii (Methanococcus jannaschi), Pyrococcus furiosus (Pyrococcus furiosus), one of the genera of Thermotogaceae (Marinicola piphilus), Aquifex aeolicus (Aquifex aeolicus), Clostridium butyricum (Clostridium butyricum), Clostridium perfringens (Clostridium perfringens), Thermomyces thermophilus (Thermus thermophilus), Bacillus salina (Natronobacter gregoryi), Enterobacter minimus (Enterobacter bartlettii), Marioticus (Kurthia maliensis), Synechococcus elongatus (Synechococcus elongatus).

In another preferred example, the mutein is a mutein based on the thermophilic nuclease Argonaute (Pfago, wild-type sequence shown in SEQ ID NO: 1) of Pyrococcus furiosus.

In another preferred example, the Ago mutein further comprises an active fragment, variant or derivative thereof, said active fragment, variant or derivative of Ago having the Y743F and/or I569Y mutation, having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity compared to said Ago mutein, and having activity for directed cleavage of a single-stranded DNA target under hyperthermal conditions.

In another preferred embodiment, the Ago mutein-derived protein has the same or essentially the same sequence as shown in SEQ ID NO. 1, except for one or more of the core amino acid mutations.

In another preferred embodiment, the ratio Q1/Q0 of the enzyme activity Q1 of the Ago mutein to the enzyme activity Q0 of the wild type is more than or equal to 1.2, preferably more than or equal to 1.5, more preferably more than or equal to 2.0, and most preferably more than or equal to 4.0.

In another preferred embodiment, the ratio Q1/Q0 is 1.0-8.0, preferably 2.0-6.5.

In another preferred embodiment, the enzymatic activity is an activity of targeted cleavage of a single-stranded DNA target.

In another preferred embodiment, the Ago mutein has double core amino acid mutations selected from the following sites:

I656Y and Y743L;

I656S and I569C;

I656Y and F769R;

Y743M and F747P;

I659Y and L626N;

Y743L and I569S;

F747W and F769Y;

a573K and F769R;

a573H and F747I;

I569Y and Y743F;

I569Y and F747W;

Y743F and L626V;

I569Y and L626V;

Y743F and F747W; or

L626V and F747W,

wherein the numbering of the mutation sites is based on SEQ ID NO 1.

In another preferred embodiment, said Ago mutein has the following core amino acid mutations compared to SEQ ID NO:1 (wild type):

I656S and I569C;

I656Y and F769R;

Y743M and F747P;

I659Y and L626N;

Y743L and I569S;

I569Y and Y743F;

I569Y and F747W;

Y743F and L626V;

wherein the numbering of the mutation sites is based on SEQ ID NO 1.

In another preferred example, said Ago mutein has 3 or 4 amino acid mutations selected from the group consisting of Z1-Z7 as compared to SEQ ID NO:1 (wild type):

(Z1)I656Y/S/C；

(Z2)Y743L/M/F；

(Z3)I569S/T/C/Y；

(Z4)A573K/H/Q；

(Z5)F769Y/R；

(Z6)F747P/I/W；

(Z7)L626P/N/V.

wherein the numbering of the mutation sites is based on SEQ ID NO 1.

In another preferred embodiment, the sequence of the Ago mutein is selected from the group consisting of:

(X1) the amino acid sequence shown in SEQ ID NO:1, and the I mutation at position 656 is Y, S, or C (I656Y/S/C);

(X2) the amino acid sequence shown in SEQ ID NO:1, and the Y at position 743 is mutated to L, M or F (Y743L/M/F);

(X3) the amino acid sequence shown in SEQ ID NO:1, and the I mutation at position 569 is S, T, C or Y (I569S/T/C/Y);

(X4) the amino acid sequence shown in SEQ ID NO:1, and the A mutation at position 573 is K, H or Q (A573K/H/Q);

(X5) the amino acid sequence shown in SEQ ID NO:1, and F at position 769 is mutated to Y or R (F769Y/R);

(X6) the amino acid sequence shown in SEQ ID NO:1, and the F mutation at the 747 position is P, I or W (F747P/I/W);

(X7) the amino acid sequence shown in SEQ ID NO:1, and the L mutation at position 626 is P, N or V (L626P/N/V);

(DX1) the amino acid sequence shown in SEQ ID NO:1, and the I mutation at position 656 is Y (I656Y) and the Y mutation at position 743 is L (Y743L);

(DX2) the amino acid sequence shown in SEQ ID NO:1 with the I mutation at position 656 to S (I656S) and the I mutation at position 569 to C (I569C);

(DX3) the amino acid sequence shown in SEQ ID NO:1, and the I mutation at position 656 is Y (I656Y) and the F mutation at position 769 is R (F769R);

(DX4) the amino acid sequence shown in SEQ ID NO:1, and the Y mutation at position 743 is M (Y743M) and the F mutation at position 747 is P (F747P);

(DX5) the amino acid sequence shown in SEQ ID NO:1, with the I mutation at position 659 being Y (I659Y) and the L mutation at position 626 being N (L626N);

(DX6) the amino acid sequence shown in SEQ ID NO:1, and the I mutation at position 569 is S (I569S) and the Y mutation at position 743 is L (Y743L);

(DX7) the amino acid sequence shown in SEQ ID NO:1, and F mutation at position 747 is W (F747W) and F mutation at position 769 is Y (F769Y);

(DX8) the amino acid sequence shown in SEQ ID NO:1, and the I mutation at position 569 is Y (I569Y) and the L mutation at position 626 is V (L626V);

(DX9) the amino acid sequence shown in SEQ ID NO:1, and the Y mutation at position 743 is F (Y743F) and the I mutation at position 569 is Y (I569Y);

(DX10) the amino acid sequence shown in SEQ ID NO:1, wherein the I mutation at the 569 th position is Y (I569Y) and the F mutation at the 747 th position is W (F747W)

In a second aspect of the invention, there is provided an isolated polynucleotide encoding an Ago mutein of the first aspect of the invention.

In a third aspect of the invention, there is provided a vector comprising the isolated polynucleotide of the second aspect of the invention.

In another preferred embodiment, the vector is an expression vector.

In a fourth aspect of the invention, there is provided a host cell comprising a vector according to the third aspect of the invention or comprising an isolated polynucleotide according to the third aspect of the invention in a nucleic acid of said host cell.

In another preferred embodiment, the host cell includes cells derived from the following microorganisms:

saccharomyces cerevisiae (Saccharomyces cerevisiae), Pichia pastoris (Pichia pastoris), Saccharomyces mansonii (Saccharomyces monasynensis), Saccharomyces bayanus (Saccharomyces bayanus), Saccharomyces pastorianus (Saccharomyces pastorianus), Saccharomyces carbergensis (Saccharomyces carbergensis), Schizosaccharomyces pombe (Saccharomyces pombe), Kluyveromyces marxianus (Kluyveromyces marxiamus), Kluyveromyces lactis (Kluyveromyces lactis), Kluyveromyces fragilis (Kluyveromyces fragilis), Pichia stipitis (Pichia stipitis), Candida shehatae (Candida shehatae), Candida tropicalis (Candida tropicalis), and Escherichia coli (Escherichia coli).

In another preferred embodiment, the host cell comprises Saccharomyces cerevisiae, Pichia pastoris, or myceliophthora thermophila.

In another preferred embodiment, the host cell expresses Ago muteins.

In a fifth aspect of the invention, there is provided a method of preparing an Ago mutein according to the first aspect of the invention, comprising the steps of:

culturing a host cell according to the fourth aspect of the invention under conditions suitable for expression, thereby expressing an Ago mutein according to the first aspect of the invention; and

isolating the expression product, thereby obtaining the Ago mutein according to the first aspect of the invention.

In a sixth aspect of the present invention, there is provided a nucleic acid cleavage system comprising:

(a) a guide dna (gdna) that targets binding to a predetermined target site; and

(b) a programmable endonuclease, argonaute (Ago), wherein the programmable endonuclease is an Ago mutein according to the first aspect of the invention.

In another preferred embodiment, the nucleic acid cleavage system further comprises:

(c) a reporter nucleic acid, wherein the reporter nucleic acid is cleaved by the Ago mutein and detected when the nucleic acid cleavage system is mixed with the nucleic acid molecule to be detected.

In another preferred embodiment, the reporter nucleic acid carries a modifying group.

In another preferred embodiment, the reporter nucleic acid does not carry a modifying group.

In another preferred embodiment, the guide DNA is a single-stranded DNA molecule phosphorylated at the 5' end.

In another preferred embodiment, the guide DNA has a length of 5 to 30nt, more preferably 15 to 21nt, and most preferably 16 to 18 nt.

In another preferred embodiment, the temperature of the nucleic acid cleavage system is 80 to 99.9 ℃, preferably 90 to 99.9 ℃, and more preferably 95 to 99.9 ℃. (the higher the temperature, the higher the cutting efficiency, and only 99.9 ℃ can be measured at present.)

In another preferred embodiment, the guide DNA is a 5' terminal phosphateAcidification, hydroxylation at the 5' end, Biotin group at the 5' end, NH at the 5' end₂C₆The radical, 5 'having a FAM group or 5' having an SHC group₆Single stranded DNA molecules of the gene (both PfAgo wild type and mutant can only utilize phosphorylated guide).

In another preferred embodiment, the guide DNA is a single-stranded DNA molecule phosphorylated at the 5' end.

In another preferred embodiment, the guide DNA has a reverse complementary fragment to the reporter nucleic acid.

In another preferred embodiment, the guide DNA has a length of 5 to 30nt, more preferably 15 to 21nt, and most preferably 16 to 18 nt.

In another preferred embodiment, the nucleotide sequence of the guide DNA is shown in SEQ ID NO 3. (in the following table, guide sequence and target sequence)

target DNA and guide DNA sequence

In another preferred embodiment, the reporter nucleic acid is single-stranded dna (ssdna).

In another preferred embodiment, the endonuclease, Argonaute, is capable of cleaving at the site of the reporter nucleic acid bound to position 10-11 from the 5' end of the gDNA.

In another preferred embodiment, when the reporter nucleic acid is cleaved, the cleavage can be detected by electrophoresis.

In another preferred embodiment, the electrophoresis is a 16% nucleic acid Urea-PAG electrophoresis detection method.

In another preferred embodiment, the reporter nucleic acid is a fluorescent reporter nucleic acid with a fluorophore and/or a quencher.

In another preferred embodiment, the fluorescent group and the quencher group are independently located at the 5 'end and the 3' end of the fluorescent reporter nucleic acid.

In another preferred embodiment, the fluorophore and the quencher are located on both sides of the complementary region of the fluorescent reporter nucleic acid and the guide DNA, respectively.

In another preferred embodiment, the fluorescent reporter nucleic acid is 10 to 100nt, preferably 20 to 70nt, more preferably 30 to 60nt, more preferably 40 to 50nt, most preferably 45nt in length.

In another preferred embodiment, the fluorescent group comprises: FAM, HEX, CY5, CY3, VIC, JOE, TET, 5-TAMRA, ROX, Texas Red-X, or a combination thereof.

In another preferred embodiment, the quenching group comprises: BHQ, TAMRA, DABCYL, DDQ, or combinations thereof.

In another preferred embodiment, the fluorescent reporter nucleic acid is a single-stranded DNA molecule having only a fluorophore, which is FAM.

In another preferred embodiment, the nucleic acid cleavage system further comprises: (d) a divalent metal ion.

In another preferred embodiment, the divalent metal ion is Mn²⁺。

In another preferred embodiment, the concentration of the divalent metal ion in the nucleic acid cleavage system is 10. mu.M-3 mM, preferably 50. mu.M-2 mM, more preferably 100. mu.M-2 mM.

In another preferred embodiment, the nucleic acid cleavage system further comprises: (e) and (4) buffering the solution.

In another preferred embodiment, the concentration of NaCl in the buffer is 500mM or less, preferably 20 to 500 mM.

In another preferred embodiment, the pH of the buffer is 7-9, preferably 8.0.

In another preferred embodiment, said significantly reducing the cleavage rate of the programmable endonuclease Argonaute is: under the same reaction condition, the shearing rate of the programmable endonuclease Argonaute is reduced by more than or equal to 80 percent, preferably reduced by more than or equal to 85 percent, and more preferably reduced by more than or equal to 90 percent.

In another preferred embodiment, the guide DNA is complementary to the sequence of the fluorescent reporter nucleic acid and directs the Ago enzyme to cleave the fluorescent reporter nucleic acid, thereby generating a detectable signal (e.g., fluorescence).

In another preferred embodiment, the concentration of the fluorescent reporter nucleic acid in the nucleic acid cleavage system is 0.4. mu.M-4. mu.M, preferably 0.6. mu.M-2. mu.M, more preferably 0.8. mu.M-1. mu.M, and most preferably 0.8. mu.M.

In another preferred embodiment, the concentration of the programmable endonuclease Ago is 10nM to 10. mu.M, preferably 100nM to 1. mu.M, more preferably 300nM to 500nM, and most preferably 400nM in the nucleic acid cleavage system.

In another preferred embodiment, the concentration of the guide DNA in the nucleic acid cleavage system is 10nM to 10. mu.M, preferably 100nM to 3. mu.M, more preferably 1. mu.M to 2.5. mu.M, and most preferably 2. mu.M.

In a seventh aspect of the present invention, a reaction system for enriching low-abundance target nucleic acid is provided, wherein the reaction system is used for simultaneously performing Polymerase Chain Reaction (PCR) and nucleic acid cleavage reaction on a nucleic acid sample, thereby obtaining an amplification-cleavage reaction product;

wherein the nucleic acid sample comprises a first nucleic acid and a second nucleic acid, wherein the first nucleic acid is the target nucleic acid and the second nucleic acid is a non-target nucleic acid;

the nucleic acid cleavage reaction is used for specifically cleaving non-target nucleic acids but not cleaving the target nucleic acids;

the amplification-cleavage reaction system comprises (i) reagents required for performing a PCR reaction and (ii) the nucleic acid cleavage system according to the sixth aspect of the present invention.

In another preferred embodiment, the reaction system contains gDNA, which comprises forward gDNA and reverse gDNA;

wherein, the forward gDNA is gDNA having the same sequence segment as the target nucleic acid, and the reverse gDNA is gDNA having a reverse complementary sequence segment with the target nucleic acid.

In another preferred embodiment, the concentration of the Ago mutein in the reaction system is 20 to 200nM, preferably 30 to 150nM, more preferably 40 to 100 nM.

In another preferred embodiment, the reagents required for performing the PCR reaction further comprise amplification primer pairs for the target nucleic acid.

In another preferred embodiment, the concentration of each primer in the amplification primer pair of the target nucleic acid is 100-300nM, preferably 150-250nM, and more preferably 200 nM.

In another preferred embodiment, the concentration of the target nucleic acid is 0.5-5nM, preferably 0.8-2nM, more preferably 1 nM.

In another preferred embodiment, the gDNA comprises forward gDNA and reverse gDNA;

wherein, the forward gDNA is gDNA having the same sequence segment as the target nucleic acid, and the reverse gDNA is gDNA having a reverse complementary sequence segment with the target nucleic acid.

In another preferred embodiment, the reaction system further comprises: a divalent metal ion.

In another preferred embodiment, the divalent metal ion is Mn²⁺。

In another preferred embodiment, the concentration of the divalent metal ion in the reaction system is 50mM-2M, preferably 100mM-1M, more preferably 0.5 mM.

In another preferred example, the reaction temperature (reaction procedure) of the reaction system is: 94 ℃ for 5 min; the number of cycles is 10-30(94 ℃, 30 s; 52 ℃, 30 s; 72 ℃, 20 s); 72 ℃ for 1 min.

In an eighth aspect of the present invention, there is provided a method for enriching a low-abundance target nucleic acid, comprising the steps of:

(a) providing a nucleic acid sample containing a first nucleic acid and a second nucleic acid, wherein the first nucleic acid is the target nucleic acid and the second nucleic acid is a non-target nucleic acid,

and the abundance of the target nucleic acid in the nucleic acid sample is F1 a;

(b) performing Polymerase Chain Reaction (PCR) and nucleic acid cleavage reaction in an amplification-cleavage reaction system by taking the nucleic acid in the nucleic acid sample as a template, thereby obtaining an amplification-cleavage reaction product;

wherein the nucleic acid cleavage reaction is used to specifically cleave non-target nucleic acids but not the nucleic acid of interest;

and, the amplification-cleavage reaction system comprises (i) reagents required for performing a PCR reaction and (ii) the nucleic acid cleavage system according to the sixth aspect of the present invention;

wherein the abundance of the target nucleic acid in the amplification-cleavage reaction product is F1b,

wherein the ratio of F1b/F1a is not less than 10.

In another preferred embodiment, the target nucleic acid and the non-target nucleic acid differ by only one base.

In another preferred embodiment, the target nucleic acid and the non-target nucleic acid differ by only one base.

In another preferred embodiment, the ratio of F1b/F1a is not less than 10 when F1a is not less than 1% and not more than 10%, the ratio of F1b/F1a is not less than 100 when F1a is not less than 0.5%, and the ratio of F1b/F1a is not less than 200 when F1a is not more than 0.1%.

In another preferred embodiment, the nucleic acid sample includes a directly heat-cleaved nucleic acid sample, a directly lyase protease-treated nucleic acid sample, an extracted nucleic acid sample, a PCR pre-amplified nucleic acid sample, or any nucleic acid-containing sample.

In another preferred embodiment, the PCR-preamplified nucleic acid sample is PCR amplification products obtained by 1-30 cycles, preferably 10-20 cycles, and more preferably 15-30 cycles.

In another preferred embodiment, the target nucleic acid is a nucleotide sequence containing mutations.

In another preferred embodiment, the mutation is selected from the group consisting of: insertions, deletions, substitutions of nucleotides, or combinations thereof.

In another preferred embodiment, the non-target nucleic acid (or second nucleic acid) is a wild-type nucleotide sequence, a highly abundant nucleotide sequence, or a combination thereof.

In another preferred embodiment, the abundance of said non-target nucleic acid in said nucleic acid sample is F2 a.

In another preferred example, F1a + F2a is 100%.

In another preferred embodiment, the ratio of F2a/F1a is 20 or more, preferably 50 or more, more preferably 100 or more, most preferably 1000 or more or 5000.

In another preferred embodiment, the abundance of said non-target nucleic acid in said amplification-cleavage reaction product is F2 b.

In another preferred example, F1b + F2b is 100%.

In another preferred embodiment, F1b/F2b is 0.5 or more, preferably 1 or more, more preferably 2 or more, most preferably 3 or 5 or more.

In another preferred embodiment, the ratio F1b/F1a is 200 or more, preferably 500 or more, more preferably 1000 or more, most preferably 2000 or more or 5000 or more.

In another preferred embodiment, F1a is 0.5% or less, preferably 0.2% or less, more preferably 0.1% or less, and most preferably 0.01% or less.

In another preferred embodiment, F1b is 10% or more, preferably 30% or more, more preferably 50% or more, most preferably 70% or less.

In another preferred embodiment, the "reagents required for performing PCR reaction" include: a DNA polymerase.

In another preferred embodiment, the "reagents required for performing PCR reaction" further include: dNTP, 1-5Mm Mg²⁺And PCR buffer solution.

In another preferred embodiment, the gDNA in the nucleic acid cleavage system forms a first complementary binding region with the nucleic acid sequence of the targeted region of the target nucleic acid (i.e., the first nucleic acid); and the gDNA in the nucleic acid cleavage system also forms a second complementary binding region with the nucleic acid sequence of the targeted region of the non-target nucleic acid (i.e., the second nucleic acid).

In another preferred embodiment, there are at least 2 mismatched base pairs in the first complementary binding region.

In another preferred embodiment, there are 0 or 1 mismatched base pairs in the second complementary binding region.

In another preferred embodiment, there are 1 mismatched base pairs in the second complementary binding region.

In another preferred embodiment, there are at least 2 mismatched base pairs in the first complementary binding region, resulting in the complex not cleaving the target nucleic acid; and 1 mismatched base pair in the second complementary binding region, thereby causing the complex to cleave the non-target nucleic acid.

In another preferred embodiment, the targeting region of the target nucleic acid (i.e., the first nucleic acid) corresponds to the targeting region of a non-target nucleic acid (i.e., the second nucleic acid).

In another preferred embodiment, in the amplification-cleavage reaction system, the nucleic acid cleavage tool enzyme is 30nM, and the DNA polymerase is a high temperature resistant polymerase, preferably Taq DNA polymerase, LA Taq DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, Phusion DNA polymerase, KOD DNA polymerase, etc., more preferably 2X PCR Precision^TM Master Mix。

In another preferred embodiment, the amount of the nucleic acid as a template in the amplification-cleavage reaction system is 0.1 to 100 nM.

In another preferred example, the method further comprises:

(c) detecting the amplification-cleavage reaction product, thereby determining the presence, absence, and/or amount of the target nucleic acid.

In another preferred embodiment, the detection in step (c) comprises quantitative detection, qualitative detection, or a combination thereof.

In another preferred embodiment, the quantitative determination is selected from the group consisting of: TaqMan fluorescent quantitative PCR, Sanger sequencing, q-PCR, ddPCR, chemiluminescence method, high-resolution melting curve method, NGS and the like; preferably selected from TaqMan fluorescent quantitative PCR, Sanger sequencing.

In another preferred embodiment, the first nucleic acid comprises n different nucleic acid sequences, wherein n is a positive integer ≧ 1.

In another preferred embodiment, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more.

In another preferred embodiment, n is 2 to 1000, preferably 3 to 100, more preferably 3 to 50.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In another preferred embodiment, said nucleic acid sample comprises nucleic acids from a test sample, wherein said test sample is selected from the group consisting of: blood, cells, serum, saliva, body fluids, plasma, urine, prostatic fluid, bronchial lavage, cerebrospinal fluid, gastric fluid, bile, lymph fluid, peritoneal fluid, stool, and the like, or combinations thereof.

In a ninth aspect of the invention, there is provided a kit for detecting a target nucleic acid molecule, the kit comprising:

(i) the thermophilic nuclease argonaute (ago) mutein of the first aspect of the invention; and (ii) instructions for use.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a schematic diagram of the structure of a mutation site;

FIG. 2 shows SDS-PAGE electrophoresis of wild-type Pfago and mutants;

FIG. 3 shows a target-sheared nucleic acid gel plot of wild-type Pfago and mutant;

FIG. 4 shows a graph of substrate cleavage efficiency versus time for wild-type PfAgo and mutant;

FIG. 5 shows a schematic diagram of the detection principle of Pfago enzyme activity;

FIG. 6 shows an enzyme map of wild-type Pfago and mutants;

FIG. 7 shows the thermostability profiles of wild-type PfAgo and mutant;

FIG. 8 shows histograms of differential splicing ability of wild-type Pfago and mutants for DNA substrate wild-type and mutant genes;

FIG. 9 shows an oncogene detection map of wild-type Pfago and mutants;

fig. 10 shows pathogen detection maps of wild-type PfAgo and mutants.

Detailed Description

The present inventors have, through extensive and intensive studies, unexpectedly obtained, for the first time, a protein having a mutant form significantly improved in Ago enzyme activity through extensive screening. Specifically, the activity of the single-stranded DNA target subjected to directional shearing is remarkably improved by the mutated Ago protein, and the activity can be improved by 50% -650%, so that the assistance is provided for pathogen detection, genotyping, disease course monitoring and the like. The present invention has been completed based on this finding.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur.

As used herein, the terms "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …" or "consisting of …".

"transduction," "transfection," "transformation" or terms as used herein refer to the process of delivering an exogenous polynucleotide into a host cell, transcription and translation to produce a polypeptide product, including the introduction of the exogenous polynucleotide into the host cell (e.g., E.coli) using a plasmid molecule.

"Gene expression" or "expression" refers to the process of transcription, translation and post-translational modification of a gene to produce the RNA or protein product of the gene.

"Polynucleotide" refers to a polymeric form of nucleotides of any length, including Deoxynucleotides (DNA), Ribonucleotides (RNA), hybrid sequences thereof, and the like. Polynucleotides may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs. The term polynucleotide as used herein refers to interchangeable single-and double-stranded molecules. Unless otherwise indicated, a polynucleotide in any of the embodiments described herein includes a double-stranded form and two complementary single strands that are known or predictable to make up the double-stranded form.

Conservative amino acid substitutions are known in the art. In some embodiments, the potential substituted amino acids are within one or more of the following groups: glycine, alanine; and valine, isoleucine, leucine and proline; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine lysine, arginine and histidine; and/or phenylalanine, tryptophan and tyrosine; methionine and cysteine. In addition, the invention also provides non-conservative amino acid substitutions that allow amino acid substitutions from different groups.

Those skilled in the art will readily understand the meaning of all parameters, dimensions, materials and configurations described herein. The actual parameters, dimensions, materials and/or configurations will depend upon the specific application for which the invention is being used. It will be understood by those skilled in the art that the embodiments or claims are given by way of example only and that within the scope of equivalents or claims, the embodiments of the invention may be covered without limitation to the specifically described and claimed scope.

All definitions, as defined and used herein, should be understood to exceed dictionary definitions or definitions in documents incorporated by reference.

All references, patents, and patent applications cited herein are hereby incorporated by reference with respect to the subject matter to which they are cited, and in some cases may contain the entire document.

It should be understood that for any method described herein that includes more than one step, the order of the steps is not necessarily limited to the order described in these embodiments.

The term "LAMP" is a Loop-mediated isothermal amplification (Loop-mediated isothermal amplification) technique, which is an isothermal nucleic acid amplification technique suitable for gene diagnosis.

The term "PCR" is a Polymerase chain reaction technique (Polymerase chain reaction), a technique suitable for rapid amplification of target nucleic acids.

As used herein, the term "secondary cleavage" refers to the cleavage of a target nucleic acid sequence by an Ago enzyme of the invention in the presence of primary guide ssDNAs in the detection methods of the invention, the cleavage product forming novel 5' phosphorylated nucleic acid sequences (secondary guide ssDNAs); the secondary guide ssDNA then continues to direct the PfAgo enzyme to cleave the target fluorescent reporter nucleic acid complementary to the secondary guide ssDNAs under the action of the PfAgo enzyme. This specific cleavage of the fluorescent reporter nucleic acid (second cleavage), followed by specific cleavage of the target nucleic acid sequence (first cleavage), is defined as "second cleavage". In the present invention, both the first cleavage and the second cleavage are specific cleavage.

Ago mutant protein

As used herein, the terms "mutein of the invention", "programmable endonuclease Argonaute mutant of the invention", "Ago enzyme mutant of the invention", "Ago mutein of the invention", "Ago nuclease mutant of the invention" are used interchangeably and refer to the mutein described in the first aspect of the invention.

Generally, the preferred working temperature for the mutant Ago enzymes of the invention is 95 ± 2 degrees.

As used herein, the terms "programmable endonuclease Pyrococcus furiosus", "nuclease Pyrococcus furiosus", "PfAgo enzyme", "PfAgo protein" are used interchangeably.

In the invention, the wild type Ago enzyme with the amino acid sequence shown as SEQ ID NO. 1 is subjected to mutation of a specific site to obtain a corresponding mutant with obviously improved activity. As used herein, the terms "mutein", "mutant" are used interchangeably and refer to an Ago mutein.

As used herein, in describing mutations, the term "I656Y/S/C" is used as an example to refer to a mutation of I at position 656 to Y or S or C based on the sequence shown in SEQ ID NO:1 (wild type). Similarly, "Y743L/M/F" means that Y at position 743 is mutated to L or M or F. Other mutations are described in a similar manner.

The amino acid sequence of the wild Pfago enzyme is shown as SEQ ID NO: 1:

MKAKVVINLVKINKKIIPDKIYVYRLFNDPEEELQKEGYSIYRLAYENVGIVIDPENLIIATTKELEYEGEFIPEGEISFSELRNDYQSKLVLRLLKENGIGEYELSKLLRKFRKPKTFGDYKVIPSVEMSVIKHDEDFYLVIHIIHQIQSMKTLWELVNKDPKELEEFLMTHKENLMLKDIASPLKTVYKPCFEEYTKKPKLDHNQEIVKYWYNYHIERYWNTPEAKLEFYRKFGQVDLKQPAILAKFASKIKKNKNYKIYLLPQLVVPTYNAEQLESDVAKEILEYTKLMPEERKELLENILAEVDSDIIDKSLSEIEVEKIAQELENKIRVRDDKGNSVPISQLNVQKSQLLLWTNYSRKYPVILPYEVPEKFRKIREIPMFIILDSGLLADIQNFATNEFRELVKSMYYSLAKKYNSLAKKARSTNEIGLPFLDFRGKEKVITEDLNSDKGIIEVVEQVSSFMKGKELGLAFIAARNKLSSEKFEEIKRRLFNLNVISQVVNEDTLKNKRDKYDRNRLDLFVRHNLLFQVLSKLGVKYYVLDYRFNYDYIIGIDVAPMKRSEGYIGGSAVMFDSQGYIRKIVPIKIGEQRGESVDMNEFFKEMVDKFKEFNIKLDNKKILLLRDGRITNNEEEGLKYISEMFDIEVVTMDVIKNHPVRAFANMKMYFNLGGAIYLIPHKLKQAKGTPIPIKLAKKRIIKNGKVEKQSITRQDVLDIFILTRLNYGSISADMRLPAPVHYAHKFANAIRNEWKIKEEFLAEGFLYFV(SEQ ID No:1)

specifically, the muteins of the present invention generally refer to core amino acid mutations engineered to have one or more sites selected from the group consisting of:

(Z1)I656Y/S/C

(Z2) Y743L/M/F; and/or

(Z3)I569S/T/C/Y；

Wherein the numbering of the mutation sites is based on the sequence shown in SEQ ID NO. 1;

and the Ago mutein has activity of directed cleavage of a single-stranded DNA target.

In addition, the Ago muteins of the present invention also have amino acid mutations at other positions, as long as the amino acid mutations do not result in loss or significant reduction of the activity of the muteins of the present invention. Preferably, other amino acid mutations include (but are not limited to):

(Z4)A573K/H/Q

(Z5)F769Y/R

(Z6) F747P/I/W; and/or

(Z7)L626P/N/V，

In view of the teachings of the present invention and the prior art, it will be appreciated by those skilled in the art that the muteins of the present invention also include active fragments, modified or unmodified variants, or derivatives thereof, of the muteins.

Specifically, the active fragment, variant or derivative protein of Ago mutein includes: the amino acid sequence formed by the mutation of Z1 and/or Z2 in the sequence shown in SEQ ID NO. 1 further has one or more (e.g. usually 1-30, preferably 1-10, more preferably 1-6, still more preferably 1-3, most preferably 1) amino acid residues deleted, inserted and/or substituted, still has a targeted single-stranded DNA cleaving activity (cA1) which is significantly higher than the corresponding activity (cA0) of the wild-type Ag enzyme shown in SEQ ID NO. 1, and significantly higher than any value of (cA1-cA0)/cA0 ≥ 10% -800%, such as ≥ 15%, ≧ 20%, ≧ 40%, ≧ 50%, ≧ 100%, ≧ 200%, or ≥ 500% or more.

Conservative variations of the mutants can be made by one skilled in the art by conservative amino acid substitutions, for example as shown in the following table.

As used herein, modified (typically without altering primary structure) forms of proteins include: chemically derivatized forms of the protein such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or to optimize solubility. These techniques are known to those skilled in the art.

In addition, the mutant protein derived proteins also include the mutant protein or its active fragments and other proteins or markers formed by fusion protein or conjugates.

The mutant protein of the invention can also contain one or more other mutations, thereby further improving the enzyme cutting activity of the Ago mutant protein.

Specifically, in one embodiment of the invention, to center on the catalytic residues of the enzyme active site, the surrounding is selected based on the crystal structure of the enzyme or homologously modeled structure aided by co-evolutionary analysis

And (3) taking the amino acid residues within as target sites, and screening through saturation mutation to obtain mutants with improved enzyme catalytic activity. The active center refers to the structural region near the enzyme-catalytic residue, as distinguished from surface residues of complex structures.

In the invention, the distance PfAgo catalytic tetranector is selected

The amino acids inside the DNA sequence are aided by coevolution analysis to determine mutation hot spots. The final selected mutation hot spots are 26 in total, and comprise positions G556, P561, M562, K563, R564, S565, I569, G570, G571, S572, A573, V586, M600, F603, L626, I631, D654, V655, I656, P740, H742, Y743, F747, N749, R752 and F769.

Furthermore, the inventors screen these candidate sites, preferably high throughput screening, including primary screening of crude enzyme solution of 96-well plate by fluorescence method, rescreening of crude enzyme solution of 96-well plate by fluorescence method, and rescreening of pure enzyme by fluorescence method.

Typically, the primary screening and secondary screening of the crude enzyme solution of the 96-well plate by the fluorescence method comprise the following steps: taking one hole from the edge and the center of each 96-well plate to culture wild type PfAgo as a control, reacting for 15min at 90 ℃ on a Q-PCR instrument, measuring the activity of the ssDNA substrate of the shearing fluorescence label, and taking the enzyme activity which is 50 percent higher than that of the wild type as a screening standard, and screening the selected clone mutant by a next 96-well plate; transferring the mutant with improved activity into a 96-deep-well plate for culturing, performing three repetitions in each well, reacting for 15min at 90 ℃ on a real-time fluorescence detector, determining the activity of the ssDNA substrate of the shearing fluorescence label, and performing the next step of rescreening of pure enzyme by using the mutant with enzyme activity higher than that of the wild type by 50 percent as a screening standard; the rescreening of the fluorescent purified enzyme comprises the following steps: sequencing the mutant with improved enzyme activity, purifying by using a protein purifier, determining the concentration of the mutant by using a BSA method, and preserving at-80 ℃ after diluting to 0.5 mg/mL.

Screening results show that seven single-point mutants of L626P/N/V, I656Y/S/C, A573K/H/Q, F747P/I/W, F769/769Y/R, I569/569S/T/C/Y, Y743L/M/F can unexpectedly improve the catalytic activity of PfAgo.

In another preferred embodiment, the inventors further obtain a mutant with higher enzyme catalytic activity by combining the mutants with the screened enzyme catalytic activity. Double-point combined mutants I656Y and Y743L with further improved activities are obtained by carrying out double-point combined mutation on activity-improving sites L626P/N/V, I656Y/S/C, A573K/H/Q, F747P/I/W, F769Y/R, I569S/T/C/Y, Y743L/M/F; I656S and I569C; I656Y and F769R; Y743M and F747P; I659Y and L626N; Y743L and I569S; F747W and F769Y; a573K and F769R; a573H and F747I; I569Y and Y743F; I569Y and F747W; Y743F and L626V; I569Y and L626V; Y743F and F747W; L626V and F747W.

Coding nucleic acids and combinations thereof

The invention also provides, on the basis of the Ago muteins of the present invention, isolated polynucleotides encoding the Ago muteins or degenerate variants thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the nucleotide sequence encoding the PfAgo mutein of the present embodiments or degenerate variants.

As used herein, "degenerate variant" refers in the present invention to a nucleic acid sequence that encodes an Ago mutein of the first aspect of the invention, but differs from the nucleotide sequence encoding the Ago mutein in the embodiments of the invention.

Vectors, host cells

The encoding polynucleotide sequence may be inserted into a recombinant expression vector or genome. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector well known in the art. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

The skilled person can use well known methods to construct expression vectors comprising DNA sequences encoding pyruvate carboxylase muteins and/or malate transporter muteins and appropriate transcription/translation control signals, including in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

The host cells described herein include host cells comprising the above-described expression vectors or having integrated into their genome the coding sequence for the muteins of the present invention. Host cells include those derived from ascomycetes hosts, which have a very high evolutionary relationship and have a high similarity in the sequence and functionality of pyruvate carboxylase and malate transporter proteins, and preferred microbial cells are as follows:

saccharomyces cerevisiae (Saccharomyces cerevisiae), Pichia pastoris (Pichia pastoris), Saccharomyces mansonii (Saccharomyces monasynensis), Saccharomyces bayanus (Saccharomyces bayanus), Saccharomyces pastorianus (Saccharomyces pastorianus), Saccharomyces carbergensis (Saccharomyces carbergensis), Schizosaccharomyces pombe (Saccharomyces pombe), Kluyveromyces marxianus (Kluyveromyces marxiamus), Kluyveromyces lactis (Kluyveromyces lactis), Kluyveromyces fragilis (Kluyveromyces fragilis), Pichia stipitis (Pichia stipitis), Candida shehatae (Candida shehatae), Candida tropicalis (Candida tropicalis), and Escherichia coli (Escherichia coli).

The mutant protein of the present invention can be produced by a recombinant transformation method which is conventional in the art, and the mutant protein can be expressed in a cell, or on a cell membrane, or secreted out of the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell disruption by osmosis, sonication, high-pressure homogenization, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof. The host cell of the invention can express the pyruvate carboxylase mutant protein and the malic acid transporter mutant protein respectively and/or simultaneously.

Preparation of muteins

The invention also provides a method of making an Ago mutein comprising culturing a host cell of the invention under conditions suitable for expression, thereby expressing the Ago mutein; and

isolating the Ago mutein.

The resulting mutant protein can also optionally be purified, thereby obtaining a purer mutant protein product.

Preferably, the conditions suitable for expression include techniques conventional in the art, and purification techniques include nickel column purification, ion exchange chromatography, and the like.

Guide ssDNA

In the detection method of the present invention, one core component is guide ssDNA, especially 2 ssDNA, and are adjacent to each other without intervening bases or intervening sequences.

In the present invention, preferred guide ssDNAs are oligonucleotides each having a length of 10 to 60nt, preferably 10 to 40nt, and more preferably 13 to 20nt, and the first 5' nucleotide is thymine (T) which may be modified by phosphorylation.

Reporter nucleic acid molecule

In the detection method of the present invention, one core component is a reporter nucleic acid carrying a reporter molecule.

In a preferred embodiment, the reporter nucleic acid molecule of the invention is a nucleic acid molecule carrying a fluorescent group and a quencher group, respectively. For example, a fluorescent group (F) is labeled at the 5 'end, and a quencher group (Q) is labeled at the 3' end.

In the present invention, the fluorescent reporter nucleic acid molecule is determined based on the location of the generation of the secondary-guided ssDNAs; the target nucleic acid sequence is cleaved by the primary guide ssDNAs to form a new 5' phosphorylated nucleic acid sequence, referred to as secondary guide ssDNAs, with the fluorescent reporter nucleic acid covering all positions of the secondary guide ssDNAs.

Detection method

The present invention also provides a nucleic acid detection method based on a gene-editing enzyme Ago, such as Pyrococcus furiosus Argonaute (Pfago).

In the method of the present invention, based on the cleavage activity of PfAgo, 3 pairs of guide ssdnas can be designed according to a target nucleic acid molecule (such as single-stranded DNA, preferably amplified target nucleic acid molecule), and these 3 pairs of guide ssdnas target different target nucleic acid molecules and mediate PfAgo to cleave the target nucleic acid molecule to form new secondary guide ssdnas. And the secondary guide ssDNA continues to guide the Pfago enzyme to shear the fluorescent reporter nucleic acid which is complementary with the secondary guide ssDNAs under the action of the Pfago enzyme, so that the detection of the target nucleic acid molecule corresponding to the fluorescent reporter nucleic acid is realized. The method of the invention can greatly improve the sensitivity and the multiplicity of the detection of the target nucleic acid.

In the invention, according to the design requirements of the guided ssDNAs, the Pfago enzyme can selectively cut the nucleic acid sequences with partial site difference through special design, thereby realizing typing detection.

In the invention, when the method is used for distinguishing different types, mutation sites corresponding to different types are arranged at the 10 th and 11 th positions of the guide ssDNAs when the guide ssDNAs are designed, and due to the selection specificity of the pfAgo enzyme, the shearing activity can be inhibited when two continuous points are mutated, thereby achieving the detection of different types.

In the invention, multiple target nucleic acid molecules and guided ssDNAs can be simultaneously added into a shearing system of Pfago enzyme, and reporter nucleic acids with different fluorescent groups are combined, so that multiple detection of target nucleic acids can be achieved.

The method of the present invention is very suitable for detecting trace nucleic acids. By combining reverse transcription LAMP and guide ssDNA with a specific sequence, the invention can detect target nucleic acid molecules with the concentration of the nucleic acid template as low as (100 copies/ml) and can stably detect the target nucleic acid molecules with the low concentration of the nucleic acid template (1000 copies/ml).

In the present invention, the amplification primers used in the amplification reaction usually have a Tm value of about 65. + -.10 degrees, and the amplified fragment size is about 90 to 200 bp. Preferably, the amplification primers are designed to avoid the segment to be detected.

Reagent kit

The invention also provides a detection method for the target nucleic acid molecule.

In a preferred embodiment, the method of the invention comprises: (a) amplification reagents for amplifying a target nucleic acid molecule, the amplification reagents comprising: a primer pair for amplifying a target nucleic acid molecule, the primer pair for performing a specific amplification reaction based on the target nucleic acid molecule, thereby producing a specific nucleic acid amplification product;

(b) a cleavage reagent or a cleavage buffer comprising said cleavage reagent, wherein said cleavage reagent comprises: 3 pairs of guide ssDNA, gene editing enzyme (Ago), and a first reporter nucleic acid, said fluorescent reporter nucleic acid carrying a fluorophore and a quencher, and said 3 pairs of guide ssDNA targeting 3 different target nucleic acid molecules.

Applications of

The invention also provides application of the Ago nuclease mutant in gene detection.

The mutant protein is particularly suitable for detecting trace target nucleic acid molecules and multiple detections, and has wide applicability.

In the present invention, the target nucleic acid molecule may be DNA or RNA. When the target nucleic acid molecule is RNA, it can be converted to cDNA by reverse transcription and then detected.

The mutant obtained by screening can exert higher catalytic activity and base distinguishing specificity at high temperature. Meanwhile, the mutant can be better applied to the nucleic acid detection technology, so that the application potential of the mutant in the nucleic acid detection industry is greatly improved, and a foundation is laid for developing a new gene operation tool.

The main advantages of the present invention include:

(1) compared with the wild type, the enzyme catalytic activity of the mutant type obtained by screening is obviously improved and can reach at least 6.5 times to the maximum;

(2) the thermal stability of the mutant is not affected by mutation, and the very good thermal stability is kept;

(3) the mutant of the invention also improves the base distinguishing specificity of the target nucleic acid wild type and mutant type genes;

(4) the mutant of the invention has application potential in nucleic acid detection technology and gene editing tool development technology.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The experimental materials referred to in the present invention are commercially available without specific reference.

In the present invention, all sequence numbers refer to specific sequences without any modifications.

Example 1 construction and Synthesis of wild type Pyrococcus furiosus Pfago plasmid vector and selection of mutation site

Searching PfAgo wild type nucleotide sequence in NCBI database, synthesizing by Kinsley biology company, after codon optimization, selecting pET28a (+) vector, selecting NdeI and XhoI double enzyme cutting sites, transforming the plasmid product synthesized by the company into escherichia coli Ecoli-BL21(DE3) competence, coating a flat plate (containing 50 ug/ml kanamycin), inverting at 37 ℃ for overnight culture, picking single colony on the next day, sequencing to verify whether the plasmid sequence is correct, and extracting the plasmid after the plasmid is aligned to be correct. The plasmid extraction procedure was as described in the specification for the Axygen plasmid miniprep kit. Plasmid concentration was determined by Nano-300.

The Wild Type (WT) amino acid sequence of the Argonaute protein (Pfago) of Pyrococcus furiosus is shown as SEQ ID No. 1.

The crystal structure of PfAgo was searched in the PDB database, and structure 1U04 with the highest resolution was selected as the study object. And then, docking the guide strand DNA and the target strand DNA into a Pfago crystal structure by using molecular docking software Amber to simulate a ternary compound structure. The periphery was analyzed centered on catalytic tetranected DEDH in selecting target sites

All amino acid residues within, and aided by co-evolutionary analysis. Amino acid residues that both correspond were selected as target sites (see table 1). The specific positions of these target amino acid sites in the structure are shown in FIG. 1. Subsequently, single point saturation mutagenesis was performed at all amino acid positions in table 1.

TABLE 1 distance of active center target site to catalytic residue D558

Example 2 construction of Gene mutation library by site-directed saturation mutagenesis

The 22 c-tribk method was used to design primer sequences to construct a library of saturating mutations. Compared with the traditional NNK method, the method effectively reduces codon redundancy and screening amount of mutants. Carrying out single-point saturation mutation on 26 amino acid sites in the table 1, and specifically comprising the following steps:

the whole plasmid PCR amplification was carried out using a wild-type gene of Pfago as a template by PrimeSTAR MAX Premix (2X) DNA polymerase (TaKaRa Co.), and the PCR product was verified by DNA agarose gel electrophoresis. The PCR system is shown below:

reaction system	50μL
		PrimeSTAR MAX Premix(2X)	25μL
10μM Forward Primer mix	2μL
		10μM Reverse Primer mix	2μL
WT Tempelate(20ng/μL)	1μL
		ddH2O	up to 50μL

The PCR amplification procedure was: each cycle comprises 20 cycles of denaturation at 98 ℃ for 10s, annealing at 55-70 ℃ for 15s and extension at 72 ℃ for 130 s.

The PCR product was digested to remove the template plasmid, and 50. mu.L of the digestion system was as follows:

reaction system	50μL
		PCR product	44μL
10×FastDigest Buffer	5μL
		Dpn I	1μL

Digestion was carried out for 2h at 37 ℃ in a 50. mu.L digestion system. The digested PCR product was then subjected to clean purification. Adding purified sample 10 μ L to 100 μ L E.coli BL21(DE3) competent cells, standing on ice for 30min, then heat-shocking in 42 deg.C water bath for 90s, standing on ice for 2min, adding 800 μ L of non-resistant LB culture medium, placing in shaker at 37 deg.C, culturing for 1h, uniformly spreading the culture solution on solid LB plate containing 50mg/ml Kana resistance, and culturing at 37 deg.C in a constant temperature incubator for overnight in an inverted manner to obtain the strain containing mutant gene. 3-5 monoclonals are picked from each plate and sent to sequencing to verify whether the saturated mutation library is correctly built.

A series of mutant strains are obtained by the method, and the amino acid sequences after mutation are shown as SEQ ID NOs: 2-27.

Example 3 screening of mutant libraries

300. mu.L of LB liquid medium containing 50. mu.g/mL Kan was added to each well of a 96-well plate, and the single colonies on the LB solid plate were picked one by one using a sterilized toothpick into the 96-well plate, and one well was taken at the edge and center of each 96-well as WT-PfAgo control, and cultured overnight at 37 ℃ and 400 rpm. The next day, 600. mu.L LB medium containing 50. mu.g/mL Kan was added to the new round of deep well plates, 50. mu.L of seed solution was transferred to the new deep well plates, and glycerol was added to the original deep well platesStoring at-80 deg.C; culturing the transferred new round of deep-hole plate at 37 ℃ and 400rpm for 2-3h to OD₆₀₀To 0.6-0.8; after cooling in a refrigerator at 4 ℃ for 1h, IPTG was added to a final concentration of 0.5mM, and the mixture was incubated at 18 ℃ and 400rpm for 20 to 22 h. Centrifuging at 4 deg.C/4000 rpm for 20min, pouring out supernatant, and repeatedly freezing and thawing in-80 deg.C ultra-low temperature refrigerator for 3 times (3 hr each time, 30min thawing in 37 deg.C incubator). After completion of freeze-thawing, 50. mu.L (250mM NaCl, 15mM Tris-HCl pH 8.0) of Reaction Buffer was added to each well, the cells were resuspended, and shaken for 30min on a micro-shaker to mix well. Each well sucks a certain amount of crude enzyme solution and adds the enzyme solution into a 96-well plate. And reacting the ssDNA substrate modified by fluorescence for 30min at 90 ℃, 495nm excitation wavelength and 520nm emission wavelength, and detecting a fluorescence signal value in real time. The enzyme activity is measured by the fluorescence signal value, and a monoclonal site with higher catalytic activity than WT is selected.

Results

The enzyme activity of some mutant proteins with single point mutation is basically unchanged or slightly reduced, such as M562F/L/T, S565D/E, F603D/Q/C, D654H/F, N749T/L.

The data of the activity improvement of the enzyme is shown in the table below, and 7 single-point mutants with improved enzyme activity, namely L626P/N/V, I656Y/S/C, A573K/H/Q, F747P/I/W, F769Y/R, I569S/T/C/Y, Y743L/M/F, are obtained through screening.

Preferred 7 single point mutants are shown in the table below.

Subsequently, the single-point mutants with improved enzyme activity are subjected to double-point combined mutation to obtain double-point combined mutants I656Y and Y743L with further improved activity; I656S and I569C; I656Y and F769R; Y743M and F747P; I659Y and L626N; Y743L and I569S; F747W and F769Y; a573K and F769R; a573H and F747I; I569Y and Y743F; I569Y and F747W; Y743F and L626V; I569Y and L626V; Y743F and F747W; L626V and F747W. The data of the increase in enzyme activity are shown in the following table.

Example 4 expression and purification of Pyrococcus furiosus Pfago nuclease wild-type and mutant proteins

And (3) recovery and activation: the E.coli BL21(DE3) -pET28a-PfAgo wild type and mutant strains were inoculated at 1% inoculum size into 50mL LB liquid medium (containing kanamycin) in small shake flasks at 220r/min and 37 ℃ for overnight culture.

Transferring: the overnight-cultured bacterial liquid was transferred to a 1L flask containing LB liquid medium (containing kanamycin) at an inoculum size of 1%, cultured at 220r/min, and cultured at 37 ℃ to OD₆₀₀The value reaches 0.8-1.0;

inducing after ice impact: taking out the shake flask from the shaking table, placing the shake flask on ice for cold shock for 15min, then adding IPTG with the final concentration of 0.5mM, inducing and expressing the mixture for 20 h-22 h at 18 ℃ at 220r/min, and centrifuging and collecting thalli.

Since the N-terminus of the target gene is designed to have a 6 XHis tag, it can be purified directly by Ni-affinity chromatography. The cells were resuspended in a resuspension buffer (containing 20mM Tris-HCl, pH8.0, 1M NaCl), then disrupted under high pressure, and centrifuged to obtain a supernatant. And (3) carrying out affinity purification on the protein by using a Ni-NTA column, and carrying out ultrafiltration concentration, desalination and the like on the eluent to obtain the purified protein. The purity of the purified protein was verified by SDS-PAGE gel chromatography as shown in FIG. 2. The purified protein was stored in a buffer containing 20mM Tris-HCl and the protein was assayed by BCA kit, the assay procedure was performed according to the protocol. Preparing a standard solution by taking BSA as a standard substance, drawing a standard curve, calculating the concentration of the purified target protein according to the standard solution, and storing the protein in a refrigerator at the temperature of-80 ℃ for later use.

Example 5 determination of cleavage Activity of Pyrococcus furiosus Pfago nuclease wild type and mutant

A target nucleic acid with 60nt single-stranded DNA and a complementary 16nt single-stranded DNA guide strand are designed and sent to the company for synthesis.

Reaction buffer (containing 15mM Tris-HCl pH8.8, 250mM NaCl) was prepared, and MnCl was added to the reaction buffer to a final concentration of 0.5mM₂100nM PfAgo, 2. mu.M synthesized gDNA and 0.8. mu.M complementary single-stranded DNA target nucleic acid, reacted at 95 ℃ for 0, 1, 3, 5, 7, 10, 15min, after the reaction, 6-10. mu.L of sample was taken, loading buffer (containing 95% (deionized) formamide, 0.5mmol/L EDTA, 0.025% bromophenol blue, 0.025% xylene blue) was added at a ratio of 1:1, and electrophoretic detection was performed under 16% nucleic acid Urea-PAGE.

The gel electrophoresis results are shown in fig. 3, and the grey scale value quantitative calculation of the protein gel Image by using Image J software is shown in fig. 4, which is a graph of the substrate DNA target shear efficiency.

The results show that the mutant has significantly higher shearing activity than the wild type after reacting for different times.

Example 6 determination of specific Activity of Pyrococcus furiosus Pfago nuclease

The reaction principle is shown in FIG. 5, and the reaction for detecting the activity of Pfago nuclease is in the reaction buffer (15mM Tris-HCl pH8.8, 250mM NaCl, 0.5mM MnCl)₂4. mu.M target DNA, 8. mu.M guide DNA).

target DNA and guide DNA sequence

The reaction procedure is as follows: reacting at 95 ℃ for 15min under the conditions of an excitation wavelength of 495nm and an emission wavelength of 520nm, collecting fluorescence signals, and repeating the steps for three times for each sample. The enzyme specific activity was calculated according to the following formula (formula 1). The enzyme activity unit (U) is defined as: the amount of enzyme required to catalyze cleavage of 0.1nM substrate per minute in the above reaction system. (Note: one unit of enzyme activity is represented by U.)

A (U/mg): the specific activity of the enzyme; c_p(μm): refers to the product concentration at time t; t (min): the time required to reach the T moment; c (mg/mL) enzyme concentration; v (mL): the volume of enzyme added; the experimental data were averaged for three replicates.

The results of the enzyme activity determination of the wild type and the mutant of Pfago nuclease are shown in FIG. 6.

Example 7 thermostability and Enzymokinetic parameters of Pyrococcus furiosus PfAgo nuclease wild type and mutants

In order to determine the change of the thermostability of the mutant, the present invention measured the change of the enzyme activity of each mutant protein after incubation at 95 ℃ for various periods of time, as in example 5, and the results are shown in FIG. 7.

The kinetic parameters were measured by measuring the maximum initial reaction rates at substrate concentrations of 0.4, 0.8, 1.2, 1.6, 2.0, 4.0, 6.0, and 8.0. mu.M at pH8.0 and a temperature of 95 ℃. Fitting a correlation curve by means of software Graphpad 8.0 and the Mie's equation (equation 2) to obtain K_mAnd V_maxThen, according to the function equation (formula 3) of the maximum rate and the enzyme concentration, the kinetic constant K is calculated_m、K_catAnd K_cat/K_m。

V＝V_max[S]/(K_m+[S]) (formula 2)

V_max＝K_cat×[E](formula 3)

Kinetic parameters of Pfago wild type and mutant (see Table 2) and a thermal stability map (see FIG. 7) were obtained by the determination method of the above method. The results show that the mutant k of Pfago nuclease_cat/K_mThe improvement is 9 times, but the thermal stability is kept unchanged.

TABLE 2 PfAgo wild type and mutant kinetic parameters

Example 8 differential cleavage of Pfago wild-type and mutant genes for DNA substrates

In order to verify the differential shearing capability of the PfAgo nuclease mutant of the invention on the wild type and mutant genes of a DNA substrate, the invention designs probe substrates (sequences are shown in Table 3) of the wild type and mutant genes, wherein the 5 'end and the 3' end of the wild type probe are respectively modified by FAM and BHQ1 fluorescent groups, the 5 'end and the 3' end of the mutant probe are respectively modified by VIC and BHQ1 fluorescent groups, and the experimental principle is shown in figure 3.

Corresponding guide DNA was designed for wild type and mutant probe substrates (sequences shown in Table 3). MnCl was added to the reaction buffer to a final concentration of 0.5mM₂200nM PfAgo, 4. mu.M synthesized gDNA and 2. mu.M single-stranded DNA wild-type and mutant gene probes were reacted at an excitation wavelength of 490nM and an emission wavelength of 550nM for 30 minutes, and the fluorescence at the end point was determined in triplicate for each experiment. A histogram is made using Graphpad 8.0 as shown in fig. 8.

The result shows that the mutant Pfago nuclease has better differential shearing capability on DNA substrate wild type and mutant genes.

TABLE 3 Probe sequences and leader chain sequences

Example 9 application of Pyrococcus furiosus PfAgo nuclease wild type and mutant in Gene detection

In order to verify the application of the PfAgo nuclease mutant in nucleic acid detection, the invention utilizes a 'low-abundance DNA mutation detection technology system' developed in a laboratory to verify the detection effect of the PfAgo nuclease mutant on oncogenes, and specific amplification primers, gDNAs and detection probe sequences are designed and screened according to the principle of the detection method aiming at the sequence characteristics of a KRAS-G12D gene fragment. Wherein the gDNAs, primers and probes were synthesized by Shanghai Bioengineering Co., Ltd. The 5' end of the gDNAs of the KRAS-G12D gene is provided with phosphorylation modification; the 5 'end of the nucleotide sequence of the mutant probe is provided with a VIC fluorescent label, and the 3' end is modified with a quenching group BHQ 1; FAM fluorescent label is arranged at the 5 'end of the nucleotide sequence of the KRAS wild type gene probe, and a quenching group BHQ2 is modified at the 3' end. The method adopts the standard substance from the cyanine good organism company to carry out verification analysis, and the mutation allele frequency (AF%) in the standard substance is 1% mut and 100% wt respectively. The enrichment detection effect of the Pfago nuclease mutant is verified by adopting 1% mut and 100% wt standard substance, and the result is shown in FIG. 9.

Meanwhile, in order to verify the application of the PfAgo nuclease mutant in the pathogen detection technology, the African swine fever (African swine virus strain BA71V) is detected by using the reverse transcription loop-mediated isothermal amplification reaction (reverse transcription LAMP) nucleic acid detection technology developed by the experiment. The detection method comprises the following specific operation steps:

(1) the amplification primer and the DNase/RNase free H for the gDNA dry powder₂Dissolving O to prepare 100 mu M storage solution; the fluorescent reporter nucleic acid dry powder uses DNase/RNase free H₂Dissolving O to obtain 10 μ M stock solution;

(2) preparing 25ul of amplification reaction premix solution by Bst enzyme and Ago enzyme digestion buffer solution amplification primers;

(3) adding 15 mu L of nucleic acid sample to be detected into the amplification reaction premixed solution, wherein the amplification reaction system is 40 mu L;

(4) preparing a 20 mu L enzyme digestion reaction system by using a Pfago mutant, an LAMP amplification buffer solution, gDNA and fluorescent reporter nucleic acid;

(5) and respectively transferring the amplification system and the enzyme digestion system into a PCR tube and a lining tube, and putting the PCR tube and the lining tube into a fluorescent quantitative PCR instrument for reaction (amplification is carried out for 30min at 62 ℃, enzyme digestion is carried out for 30min at 95 ℃, and a signal is detected every minute).

The detection results of the Pfago mutant on the pathogen African swine fever are shown in FIG. 10.

The result shows that the detection effect of the Pfago nuclease mutant on oncogenes and pathogens is faster and more accurate and the enrichment effect is better than that of the wild type.

The foregoing is merely exemplary and illustrative of the present invention and it is within the purview of one skilled in the art to modify or supplement the embodiments described or to substitute similar ones without the exercise of inventive faculty, and still fall within the scope of the claims.

Sequence listing

<110> Shanghai university of transportation

Zenghong Biotechnology (Shanghai) Co Ltd

<120> novel thermophilic endonuclease mutant and preparation method and application thereof

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Claims (10)

1. A thermophilic nuclease Argonaute (Ago) mutein characterized in that the Ago mutein has a core amino acid mutation at one or more positions selected from the group consisting of:

(Z1)I656Y/S/C

(Z2) Y743L/M/F; and/or

(Z3)I569S/T/C/Y；

Wherein the numbering of the mutation sites is based on the sequence shown in SEQ ID NO. 1;

and the Ago mutein has the activity of directed cleavage of a single-stranded DNA target.

2. The Ago mutein according to claim 1, wherein the Ago mutein has a double core amino acid mutation selected from the group consisting of:

I656Y and Y743L;

I656S and I569C;

I656Y and F769R;

Y743M and F747P;

I659Y and L626N;

Y743L and I569S;

F747W and F769Y;

a573K and F769R;

a573H and F747I;

I569Y and Y743F;

I569Y and F747W;

Y743F and L626V;

I569Y and L626V;

Y743F and F747W; or

L626V and F747W,

wherein the numbering of the mutation sites is based on SEQ ID NO 1.

3. The Ago mutein according to claim 1, characterized in that the sequence of the Ago mutein is selected from the group consisting of:

(X1) the amino acid sequence shown in SEQ ID NO:1, and the I mutation at position 656 is Y, S or C (I656Y/S/C);

(X2) the amino acid sequence shown in SEQ ID NO:1, and the Y mutation at position 743 is L, M or F (Y743L/M/F);

(X3) the amino acid sequence shown in SEQ ID NO:1, and the I mutation at position 569 is S, T, C or Y (I569S/T/C/Y);

(X4) the amino acid sequence shown in SEQ ID NO:1, and the A mutation at position 573 is K, H or Q (A573K/H/Q);

(X5) the amino acid sequence shown in SEQ ID NO:1, and F769 position is mutated into Y or R (F769Y/R);

(X6) the amino acid sequence shown in SEQ ID NO:1, and the F mutation at the 747 position is P, I or W (F747P/I/W);

(X7) the amino acid sequence shown in SEQ ID NO:1, and the L mutation at position 626 is P, N or V (L626P/N/V);

(DX1) the amino acid sequence shown in SEQ ID NO:1, and the I mutation at position 656 is Y (I656Y) and the Y mutation at position 743 is L (Y743L);

(DX2) the amino acid sequence shown in SEQ ID NO:1 with the I mutation at position 656 to S (I656S) and the I mutation at position 569 to C (I569C);

(DX3) the amino acid sequence shown in SEQ ID NO:1, and the I mutation at position 656 is Y (I656Y) and the F mutation at position 769 is R (F769R);

(DX4) the amino acid sequence shown in SEQ ID NO:1, and the Y mutation at position 743 is M (Y743M) and the F mutation at position 747 is P (F747P);

(DX5) the amino acid sequence shown in SEQ ID NO:1, and the I mutation at position 659 is Y (I659Y) and the L mutation at position 626 is N (L626N);

(DX6) the amino acid sequence shown in SEQ ID NO:1, and the I mutation at position 569 is S (I569S) and the Y mutation at position 743 is L (Y743L);

(DX7) the amino acid sequence shown in SEQ ID NO:1, and F mutation at position 747 is W (F747W) and F mutation at position 769 is Y (F769Y);

(DX8) the amino acid sequence shown in SEQ ID NO:1, and the I mutation at position 569 is Y (I569Y) and the L mutation at position 626 is V (L626V);

(DX9) the amino acid sequence shown in SEQ ID NO:1, and the Y mutation at position 743 is F (Y743F) and the I mutation at position 569 is Y (I569Y);

(DX10) the amino acid sequence shown in SEQ ID NO:1, and the I mutation at the 569 th position is Y (I569Y) and the F mutation at the 747 th position is W (F747W).

4. An isolated polynucleotide encoding the Ago mutein of claim 1.

5. A vector comprising the isolated polynucleotide of claim 4.

6. A host cell comprising the vector of claim 5, or wherein the nucleic acid of the host cell comprises the isolated polynucleotide of claim 4.

7. A method of preparing the Ago mutein of claim 1 comprising the steps of:

culturing the host cell of claim 6 under conditions suitable for expression, thereby expressing the Ago mutein of claim 1; and

isolating the expression product, thereby obtaining the Ago mutein of claim 1.

8. A nucleic acid cleavage system, comprising:

(a) a guide dna (gdna) that targets binding to a predetermined target site; and

(b) a programmable endonuclease, Argonaute (Ago), wherein the programmable endonuclease is the Ago mutein of claim 1.

9. A reaction system for enriching low-abundance target nucleic acid, wherein the reaction system is used for simultaneously performing Polymerase Chain Reaction (PCR) and nucleic acid cleavage reaction on a nucleic acid sample, thereby obtaining an amplification-cleavage reaction product;

wherein the nucleic acid sample comprises a first nucleic acid and a second nucleic acid, wherein the first nucleic acid is the target nucleic acid and the second nucleic acid is a non-target nucleic acid;

the nucleic acid cleavage reaction is used for specifically cleaving non-target nucleic acids but not cleaving the target nucleic acids;

the amplification-cleavage reaction system comprises (i) reagents required for performing a PCR reaction and (ii) the nucleic acid cleavage system of claim 8.

10. A method for enriching a low abundance target nucleic acid, comprising the steps of:

(a) providing a nucleic acid sample containing a first nucleic acid and a second nucleic acid, wherein the first nucleic acid is the target nucleic acid and the second nucleic acid is a non-target nucleic acid,

and the abundance of the target nucleic acid in the nucleic acid sample is F1 a;

(b) performing Polymerase Chain Reaction (PCR) and nucleic acid cleavage reaction in an amplification-cleavage reaction system by taking the nucleic acid in the nucleic acid sample as a template, thereby obtaining an amplification-cleavage reaction product;

wherein the nucleic acid cleavage reaction is used to specifically cleave non-target nucleic acids but not the nucleic acid of interest;

and, the amplification-cleavage reaction system comprises (i) reagents required for performing a PCR reaction and (ii) the nucleic acid cleavage system of claim 8;

wherein the abundance of the target nucleic acid in the amplification-cleavage reaction product is F1b,

wherein the ratio of F1b/F1a is not less than 10.

Images