WO2023169228A1 - Novel thermophilic endonuclease mutant, and preparation method therefor and application thereof - Google Patents

Abstract

The present invention provides a mutant of a thermophilic nuclease Argonaute (PfAgo) protein, and a preparation method for and an application of the mutant. The Ago mutant in the present invention has the enzyme catalytic activity remarkably improved compared with a wild type, and has better high-temperature thermal stability; moreover, the mutant has a better capability of distinguishing between wild and mutant bases in the target DNA substrate selectivity, thus, the application potential of the mutant in the technical field of nucleic acid tests is improved, and a foundation is laid for developing new genetic manipulation tools.

Description

A novel thermophilic endonuclease mutant and its preparation method and application Technical field

The invention belongs to the technical field of protein engineering and specifically relates to mutants of Pyrococcus furiosus PfAgo, specifically, obtaining mutants with improved enzyme activity, thermal stability and substrate specificity, and preparation methods and applications thereof.

Background technique

Argonaute (Ago) protein, as an important component of the RNA-induced silencing complex (RISC), plays a very critical role in the shearing and recruitment of small RNAs. It is a protein that has attracted much attention after the discovery of RNA interference (RNAi). protein family. It is currently found that Argonaute proteins are widely present in eukaryotes and prokaryotes, and have different functions in eukaryotes and prokaryotes. In eukaryotes, Ago proteins are mainly involved in the RNAi process and play an important role in the formation of the RISC complex. In prokaryotes, Ago protein mainly plays a host defense function, participating in the bacterial defense mechanism against foreign gene invasion and participating in host replication and damage repair.

The in vitro cleavage experiment of TtAgo proved that prokaryotic pAgos can use ssDNA as a guide (guide strand) to target ssDNA or RNA (target strand). The Ago protein first binds to the guide-DNA to form a binary complex, in which the MID domain of the Ago protein serves as the 5' end binding site of the guide, and the PAZ domain serves as the 3' end binding site, and then binds to the bases of the target through the guide Complementary pairing targets the target DNA to form a guide-Ago-target ternary complex, and then the PIWI domain cleaves between the 10th and 11th base of the guide, breaking the target chain, and finally releases the guide and the target through the N domain Cut off target. PfAgo is also a DNA-guided endonuclease that can directionally cleave single-stranded DNA targets under ultra-high temperature conditions.

Since the Han Chunyu incident in 2016, the applications of Ago proteins have been gradually discovered by many biologists. Since then, between 2017 and 2021, a variety of Ago proteins have been characterized, such as CpAgo/IbAgo among normal-temperature Ago proteins and CbAgo/LrAgo extracted from intestinal microorganisms. As multiple Ago proteins have been characterized, several applications have emerged. From 2018 to 2019, Professor Feng Yan's team (the team of this research group) used PfAgo to successively develop RADAR (Renewed gDNA Assisted DNA cleavage with Argonaute) and MULAN (MULtiplex Argonaute-based Nucleic acid detection) technologies for multiple pathogen nucleic acid detection, and In 2019, Ma Lixin's team developed the PAND nucleic acid detection technology for the A-STAR (Ago-directed Specific Target enrichment and detection) technology for enrichment detection of tumor-related SNVs (Single nucleotide variants). In 2020, the Oost team used TtAgo to invent the NAVIGATER nucleic acid-specific enrichment technology. In the same year, the research group invented the LAMP-RADAR viral nucleic acid detection technology as well as one-tube device design and reaction tube design. In 2021, Huimin Zhao and others developed a fast, accurate, and portable SPOT (Scalable and Portable Testing) technology for COVID-19 detection based on PfAgo.

Similar to the now commonly used CRISPR-Cas9 and CRISPR-Cas12a/13a enzymes, pAgos has also been proposed as a next-generation genome editing tool. However, the well-studied TtAgo, PfAgo and MjAgo are less suitable for use in Gene editing.

At present, compared with normal temperature NgAgo, CbAgo, etc., high temperature TtAgo and PfAgo have wider application prospects, because the biggest disadvantage of Ago protein compared with CRISPR/Cas system is that it cannot open the DNA double strand and does not have unwinding function. This is also the most important reason why Ago proteins cannot be used in gene editing. However, under high temperature conditions, DNA can open the double strands on its own, which allows the Ago protein to play the role of nuclease shearing, hence the nucleic acid detection technology.

However, the current nucleic acid detection technologies developed using PfAgo protein have found that the low catalytic activity of the enzyme limits the sensitivity and detection time of the detection. Therefore, there is an urgent need in the art to develop a method to obtain PfAgo mutants with high catalytic activity. And the modification of the catalytic activity of PfAgo enzyme will further reveal its structure and function relationship.

Contents of the invention

The object of the present invention is to provide mutants of Pyrococcus furiosus PfAgo nuclease, especially mutants with high enzymatic catalytic activity and mutants with improved specificity for base discrimination between wild-type and mutant genes.

In a first aspect of the present invention, a thermophilic nuclease Argonaute (Ago) mutant protein is provided, characterized in that the Ago mutant protein has core amino acid mutations at one or more sites selected from the following group:

(Z1)I656Y/S/C

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

(Z3)I569S/T/C/Y;

Wherein, the numbering of the mutation site is based on the sequence shown in SEQ ID NO:1;

And the Ago mutant protein has the activity of directional shearing of single-stranded DNA targets.

In another preferred embodiment, the Ago mutant protein also has core amino acid mutations at one or more sites selected from the following group:

(Z4)A573K/H/Q

(Z5)F769Y/R

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

(Z7)L626P/N/V,

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

In another preferred example, the Ago mutant protein has the activity of directional shearing of single-stranded DNA targets under ultra-high temperature conditions and under the guidance of guide DNA.

In another preferred example, the Ago mutant protein has the activity of directional shearing of single-stranded DNA targets under ultrahigh temperature conditions.

In another preferred example, the ultra-high temperature condition refers to 80-99.9°C, preferably 90-99.9°C, more preferably 94-96°C, and more preferably 95°C.

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

In another preferred embodiment, the protein is derived from the endonuclease Argonaute of any species selected from the following group: prokaryotes Thermococcus eurythermalis, Methanocaldococcus fervens, Methanococcus jannaschii Methanocaldococcus jannaschii, Pyrococcus furious, Marinitoga piezophila, Aquifex aeolicus, Clostridium butyricum, Clostridium perfringens (Clostridium perfringens), Thermus thermophilus, Natronobacterium gregoryi, Intestinibacter bartlettii, Kurthia massiliensis, Synechococcus elongatus).

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

In another preferred embodiment, the Ago mutant protein also includes active fragments, mutant forms or derivative proteins thereof, and the active fragments, mutant forms or derivative proteins of the Ago mutant protein have Y743F and/or I569Y mutations, which are the same as the Ago mutant protein. The mutant proteins have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, and have the ability to orient under ultrahigh temperature conditions Activity in cleaving single-stranded DNA targets.

In another preferred embodiment, except for one or more of the core amino acid mutations, the Ago mutant protein derivative protein has the same or substantially the same sequence as the sequence shown in SEQ ID NO: 1.

In another preferred example, the ratio Q1/Q0 of the enzyme activity Q1 of the Ago mutant protein to the wild-type enzyme activity Q0 is ≥1.2, preferably ≥1.5, more preferably ≥2.0, optimally ≥ 4.0.

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

In another preferred embodiment, the enzyme activity is the activity of directional shearing of single-stranded DNA targets.

In another preferred example, the Ago mutant protein 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 site is based on SEQ ID NO:1.

In another preferred example, the Ago mutant protein has core amino acid mutations at the following sites compared with 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 site is based on SEQ ID NO:1.

In another preferred example, compared with SEQ ID NO: 1 (wild type), the Ago mutant protein has amino acid mutations at 3 sites or 4 sites selected from Z1 to Z7:

(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 site is based on SEQ ID NO:1.

In another preferred embodiment, the sequence of the Ago mutant protein is selected from the following group:

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

(X2) The amino acid sequence shown in SEQ ID NO:1, and 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 at position 569 is mutated to S, T, C or Y (I569S/T/C/Y);

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

(X5) The amino acid sequence shown in SEQ ID NO:1, and the 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 at position 747 is mutated to P, I or W (F747P/I/W);

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

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

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

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

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

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

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

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

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

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

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

A second aspect of the invention provides an isolated polynucleotide encoding the Ago mutant protein of the first aspect of the invention.

The third aspect of the present invention provides a carrier, the carrier contains the second aspect of the present invention. of isolated polynucleotides.

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

The fourth aspect of the present invention provides a host cell, the host cell contains the vector according to the third aspect of the present invention, or the nucleic acid of the host cell contains the isolated vector according to the third aspect of the present invention. Polynucleotides.

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

Saccharomyces cerevisiae, Pichia pastoris, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces carlsbergensis, Saccharomyces carlsbergensis Yeast (Saccharomyces pombe), Kluyveromyces marxiamus (Kluyveromyces marxiamus), Kluyveromyces lactis (Kluyveromyces lactis), Kluyveromyces fragilis (Kluyveromyces fragilis), Pichia stipites, Huata Candida shehatae, Candida tropicalis, Escherichia coli.

In another preferred embodiment, the host cell includes Saccharomyces cerevisiae, Pichia pastoris or Myceliophthora thermophila.

In another preferred embodiment, the host cell expresses Ago mutant protein.

The fifth aspect of the present invention provides a method for preparing the Ago mutant protein according to the first aspect of the present invention, including the steps:

Under suitable expression conditions, culture the host cell according to the fourth aspect of the present invention to express the Ago mutant protein according to the first aspect of the present invention; and

The expression product is isolated to obtain the Ago mutant protein according to the first aspect of the present invention.

A sixth aspect of the present invention provides a nucleic acid cleavage system, which includes:

(a) guide DNA (gDNA) that targets and binds to a predetermined target site; and

(b) Programmable endonuclease Argonaute (Ago), wherein the programmable endonuclease is the Ago mutant protein according to the first aspect of the present invention.

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

(c) Reporter nucleic acid, wherein when the nucleic acid cleavage system is mixed with the nucleic acid molecule to be detected, the reporter nucleic acid will be cleaved by the Ago mutant protein and 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 example, the length of the guide DNA is 5-30nt, more preferably 15-21nt, most preferably 16-18nt.

In another preferred embodiment, the temperature of the nucleic acid cutting system is 80-99.9°C, preferably 90-99.9°C, more preferably 95-99.9°C. (The higher the temperature, the higher the cutting efficiency. Currently, the highest temperature can only be measured at 99.9°)

In another preferred example, the guide DNA is phosphorylated at the 5' end, hydroxylated at the 5' end, has a Biotin group at the 5' end, an NH 2 C 6 group at the 5' end, and a NH ₂ C ₆ group at the 5' end. Single-stranded DNA molecules with a FAM group or a SHC ₆ group at the 5' end (both PfAgo wild type and mutants can only use phosphorylation 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 and the reporter nucleic acid have reverse complementary fragments.

In another preferred example, the length of the guide DNA is 5-30nt, more preferably 15-21nt, most preferably 16-18nt.

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

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

In another preferred embodiment, the endonuclease Argonaute can cleave at the site of the reporter nucleic acid bound to positions 10-11 from the 5' end of the gDNA.

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

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

In another preferred embodiment, the reporter nucleic acid is a fluorescent reporter nucleic acid, and the fluorescent reporter nucleic acid carries a fluorescent group and/or a quenching group.

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

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

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

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

In another preferred example, the quenching group includes: BHQ, TAMRA, DABCYL, DDQ, or a combination thereof.

In another preferred embodiment, the fluorescent reporter nucleic acid is a single-stranded DNA molecule with only a fluorescent group, and the fluorescent group is FAM.

In another preferred embodiment, the nucleic acid cleavage system also includes: (d) divalent metal ions.

In another preferred example, the divalent metal ion is Mn ²⁺ .

In another preferred example, in the nucleic acid cleavage system, the concentration of divalent metal ions is 10 μM-3mM, preferably 50 μM-2mM, more preferably 100 μM-2mM.

In another preferred embodiment, the nucleic acid cleavage system further includes: (e) buffer.

In another preferred embodiment, the concentration of NaCl in the buffer is ≤500mM, preferably 20-500mM.

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

In another preferred example, the significant reduction in the shearing rate of the programmable endonuclease Argonaute means: under the same reaction conditions, the shearing rate of the programmable endonuclease Argonaute is reduced by ≥80%. , preferably reduced by ≥85%, more preferably reduced by ≥90%.

In another preferred embodiment, after the guide DNA is complementary to the sequence of the fluorescent reporter nucleic acid, it guides the Ago enzyme to cleave the fluorescent reporter nucleic acid, thereby generating a detectable signal (such as fluorescence).

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

In another preferred example, in the nucleic acid cleavage system, the concentration of the programmable endonuclease Ago is 10nM-10μM, preferably 100nM-1μM, more preferably 300nM-500nM, most preferably 400nM .

In another preferred example, in the nucleic acid cleavage system, the concentration of the guide DNA is 10 nM-10 μM, preferably 100 nM-3 μM, more preferably 1 μM-2.5 μM, and most preferably 2 μM.

A seventh aspect of the present invention provides a reaction system for enriching low-abundance target nucleic acids. The reaction system is used to simultaneously perform polymerase chain reaction (PCR) and nucleic acid cleavage reaction on a nucleic acid sample, thereby obtaining amplification. Addition-cleavage reaction products;

Wherein, the nucleic acid sample contains 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 to specifically cleave non-target nucleic acids, but does not cleave the target nucleic acid;

The amplification-cleavage reaction system contains (i) reagents required for PCR reaction and (ii) the nucleic acid cleavage system as described in the sixth aspect of the present invention.

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

Wherein, the forward gDNA refers to the gDNA having the same sequence fragment as the target nucleic acid, and the reverse gDNA refers to the gDNA having the reverse complementary sequence fragment to the target nucleic acid.

In another preferred example, the concentration of the Ago mutant protein in the reaction system is 20-200 nM, preferably 30-150 nM, and more preferably 40-100 nM.

In another preferred embodiment, the reagents required for the PCR reaction also include a primer pair for amplifying the target nucleic acid.

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

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

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

Wherein, the forward gDNA refers to the gDNA having the same sequence fragment as the target nucleic acid, and the reverse gDNA refers to the gDNA having the reverse complementary sequence fragment to the target nucleic acid.

In another preferred embodiment, the reaction system further includes: divalent metal ions.

In another preferred example, the divalent metal ion is Mn ²⁺ .

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

In another preferred example, the reaction temperature (reaction program) of the reaction system is: 94°C, 5min; cycle number 10-30 (94°C, 30s; 52°C, 30s; 72°C, 20s); 72°C, 1min.

The eighth aspect of the present invention provides a method for enriching low-abundance target nucleic acids, which is characterized by including the steps:

(a) providing a nucleic acid sample, the 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,

Moreover, the abundance of the target nucleic acid in the nucleic acid sample is F1a;

(b) using the nucleic acid in the nucleic acid sample as a template, performing polymerase chain reaction (PCR) and nucleic acid cleavage reaction in an amplification-cleavage reaction system, thereby obtaining an amplification-cleavage reaction product;

Wherein, the nucleic acid cleavage reaction is used to specifically cleave non-target nucleic acids, but does not cleave the target nucleic acid;

Furthermore, the amplification-cleavage reaction system contains (i) reagents required for PCR reaction and (ii) the nucleic acid cleavage system as described in the sixth aspect of the present invention;

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

Among them, the ratio of F1b/F1a is ≥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 example, when 1%≤F1a≤10%, the ratio of F1b/F1a≥10, when 0.1%≤F1a≤0.5%, the ratio of F1b/F1a≥100, when F1a≤0.1%, The ratio of F1b/F1a is ≥200.

In another preferred embodiment, the nucleic acid sample includes a nucleic acid sample directly heated to cleave, a nucleic acid sample treated with a direct cleavage enzyme protease, an extracted nucleic acid sample, a nucleic acid sample pre-amplified by PCR, or any sample containing nucleic acid. .

In another preferred embodiment, the nucleic acid sample pre-amplified by PCR is a PCR amplification product of 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 following group: nucleotide insertion, deletion, substitution, or combinations thereof.

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

In another preferred embodiment, the abundance of the non-target nucleic acid in the nucleic acid sample is F2a.

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

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

In another preferred embodiment, the abundance of the non-target nucleic acid in the amplification-cleavage reaction product is F2b.

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

In another preferred example, the F1b/F2b≥0.5, preferably ≥1, more preferably ≥2, optimally ≥3 or ≥5.

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

In another preferred example, F1a≤0.5%, preferably ≤0.2%, more preferably ≤0.1%, most preferably ≤0.01%.

In another preferred example, F1b ≥ 10%, preferably ≥ 30%, more preferably ≥ 50%, most preferably ≤ 70%.

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

In another preferred example, the "reagents required for PCR reaction" also include: dNTP, 1-5Mm Mg ²⁺ , and PCR buffer.

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 interacts with a non-nucleic acid cleavage system. The nucleic acid sequence of the targeted region of the target nucleic acid (ie, the second nucleic acid) forms a second complementary binding region.

In another preferred embodiment, the first complementary binding region contains at least 2 unmatched base pairs.

In another preferred embodiment, the second complementary binding region contains 0 or 1 unmatched base pairs.

In another preferred embodiment, the second complementary binding region contains one unmatched base pair.

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

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

In another preferred example, in the amplification-cleavage reaction system, the nucleic acid cutting tool enzyme is 30 nM, 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., preferably 2X PCR Precision ^TM Master Mix.

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

In another preferred embodiment, the method further includes:

(c) detecting the amplification-cleavage reaction product to determine the presence and/or quantity of the target nucleic acid.

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

In another preferred example, the quantitative detection is selected from the following group: TaqMan fluorescence quantitative PCR, Sanger sequencing, q-PCR, ddPCR, chemiluminescence method, high-resolution melting curve method, NGS, etc.; preferably From TaqMan fluorescence quantitative PCR, Sanger sequencing.

In another preferred embodiment, the first nucleic acid includes n different nucleic acid sequences, where n is a positive integer ≥1.

In another preferred example, 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 greater.

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

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

In another preferred embodiment, the nucleic acid sample includes nucleic acid from a sample, wherein the sample is selected from the following group: blood, cells, serum, saliva, body fluids, plasma, urine, prostatic fluid, bronchial lavage fluid, cerebrospinal fluid, gastric juice, bile, lymph fluid, peritoneal fluid and feces, etc. or a combination thereof.

A ninth aspect of the present invention provides a kit for detecting target nucleic acid molecules, the kit comprising:

(i) The thermophilic nuclease Argonaute (Ago) mutant protein according to the first aspect of the present invention; and (ii) instructions for use.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the following (such as embodiments) Each of the technical features specifically described in can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

Description of the drawings

Figure 1 shows a schematic diagram of the mutation site structure;

Figure 2 shows the SDS-PAGE electrophoresis patterns of wild-type PfAgo and mutants;

Figure 3 shows the target cleavage nucleic acid gel image of wild-type PfAgo and mutants;

Figure 4 shows the substrate cleavage efficiency versus time plot of wild-type PfAgo and mutants;

Figure 5 shows a schematic diagram of the PfAgo enzyme activity detection principle;

Figure 6 shows the enzyme activity graphs of wild-type PfAgo and mutants;

Figure 7 shows the thermal stability curves of wild-type PfAgo and mutants;

Figure 8 shows a bar graph showing the differential cleavage ability of wild-type PfAgo and mutants on DNA substrate wild-type and mutant genes;

Figure 9 shows the oncogene detection graph of wild-type PfAgo and mutants;

Figure 10 shows pathogen detection profiles of wild-type PfAgo and mutants.

Detailed ways

Through extensive and in-depth research and extensive screening, the inventors unexpectedly obtained for the first time a mutant protein with significantly improved Ago enzyme activity. Specifically, the mutated Ago protein significantly improves the activity of directional shearing of single-stranded DNA targets, and the activity can be increased by 50% to 650%, thus providing assistance for pathogen detection, genotyping, disease course monitoring, etc. On this basis, the present invention was completed.

the term

Unless otherwise defined, 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 all values between 99 and 101 and between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the terms "optionally" or "optionally" mean that the subsequently described event or circumstance may occur but does not necessarily occur.

As used herein, the term "contains" or "includes" can mean open, semi-closed, and closed. In other words, the term also includes "consisting essentially of" or "consisting of."

"Transduction", "transfection", "transformation" or terms used herein refer to the process of delivering exogenous polynucleotides into host cells, transcribing and translating them to produce polypeptide products, including the use of plasmid molecules to convert exogenous polynucleotides into host cells. The polynucleotide is introduced into a host cell (eg, E. coli).

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

"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. As used herein, the term polynucleotide refers to interchangeable single- and double-stranded molecules. Unless otherwise stated, polynucleotides in any embodiment described herein include double-stranded forms and two complementary single strands known or predicted to constitute the double-stranded form.

Conservative amino acid substitutions are known in the art. In some embodiments, 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 Acid; asparagine, glutamine; serine, threonine, lysine, arginine and histidine; and/or phenylalanine, tryptophan and tyrosine; methionine and cysteine . In addition, the present 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 construction described herein. Actual parameters, dimensions, materials and/or configurations will depend on the particular application described using this invention. It will be understood by those skilled in the art that the embodiments or claims are given by way of example only, and within the scope of equivalents or claims, the scope of the embodiments of the present invention may not be limited to the specific description and claims. scope.

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

All references, patents and patent applications cited herein are 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 examples.

The term "LAMP" stands for Loop-mediated isothermal amplification, a isothermal nucleic acid amplification technology suitable for genetic diagnosis.

The term "PCR" refers to polymerase chain reaction technology (Polymerase chain reaction), which is a technology suitable for rapid amplification of target nucleic acids.

As used herein, the term "secondary cleavage" means that in the detection method of the present invention, in the presence of primary guide ssDNAs, the Ago enzyme of the present invention cuts the target nucleic acid sequence, and the sheared product forms a new 5' phosphorylated nucleic acid sequence. (Secondary guide ssDNAs); then, the secondary guide ssDNA continues to guide the PfAgo enzyme to cut the target fluorescent reporter nucleic acid complementary to the secondary guide ssDNAs under the action of PfAgo enzyme. This kind of specific cleavage of the target nucleic acid sequence first (first cleavage), and then specific cleavage of the fluorescent reporter nucleic acid (second cleavage) is defined as "secondary cleavage". In the present invention, both the first cleavage and the second cleavage are specific cleavages.

Ago mutant protein

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

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

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

In the present invention, the wild-type Ago enzyme whose amino acid sequence is shown in SEQ ID NO: 1 is mutated at specific sites, and a corresponding mutant with significantly improved activity is obtained. As used herein, the terms "mutatein" and "mutant" are used interchangeably to refer to the Ago mutein.

As used herein, when describing mutations, the term "I656Y/S/C" is used as an example to refer to the sequence shown in SEQ ID NO: 1 (wild type), in which position I at position 656 is mutated to Y or S or C. Similarly, "Y743L/M/F" means that the 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 wild-type PfAgo enzyme is shown in SEQ ID NO:1:

Specifically, the mutant protein of the present invention generally refers to a core amino acid mutation that has one or more sites selected from the following group after modification based on the sequence shown in SEQ ID NO: 1 (wild type):

(Z1)I656Y/S/C

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

(Z3)I569S/T/C/Y;

Wherein, the numbering of the mutation site is based on the sequence shown in SEQ ID NO:1;

And the Ago mutant protein has the activity of directional shearing of single-stranded DNA targets.

In addition, the Ago mutant protein of the present invention also has amino acid mutations at other sites, as long as the amino acid mutation does not cause the loss or significant decrease of the activity of the mutant protein 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, those skilled in the art will understand that the mutein of the present invention should also include active fragments, modified or unmodified variant forms of the mutein, or derivative proteins thereof.

Specifically, the active fragment, variant form or derivative protein of the Ago mutant protein includes: an amino acid sequence formed by Z1 and/or Z2 mutations on the sequence shown in SEQ ID NO:1, and further has one or several ( For example, usually 1-30, preferably 1-10, more preferably 1-6, more preferably 1-3, most preferably 1) after deletion, insertion and/or substitution of amino acid residues , still has directional shearing single-stranded DNA activity (cA1), which is significantly higher than the corresponding activity (cA0) of the wild-type Ag enzyme shown in SEQ ID NO:1, which is significantly higher than (cA1-cA0)/cA0 Any value from ≥10% to 800%, such as ≥15%, ≥20%, ≥40%, ≥50%, ≥100%, ≥200%, or ≥500% or higher.

Those skilled in the art can perform conservative amino acid substitutions according to, for example, the table below to generate conservatively modified mutants.

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

In addition, derivatives of the mutant protein of the present invention also include fusion proteins or conjugates formed by the mutant protein of the present invention or active fragments thereof and other proteins or markers.

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

Specifically, in one embodiment of the present invention, the catalytic residue in the active site of the enzyme is centered, and the surrounding residues are selected based on the crystal structure of the enzyme or the structure of homology modeling supplemented by co-evolution analysis. The amino acid residues within are used as target sites, and mutants with improved enzyme catalytic activity are obtained through saturation mutation screening. The active center refers to the structural region near the enzyme catalytic residues, which is distinguished from the surface residues of complex structures.

In the present invention, the selected distance PfAgo catalytic quadruplex The amino acids within are supplemented by co-evolution analysis to identify mutation hotspots. A total of 26 mutation hotspots were finally selected, including sites 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, F769.

Further, the inventors screen these candidate sites, preferably high-throughput screening, including preliminary screening of the crude enzyme solution in a 96-well plate using the fluorescence method, re-screening of the crude enzyme solution in a 96-well plate using the fluorescence method, and screening of the pure enzyme using the fluorescence method. Screen again.

Typically, the preliminary screening and re-screening of the crude enzyme solution in a 96-well plate using the fluorescence method includes: cultivating wild-type PfAgo in one well from the edge and the center of each 96-well plate as a control, and reacting at 90°C on a Q-PCR instrument After 15 minutes, the activity of sheared fluorescently labeled ssDNA substrate was measured, and the enzyme activity was 50% higher than the wild type as the screening Select the standard and screen the cloned mutants into the next step of 96-well plate screening; transfer the mutants with increased activity to a 96-deep well plate for culture, make three replicates for each well, and react at 90°C for 15 minutes on a real-time fluorescence detector. After that, the activity of sheared fluorescently labeled ssDNA substrate is measured, and the enzyme activity is 50% higher than the wild type as the screening standard. The screened mutants enter the next step of re-screening of pure enzyme by fluorescence method; re-screening of pure enzyme by fluorescence method Including: sequencing the mutant with increased enzyme activity, purifying with a protein purifier, and measuring the concentration of the mutant with the BSA method. After diluting to 0.5 mg/mL, store at -80°C.

The screening results showed that seven single points: L626P/N/V, I656Y/S/C, A573K/H/Q, F747P/I/W, F769Y/R, I569S/T/C/Y, Y743L/M/F Mutants can unexpectedly enhance the enzyme catalytic activity of PfAgo.

In another preferred embodiment, the inventor also mutates the selected mutants with improved enzymatic catalytic activity, and obtains mutants with higher enzymatic catalytic activity through combined mutations. By double-pointing the activity-increasing sites L626P/N/V, I656Y/S/C, A573K/H/Q, F747P/I/W, F769Y/R, I569S/T/C/Y, and Y743L/M/F Combination mutations were used to obtain double-point combination mutants with further improved activity: I656Y and Y743L; I656S and I569C; I656Y and F769R; Y743M and F747P; I659Y and L626N; Y743L and I569S; ; I569Y and Y743F; I569Y and F747W; Y743F and L626V; I569Y and L626V; Y743F and F747W; L626V and F747W.

Coding nucleic acids and combinations thereof

Based on the Ago mutant protein of the present invention, the present invention also provides an isolated polynucleotide encoding the Ago mutant protein or a degenerate variant thereof. The polynucleotides of the invention may be in DNA form or RNA form. Forms of DNA include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the nucleotide sequence encoding the PfAgo mutant protein in the embodiment of the present invention, or may be a degenerate variant.

As used herein, "degenerate variant" in the present invention refers to a nucleic acid encoding the Ago mutant protein in the first aspect of the present invention, but which is different from the nucleotide sequence encoding the Ago mutant protein in the embodiments of the present invention. sequence.

vector, host cell

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

Those skilled in the art can use well-known methods to construct expression vectors containing the DNA sequence encoding "pyruvate carboxylase mutant protein and/or malate transporter mutant protein" and appropriate transcription/translation control signals, including in vitro Recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA The sequence can be operably linked to an appropriate 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 containing the above-mentioned expression vector or the genome integrating the coding sequence of the mutant protein of the present invention. The host cells include hosts derived from Ascomycetes, which have a very high evolutionary relationship and have high similarity in the sequences and functions of pyruvate carboxylase and malate transporter. Preferred microbial cells are as follows:

Saccharomyces cerevisiae, Pichia pastoris, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces carlsbergensis, Saccharomyces carlsbergensis Yeast (Saccharomyces pombe), Kluyveromyces marxiamus (Kluyveromyces marxiamus), Kluyveromyces lactis (Kluyveromyces lactis), Kluyveromyces fragilis (Kluyveromyces fragilis), Pichia stipites, Huata Candida shehatae, Candida tropicalis, Escherichia coli.

The mutant protein of the present invention can be transformed by conventional recombinant transformation methods in the art, and the mutant protein can be expressed within the cell, on the cell membrane, or secreted out of the cell. If desired, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic sterilization, sonication, high-pressure homogenization, ultracentrifugation, molecular sieve chromatography (gel filtration) ), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods. The host cells of the present invention can be obtained by expressing pyruvate carboxylase mutant protein and malate transporter mutant protein separately and/or simultaneously.

Preparation of mutant proteins

The present invention also provides a method for preparing Ago mutant protein, which includes culturing the host cell of the present invention under suitable expression conditions to express the Ago mutant protein; and

The Ago mutant protein is isolated.

The obtained mutein may optionally be purified to obtain a purer mutein product.

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

WizardssDNA

In the detection method of the present invention, a core component is guide ssDNA, especially two ssDNAs, which are adjacent to each other and have no spacer bases or spacer sequences between each other.

In the present invention, the preferred guide ssDNAs are all oligonucleotides with a length of 10-60nt, preferably 10-40nt, more preferably, 13-20nt, and the 5' first nucleotide is Thymine (T), can be phosphated chemical modification.

Reporting nucleic acid molecules

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

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

In the present invention, the fluorescent reporter nucleic acid molecule is determined based on the production position of the secondary guide ssDNAs; the target nucleic acid sequence is cut by the primary guide ssDNAs to form a new 5' phosphorylated nucleic acid sequence, which is called the secondary Guide ssDNAs, fluorescent reporter nucleic acid covers all positions of the secondary guide ssDNAs.

Detection method

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

In the method of the present invention, based on the shearing activity of the PfAgo enzyme, three pairs of guide ssDNA can be designed according to the target nucleic acid molecule (such as single-stranded DNA, preferably the amplified target nucleic acid molecule). These three pairs of guide ssDNA are targeted to Different target nucleic acid molecules mediate the PfAgo enzyme to cleave the target nucleic acid molecules to form new secondary guide ssDNA. The secondary guide ssDNA continues to guide the PfAgo enzyme to cut the fluorescent reporter nucleic acid complementary to the secondary guide ssDNAs under the action of the PfAgo enzyme, thereby achieving the detection of the target nucleic acid molecule corresponding to the fluorescent reporter nucleic acid. The method of the present invention can greatly improve the sensitivity and multiplicity of target nucleic acid detection.

In the present invention, according to the design requirements of the guide ssDNAs, the PfAgo enzyme can be specially designed to selectively cut nucleic acid sequences that differ at some sites, thereby realizing typing detection.

In the present invention, when used to distinguish different types, when designing guide ssDNAs, the mutation sites corresponding to different types are placed at the 10th and 11th digits of the guide ssDNAs. Due to the selection specificity of the pfAgo enzyme, two consecutive Point mutations can inhibit the cleavage activity, thereby achieving the detection of different types.

In the present invention, multiple target nucleic acid molecules and guide ssDNAs can be added simultaneously to the shearing system of PfAgo enzyme, and combined with reporter nucleic acids with different fluorescent groups, multiple detection of target nucleic acids can be achieved.

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

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

Reagent test kit

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

In a preferred embodiment, the method of the present invention includes: (a) an amplification reagent for amplifying a target nucleic acid molecule, the amplification reagent comprising: a primer pair for amplifying the 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) Cutting reagent or cutting buffer containing the cutting reagent, wherein the cutting reagent includes: 3 pairs of guide ssDNA, gene editing enzyme (Ago), and the first reporter nucleic acid, the fluorescent reporter nucleic acid has fluorescence group and quenching group, and the three pairs of guide ssDNA target three different target nucleic acid molecules.

application

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

The mutant protein of the present invention is particularly suitable for detecting trace amounts of target nucleic acid molecules and multiplex detection, and has wide applicability.

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

The mutants screened by the present invention can exhibit higher catalytic activity and base discrimination specificity at high temperatures. At the same time, this mutant can be better used in nucleic acid detection technology, which greatly improves its application potential in the nucleic acid detection industry and also lays the foundation for the development of new genetic manipulation tools.

The main advantages of the present invention include:

(1) Compared with the wild type, the mutant type obtained by screening in the present invention has significantly improved enzyme catalytic activity, up to at least 6.5 times;

(2) The thermal stability of the mutant of the present invention is not adversely affected by the mutation and maintains very good thermal stability;

(3) The mutant of the present invention also improves the specificity of base discrimination between the wild-type and mutant genes of the target nucleic acid;

(4) The mutants of the present invention are useful in nucleic acid detection technology and gene editing tool development technology. application potential.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing Conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

The experimental materials involved in the present invention can be obtained from commercial channels unless otherwise specified.

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

Example 1 Construction and synthesis of wild-type Pyrococcus furiosus PfAgo plasmid vector and selection of mutation sites

Search for the nucleotide sequence of PfAgo wild type in the NCBI database and send it to GenScript Biologics for synthesis. After codon optimization, select the pET28a(+) vector, select NdeI and XhoI double enzyme cutting sites, and convert the plasmid product synthesized by the company. Transform into the competent E. coli Ecoli-BL21 (DE3), spread on a plate (containing 50 μg/ml kanamycin), and incubate overnight at 37°C. The next day, a single bacterial colony will be picked and sent for sequencing to verify whether the plasmid sequence is correct. After the comparison is correct, extract the plasmid. For plasmid extraction steps, refer to the instruction manual of 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 involved in the present invention is shown in SEQ ID No: 1.

Search the PDB database for the crystal structure of PfAgo and select the structure with the highest resolution, 1U04, as the research object. Later, the molecular docking software Amber was used to dock the guide strand DNA and the target strand DNA into the PfAgo crystal structure to simulate the ternary complex structure. When selecting the target site, take the catalytic quadruple DEDH as the center and analyze the surrounding All amino acid residues within the range, supplemented by co-evolution analysis. Select the amino acid residue that matches both as the target site (see Table 1). The specific positions of these target amino acid sites in the structure are shown in Figure 1. Subsequently, single-point saturation mutations were performed on all amino acid positions in Table 1.

Table 1 Distance from active center target site to catalytic residue D558

Example 2 Site-directed saturation mutagenesis to construct a gene mutation library

The 22c-trick method was used to design primer sequences to construct a saturated mutation library. Compared with the traditional NNK method, it effectively reduces codon redundancy and the amount of mutant screening. Perform single-point saturation mutations on the 26 amino acid sites in Table 1. The specific steps are as follows:

Under the action of PrimeSTAR MAX Premix (2×) DNA polymerase (TaKaRa Company), the wild-type gene of PfAgo was used as a template to perform whole plasmid PCR amplification, and DNA agarose gel electrophoresis was used to verify the PCR product. The PCR system is as follows:

The PCR amplification program is: denaturation at 98°C for 10 seconds per cycle, annealing at 55-70°C for 15 seconds, and extension at 72°C for 130 seconds, for a total of 20 cycles.

Digest the PCR product to remove the template plasmid in the system. The 50 μL digestion system is as follows:

Digest in 50 μL digestion system at 37°C for 2 hours. The digested PCR products are then cleaned and purified. Add 10 μL of the purified sample to 100 μL of E.coli BL21 (DE3) competent cells, place on ice for 30 minutes, then heat shock in a 42°C water bath for 90 seconds, leave on ice for 2 minutes, add 800 μL of anti-antibody LB culture medium and add After culturing for 1 hour at 37°C on a shaking table, the culture solution was evenly spread on a solid LB plate containing 50 mg/ml Kana resistance, and the culture was inverted in a 37°C constant-temperature incubator overnight to obtain a strain containing the mutant gene. Pick 3-5 single clones from each plate and send them for sequencing to verify whether the saturated mutation library construction is correct.

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

Example 3 Screening of mutation library

Add 300 μL of LB liquid culture medium containing 50 μg/mL Kan to each well of the 96-deep well plate, and use a sterilized toothpick to pick out the single colonies on the LB solid plate one by one into the 96-deep well plate. Take one hole each from the edge and the middle as a WT-PfAgo control, and culture it overnight at 37°C and 400rpm. The next day, add 600 μL of LB medium containing 50 μg/mL Kan to a new round of deep well plates, transfer 50 μL of seed solution to the new deep well plate, add glycerol to the original deep well plate and store it at –80°C; after transfer Incubate a new round of deep well plate at 37°C and 400rpm for 2-3h until the OD ₆₀₀ reaches 0.6-0.8; place it in a 4°C refrigerator to cool for 1 hour, add IPTG with a final concentration of 0.5mM, and incubate at 18°C and 400rpm for 20-22h. Centrifuge at 4°C/4000rpm for 20 minutes, discard the supernatant, and place in a -80°C ultra-low temperature refrigerator to repeatedly freeze and thaw 3 times (freezing for 3 hours each time, thawing in a 37°C incubator for 30 minutes). After freezing and thawing, add 50 μL (250mM NaCl, 15mM Tris-HCl pH 8.0) of Reaction Buffer to each well, resuspend the cells, and shake on a micro-oscillator for 30 minutes to mix thoroughly. Draw a certain amount of crude enzyme solution from each well and add it to the 96-well plate. Use the fluorescently modified ssDNA substrate to react for 30 minutes at 90°C, an excitation wavelength of 495nm, and an emission wavelength of 520nm, and detect the fluorescence signal value in real time. The enzyme activity was measured by fluorescence signal value, and single cloning sites with higher catalytic activity than WT were selected.

result

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

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

The seven preferred single point mutants are shown in the table below.

Subsequently, these single-point mutants with improved enzyme activity were subjected to double-point combination mutations, and double-point combination mutants with further improved activities were obtained: 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; L626V and F74777 and F74777; W. The enzyme activity improvement data is shown in the table below.

Example 4 Expression and purification of wild-type and mutant proteins of Pyrococcus furiosus PfAgo nuclease

Recovery and activation: Inoculate E.coli BL21(DE3)-pET28a-PfAgo wild-type and mutant bacterial liquid with 1% inoculum volume into a small shake flask of 50mL LB liquid medium (containing kanamycin), 220r/min , incubate overnight at 37°C.

Transfer: transfer the bacterial solution cultured overnight into a shake flask of 1L LB liquid medium (containing kanamycin) at an inoculation volume of 1%, and culture at 220r/min and 37°C until the OD ₆₀₀ value reaches 0.8-1.0;

Induction after ice shock: Take out the shake flask from the shaker, place it on ice for 15 minutes, then add IPTG with a final concentration of 0.5mM, induce expression at 18°C, 220r/min for 20h to 22h, and then centrifuge to collect the cells.

Since the N-terminus of the target gene is designed with a 6×His tag, it can be purified directly by Ni-affinity chromatography. Use resuspension buffer (containing 20mM Tris-HCl, pH around 8.0, 1M NaCl) to resuspend the bacterial cells, then high-pressure disrupt the bacterial cells, and centrifuge to obtain the supernatant. Ni-NTA column is used to affinity purify the protein, and the eluate is concentrated by ultrafiltration, desalted and other steps to obtain the purified protein. The purity of the purified protein was verified by SDS-PAGE gel chart, as shown in Figure 2. The purified protein was stored in a buffer containing 20mM Tris-HCl, and the protein was measured by a BCA kit. The measurement steps were carried out according to the operating instructions. Use BSA as a standard, prepare a standard solution, draw a standard curve, and calculate the concentration of the purified target protein accordingly. Store the protein in a -80°C refrigerator for later use.

Example 5 Determination of shearing activity of Pyrococcus furiosus PfAgo nuclease wild type and mutants

Design a 60nt single-stranded DNA target nucleic acid and a complementary 16nt single-stranded DNA guide strand, and send them to the company for synthesis.

Prepare a reaction buffer (containing 15mM Tris-HCl pH 8.8, 250mM NaCl), add a final concentration of 0.5mM MnCl ₂ , 100nM PfAgo, 2μM synthesized gDNA and 0.8μM complementary single-stranded DNA target nucleic acid into the reaction buffer. React at 95°C for 0, 1, 3, 5, 7, 10, and 15 minutes. After the reaction is completed, take 6-10 μL of sample and add loading buffer (containing 95% (deionized) formamide, 0.5 mmol) in a 1:1 ratio. /L EDTA, 0.025% bromophenol blue, 0.025% xylene blue), run under 16% nucleic acid Urea-PAGE detection.

The gel electrophoresis results are shown in Figure 3. Image J software was used to quantitatively calculate the gray value of the protein gel image, and the obtained substrate DNA target shearing efficiency line chart is shown in Figure 4.

The results showed that after different reaction times, the shearing activity of the mutant was significantly higher than that of the wild type.

Example 6 Determination of specific activity of Pyrococcus furiosus PfAgo nuclease enzyme

The reaction principle is shown in Figure 5. The reactions for PfAgo nuclease activity detection are all carried out in reaction buffer (15mM Tris-HCl pH8.8, 250mM NaCl, 0.5mM MnCl ₂ , 4μM target DNA, 8μM guide DNA).

The reaction procedure is: under the conditions of excitation wavelength of 495nm and emission wavelength of 520nm, react at 95°C for 15 minutes, collect fluorescence signals, and repeat three times for each sample. Calculate the enzyme specific activity according to the following formula (Formula 1). Enzyme activity unit (U) is defined as: the amount of enzyme required to catalyze the cleavage of 0.1 nM substrate per minute under the above reaction system. (Note: One unit of enzyme activity is represented by U.)

A (U/mg): specific activity of the enzyme; C _p (μm): refers to the product concentration at time t; T (min): the time required to reach time T; C (mg/mL): concentration of the enzyme; V (mL): the volume of enzyme added; experimental data are the average of three replicates.

The enzyme activity measurement results of PfAgo nuclease wild type and mutants are shown in Figure 6.

Example 7 Thermal stability and enzyme kinetic parameters of Pyrococcus furiosus PfAgo nuclease wild type and mutants

In order to measure the changes in thermal stability of the mutants, the present invention incubated each mutant protein at 95°C for different times, and then measured changes in its enzyme activity. The measurement method was as in Example 5, and the results are shown in Figure 7.

The kinetic parameter measurement is to measure the maximum initial reaction velocity corresponding to substrate concentrations of 0.4, 0.8, 1.2, 1.6, 2.0, 4.0, 6.0, and 8.0 μM under the conditions of pH 8.0 and temperature 95°C. Use the software Graphpad 8.0 and the Michaelis-Menten equation (Formula 2) to fit the relevant curve to obtain K _m and V _max , and then calculate the kinetic constants K _m and K according to the functional equation of the maximum rate and enzyme concentration (Formula 3) _cat and K _cat /K _m .
V＝V _max [S]/(K _m +[S]) (Formula 2)
V _max =K _cat ×[E] (Formula 3)

Through the above methods, the kinetic parameters of PfAgo wild type and mutants were obtained (see Table 2) and thermal stability diagram (see Figure 7). The results showed that the mutant k _cat /K _m of PfAgo nuclease increased 9 times, but the thermal stability remained unchanged.

Table 2 Kinetic parameters of PfAgo wild type and mutants

Example 8 Differential cleavage of wild-type and mutant genes of DNA substrate by PfAgo wild type and mutants

In order to verify the ability of the PfAgo nuclease mutant in the present invention to differentiate between wild-type and mutant genes on DNA substrates, the present invention designed probe substrates for wild-type and mutant genes (sequences are shown in Table 3). The 5' and 3' ends of the wild-type probe are modified by FAM and BHQ1 fluorophores respectively, and the 5' and 3' ends of the mutant probe are modified by VIC and BHQ1 fluorophores respectively. The experimental principle is shown in Figure 3 .

Design corresponding guide DNA for wild-type and mutant probe substrates (sequences are shown in Table 3). Add a final concentration of 0.5mM MnCl ₂ , 200nM PfAgo, 4μM synthesized gDNA and 2μM single-stranded DNA wild-type and mutant gene probes to the reaction buffer, and react for 30 minutes at an excitation wavelength of 490nm and an emission wavelength of 550nm. , the fluorescence value at the end point was measured, and each experiment was repeated three times. Use Graphpad 8.0 to make a column chart, as shown in Figure 8.

The results showed that the mutant PfAgo nuclease had better ability to differentiate between wild-type and mutant genes of DNA substrates.

Table 3 Probe sequences and guide strand sequences

Example 9 Application of Pyrococcus furiosus PfAgo nuclease wild type and mutants in genetic detection

In order to verify the application of PfAgo nuclease mutants in nucleic acid detection in the present invention, the present invention uses the 'low-abundance DNA mutation detection technology system' developed by our laboratory to verify the detection effect of PfAgo nuclease mutants on oncogenes. According to The principle of this detection method is to design and screen specific amplification primers, gDNAs and detection probe sequences based on the sequence characteristics of the KRAS-G12D gene fragment. The gDNAs, primers and probes were synthesized by Shanghai Sangon Bioengineering Co., Ltd. The 5' end of KRAS-G12D gene gDNAs is phosphorylated Modification; the 5' end of the nucleotide sequence of the mutant probe is equipped with a VIC fluorescent label, and the 3' end is modified with the quenching group BHQ1; the 5' end of the KRAS wild-type gene probe's nucleotide sequence is equipped with a FAM fluorescent label, The 3' end is modified with the quenching group BHQ2. This method was verified and analyzed using standards from Jingliang Biological Company. The mutation allele frequencies (AF%) in the standards were 1% mut and 100% wt respectively. 1% mut and 100% wt standards were used to verify the enrichment detection effect of PfAgo nuclease mutants. The results are shown in Figure 9.

At the same time, in order to verify the application of PfAgo nuclease mutants in pathogen detection technology, the present invention uses the reverse transcription loop-mediated isothermal amplification reaction (reverse transcription LAMP) nucleic acid detection technology developed in this experiment to detect African swine fever (African swine fever virus). strain BA71V) for testing. The specific steps for this detection method are as follows:

(1) The amplification primer and gDNA dry powder are dissolved with DNase/RNase free H ₂ O to make a 100 μM storage solution; the fluorescent reporter nucleic acid dry powder is dissolved with DNase/RNase free H ₂ O to make a 10 μM storage solution. liquid;

(2) Use Bst enzyme and Ago digestion buffer amplification primer to prepare 25ul amplification reaction master mix;

(3) Add 15 μL of the nucleic acid sample to be tested to the amplification reaction premix, and the amplification reaction system is 40 μL;

(4) Use PfAgo mutant, LAMP amplification buffer, gDNA, and fluorescent reporter nucleic acid to prepare a 20 μL enzyme digestion reaction system;

(5) Transfer the amplification system and enzyme digestion system to PCR tubes and lined tubes respectively, and put them into a fluorescence quantitative PCR instrument for reaction (amplification at 62°C for 30 minutes, enzyme digestion at 95°C for 30 minutes, and detect the signal every minute) ).

The detection results of PfAgo mutants against the pathogen African swine fever are shown in Figure 10.

The results show that the PfAgo nuclease mutant is more rapid and accurate in detecting oncogenes and pathogens and has better enrichment effects than the wild type.

The above contents are only examples and descriptions of the structure of the present invention. Modifications or additions to the described specific embodiments or substitutions in similar ways by those skilled in the art without creative efforts will still fall within the scope of protection of this patent.

Claims (20)

A thermophilic nuclease Argonaute (Ago) mutant protein, characterized in that the Ago mutant protein has core amino acid mutations at one or more sites selected from the following group: (Z1)I656Y/S/C (Z2)Y743L/M/F; and/or (Z3)I569S/T/C/Y; Wherein, the numbering of the mutation site is based on the sequence shown in SEQ ID NO:1; And the Ago mutant protein has the activity of directional shearing of single-stranded DNA targets. The Ago mutant protein of claim 1, wherein the Ago mutant protein also has core amino acid mutations at one or more sites selected from the following group: (Z4)A573K/H/Q (Z5)F769Y/R (Z6)F747P/I/W; and/or (Z7)L626P/N/V, Wherein, the numbering of the mutation site is based on the sequence shown in SEQ ID NO:1. The Ago mutant protein according to claim 1, characterized in that the Ago mutant protein has the activity of directional shearing of single-stranded DNA targets under ultra-high temperature conditions and under the guidance of guide DNA. The Ago mutant protein according to claim 1, characterized in that the Ago mutant protein has the activity of directional shearing of single-stranded DNA targets under ultra-high temperature conditions. The Ago mutant protein according to claim 4, characterized in that the ultra-high temperature condition refers to 80-99.9°C, preferably 90-99.9°C, more preferably 94-96°C, more preferably 95 ℃. The Ago mutant protein of claim 1, wherein the ratio Q1/Q0 of the enzyme activity Q1 of the Ago mutant protein to the wild-type enzyme activity Q0 is ≥1.2, preferably ≥1.5, more preferably Ground ≥ 2.0, optimal ground ≥ 4.0. The Ago mutant protein of claim 6, wherein the enzyme activity is the activity of directional shearing of single-stranded DNA targets. The Ago mutant protein according to claim 1, characterized in that the Ago mutant protein has There are dual core amino acid mutations selected from the following positions: 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 site is based on SEQ ID NO:1. The Ago mutant protein according to claim 1, characterized in that the sequence of the Ago mutant protein is selected from the following group: (X1) The amino acid sequence shown in SEQ ID NO:1, and the I at position 656 is mutated to Y, S or C (I656Y/S/C); (X2) The amino acid sequence shown in SEQ ID NO:1, and 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 at position 569 is mutated to S, T, C or Y (I569S/T/C/Y); (X4) The amino acid sequence shown in SEQ ID NO:1, and the A at position 573 is mutated to K, H or Q (A573K/H/Q); (X5) The amino acid sequence shown in SEQ ID NO:1, and the 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 at position 747 is mutated to P, I or W (F747P/I/W); (X7) The amino acid sequence shown in SEQ ID NO:1, and the L at position 626 is mutated to P, N or V (L626P/N/V); (DX1) The amino acid sequence shown in SEQ ID NO:1, and the I at position 656 is mutated to Y (I656Y) And Y at position 743 is mutated to L (Y743L); (DX2) The amino acid sequence shown in SEQ ID NO:1, and the I at position 656 is mutated to S (I656S) and the I at position 569 is mutated to C (I569C); (DX3) The amino acid sequence shown in SEQ ID NO:1, and the I at position 656 is mutated to Y (I656Y) and the F at position 769 is mutated to R (F769R); (DX4) The amino acid sequence shown in SEQ ID NO:1, and the Y at position 743 is mutated to M (Y743M) and the F at position 747 is mutated to P (F747P); (DX5) The amino acid sequence shown in SEQ ID NO:1, and the I at position 659 is mutated to Y (I659Y) and the L at position 626 is mutated to N (L626N); (DX6) The amino acid sequence shown in SEQ ID NO:1, and the I at position 569 is mutated to S (I569S) and the Y at position 743 is mutated to L (Y743L); (DX7) The amino acid sequence shown in SEQ ID NO:1, and the F at position 747 is mutated to W (F747W) and the F at position 769 is mutated to Y (F769Y); (DX8) The amino acid sequence shown in SEQ ID NO:1, and the I at position 569 is mutated to Y (I569Y) and the L at position 626 is mutated to V (L626V); (DX9) The amino acid sequence shown in SEQ ID NO:1, and the Y at position 743 is mutated to F (Y743F) and the I at position 569 is mutated to Y (I569Y); (DX10) The amino acid sequence shown in SEQ ID NO:1, and the I at position 569 is mutated to Y (I569Y) and the F at position 747 is mutated to W (F747W) An isolated polynucleotide, characterized in that the polynucleotide encodes the Ago mutant protein of claim 1. A vector, characterized in that the vector contains the isolated polynucleotide of claim 10. A host cell, characterized in that the host cell contains the vector of claim 11, or the nucleic acid of the host cell contains the isolated polynucleotide of claim 10. A method for preparing the Ago mutant protein of claim 1, characterized in that it includes the steps: Under conditions suitable for expression, culturing the host cell of claim 12 to express the Ago mutant protein of claim 1; and The expression product is isolated to obtain the Ago mutant protein according to claim 1. A nucleic acid cleavage system, characterized in that the nucleic acid cleavage system includes: (a) guide DNA (gDNA) that targets and binds to a predetermined target site; and (b) Programmable endonuclease Argonaute (Ago), wherein the programmable endonuclease is the Ago mutant protein of claim 1. The nucleic acid cleavage system of claim 14, wherein the nucleic acid cleavage system further includes: (c) Reporter nucleic acid, wherein when the nucleic acid cleavage system is mixed with the nucleic acid molecule to be detected, the reporter nucleic acid will be cleaved by the Ago mutant protein and detected. The nucleic acid cutting system of claim 15, wherein the reporter nucleic acid is single-stranded DNA (ssDNA). A reaction system for enriching low-abundance target nucleic acids, characterized in that the reaction system is used to simultaneously perform 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 contains 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 to specifically cleave non-target nucleic acids, but does not cleave the target nucleic acid; The amplification-cleavage reaction system contains (i) reagents required for PCR reaction and (ii) the nucleic acid cleavage system as claimed in claim 14. The reaction system of claim 17, wherein the reaction system further includes: divalent metal ions. The reaction system of claim 18, wherein the divalent metal ion is Mn ²⁺ . A method for enriching low-abundance target nucleic acids, which is characterized by including the steps: (a) providing a nucleic acid sample, the 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, Moreover, the abundance of the target nucleic acid in the nucleic acid sample is F1a; (b) using the nucleic acid in the nucleic acid sample as a template, performing polymerase chain reaction (PCR) and nucleic acid cleavage reaction in an amplification-cleavage reaction system, thereby obtaining an amplification-cleavage reaction product; Wherein, the nucleic acid cleavage reaction is used to specifically cleave non-target nucleic acids, but does not cleave the target nucleic acid. nucleic acid; Moreover, the amplification-cleavage reaction system contains (i) reagents required for PCR reaction and (ii) the nucleic acid cleavage system as claimed in claim 8; Wherein, the abundance of the target nucleic acid in the amplification-cleavage reaction product is F1b, Among them, the ratio of F1b/F1a is ≥10.