PfAgo mutant with enhanced target nucleic acid cleavage activity and application thereof

Abstract

The invention discloses a PfAgo mutant with enhanced target nucleic acid cleavage activity and application thereof, the PfAgo mutant protein is a protein obtained by mutation of the following (1) or (2) on the basis of a wild type PfAgo protein with an amino acid sequence as shown in SEQ ID NO.1: (1) V142A, Y413C, K417R and I623F; and (2) on the basis of (1), the 623rd amino acid is further mutated into methionine. The PfAgo mutant protein provided by the invention realizes significant breakthrough in target nucleic acid cleavage activity and practical performance. According to the M2A4 obtained through random mutation screening, the nucleic acid cleavage activity and the protein expression quantity are greatly improved compared with those of a wild type, the key functional site-I623F mutation is determined through site-specific mutagenesis verification, the catalytic efficiency and the high salt tolerance are directly improved through I623F mutation, the protein expression quantity is remarkably optimized through Y413C mutation, the discovery reveals a PfAgo structure and function association mechanism, and the application prospect is broad. And a molecular target is provided for accurate transformation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of programmable nuclease, in particular to a PfAgo mutant with enhanced target nucleic acid cleavage activity and application thereof. BACKGROUND

[0002] Argonaute (Ago) is a new type of programmable nuclease, widely distributed in eukaryotes, true bacteria and archaea, and its function varies significantly depending on the source. Eukaryotic Argonaute (eAgo) protein is an important part of the RNA interference mechanism and plays an important role in the formation of RISC complex; while prokaryotic Ago (pAgo) protein mainly plays a host defense function, participates in the defense mechanism of bacteria against exogenous gene invasion and participates in host replication and damage repair. Compared with the widely used CRISPR / Cas system, the pAgo system is considered as a potential tool for the next generation of nucleic acid sensing technology due to its flexible guide chain design (without relying on PAM sequence) and high target recognition specificity, and shows unique advantages in clinical diagnosis, environmental monitoring, food safety and other fields. Among them, PfAgo derived from Pyrococcus furiosus, as a typical representative of the pAgo family, has precise target DNA cleavage ability by relying on 5' phosphate guide DNA (5'P-gDNA), as well as high activity characteristics at high temperature of 87~99.99℃, and has become the focus of early nucleic acid detection technology development.

[0003] Although PfAgo has been initially applied to nucleic acid detection due to its excellent target sequence specificity and high temperature activity, in actual detection scenarios, the abundance of target nucleic acids in real samples (such as clinical specimens, environmental samples) is often extremely low, and direct detection of trace amounts of DNA or RNA sequences faces great challenges. The existing technology relies on nucleic acid amplification technology (such as PCR, LAMP) to expand the target concentration to improve the detection sensitivity, but such methods have problems such as complex procedures, long time-consuming and high risk of aerosol pollution, which seriously restricts the realization of on-site rapid detection. Further research has found that the low catalytic activity of PfAgo not only leads to insufficient detection sensitivity (difficult to capture trace amounts of targets), but also prolongs the detection reaction time, making it unable to meet the efficiency requirements of clinical point-of-care testing (POCT) and other scenarios. Therefore, developing a PfAgo mutant that can significantly improve the target nucleic acid cleavage activity has become a key to breaking through the bottleneck of existing technology and promoting its application from the laboratory to practical application. SUMMARY

[0004] The main objective of this invention is to propose a PfAgo mutant with enhanced target nucleic acid cleavage activity and its applications. The aim is to improve the catalytic activity, expression level, and stability of PfAgo and promote its industrial application by performing directed evolution of the PfAgo protein and virtual saturation mutations at core sites.

[0005] To achieve the above objectives, this invention proposes a PfAgo mutant protein with enhanced target nucleic acid cleavage activity. The PfAgo mutant protein is obtained by mutating the wild-type PfAgo protein, whose amino acid sequence is shown in SEQ ID NO.1, by the following (1) or (2): (1) V142A, Y413C, K417R, I623F (denoted as M2A4); (2) Based on (1), the 623rd amino acid is further mutated to methionine (denoted as M2A4-M).

[0006] The amino acid sequence of the mutant M2A4 protein is shown in SEQ ID NO.2; the amino acid sequence of the mutant M2A4-M protein is shown in SEQ ID NO.3.

[0007] This invention constructs a random mutant library using the PfAgo-pET28a plasmid as a template, and obtains the dominant random mutant M2A4 (sequence shown in SEQ ID NO.2) through high-throughput screening. Specifically, valine at position 142 is mutated to alanine (Ala, A), denoted as V142A; tyrosine at position 413 is mutated to cysteine ​​(Cys, C), denoted as V413C; lysine at position 417 is mutated to arginine (Lys, K), denoted as K417R; and isoleucine at position 623 is mutated to phenylalanine (Phe, F), denoted as I623F.

[0008] Preferably, in the PfAgo mutant protein provided by the present invention, valine at position 142 is mutated to alanine (Ala, A), denoted as V142A; tyrosine at position 413 is mutated to cysteine ​​(Cys, C), denoted as V413C; lysine at position 417 is mutated to arginine (Lys, K), denoted as K417R; and isoleucine at position 623 is mutated to phenylalanine (Phe, F) and methionine (Met, M), denoted as I623F / M.

[0009] Data from the embodiments of this invention show that: (1) When the tyrosine at position 413 is mutated to cysteine, the expression level of the mutant protein is significantly increased.

[0010] (2) Compared with wild-type PfAgo protein, the PfAgo mutant protein M2A4 provided by the present invention has a catalytic efficiency of 5.76 times higher than that of target nucleic acid cleavage activity under optimal reaction conditions, and the catalytic efficiency of mutant protein M2A4-M has an 8.42 times higher than that of wild-type PfAgo protein.

[0011] (3) Among the PfAgo mutant proteins provided by this invention, the mutant protein M2A4 not only has a significantly enhanced activity compared to the wild-type protein, but also has an ionic strength range of (200-400 mmol L) -1 It maintains a relative activity of >97% even under NaCl conditions, demonstrating significantly enhanced salt tolerance.

[0012] The present invention also proposes a polynucleotide that encodes the PfAgo mutant protein as described above.

[0013] The sequences of the polynucleotides are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0014] The present invention also proposes an expression cassette, vector or transformant comprising the polynucleotides described above.

[0015] The present invention also proposes a host cell containing the polynucleotides described above or containing the expression cassette, vector or transformant described above.

[0016] The present invention also proposes a nucleic acid cleavage system, the nucleic acid cleavage system comprising: (a) Single-stranded guide nucleic acid; (b) The PfAgo mutant protein as described above, wherein the PfAgo mutant protein is a programmable endonuclease.

[0017] Preferably, the nucleic acid cleavage system further includes a reporter nucleic acid, wherein when the nucleic acid cleavage system is mixed with the nucleic acid molecule to be detected, the reporter nucleic acid is cleaved by the PfAgo mutant protein and detected.

[0018] Preferably, the reporter nucleic acid has a modifying group.

[0019] Preferably, the reporter nucleic acid is a fluorescent reporter nucleic acid, which has a fluorescent group and / or a quencher group.

[0020] Preferably, the fluorescent group and the quenching group are located on both sides of the complementary regions of the fluorescent reporter nucleic acid and the single-stranded guide nucleic acid, respectively.

[0021] Preferably, the length of the fluorescent reporter nucleic acid is 10-100 nt, more preferably 20-70 nt, more preferably 30-60 nt, more preferably 40-50 nt, and more preferably 45 nt.

[0022] Preferably, the fluorescent group includes at least one of FAM, HEX, CY5, CY3, VIC, JOE, TET, 5-TAMRA, ROX, and TexasRed-X.

[0023] Preferably, the quenching group includes at least one of BHQ, TAMRA, DABCYL, and DDQ.

[0024] Preferably, the single-stranded guide nucleic acid has a length of 12-64 nt, and the single-stranded guide nucleic acid is 5'P-gDNA or 5'OH-gDNA.

[0025] Preferably, the temperature of the nucleic acid cleavage system is 80-99.9℃, and more preferably 97℃.

[0026] This invention also proposes an application of the nucleic acid cleavage system described above in the specific cleavage of target DNA. Target DNA is added to the nucleic acid cleavage system, and the target DNA is complementary to the single-stranded guide nucleic acid sequence. The PfAgo mutant protein specifically cleaves the target DNA under the guidance of the single-stranded guide nucleic acid. Specifically, complementary pairing refers to: The target DNA has a nucleotide sequence that is completely complementary to the single-stranded guide nucleic acid sequence; Alternatively, the target DNA may have a nucleotide sequence with a single, two, three, four, or five base mismatches with the single-stranded guide nucleic acid sequence.

[0027] Preferably, the nucleic acid cleavage system contains Mn 2+ Mg 2+ Co 2+ At least one divalent metal ion.

[0028] Preferably, the divalent metal cation is Mn. 2+ and Mg 2+ More preferably, the divalent metal cation is Mn. 2+ .

[0029] Preferably, in the nucleic acid cleavage system, Mn 2+ Mg 2+ Co 2+ The concentration is 0.25-10 mmol / L. -1 .

[0030] Preferably, in the nucleic acid cleavage system, the final concentration of the divalent metal cation is 0.5-10 mmol / L. -1 Example data shows that the mutant M2A4 in Mn 2+ Concentration of 1-10 mmol / L-1 The relative activity is above 82%.

[0031] Preferably, the concentration of NaCl in the cutting system is ≤1000 mmol / L. -1 More preferably, it is 20-600 mmol / L. -1 .

[0032] This invention also proposes the application of the PfAgo mutant protein as described above in the preparation of nucleic acid detection kits.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The PfAgo mutant protein provided by this invention has achieved significant breakthroughs in target nucleic acid cleavage activity and practical performance. The M2A4 obtained through random mutation screening not only has significantly enhanced nucleic acid cleavage activity and protein expression level compared with the wild type, but also, through site-directed mutagenesis verification, it has been clarified that the key functional sites—the I623F mutation directly improves catalytic efficiency and high salt tolerance, and the Y413C mutation significantly optimizes protein expression level. This discovery reveals the correlation mechanism between the structure and function of PfAgo and provides molecular targets for precise modification. On this basis, the M2A4-M obtained through saturation mutation optimization has an 8.42-fold increase in catalytic efficiency compared with the wild type, completely breaking through the bottleneck of low catalytic activity of natural PfAgo, solving the core problem of "weak signal and slow reaction" in trace nucleic acid detection, and laying the enzymatic basis for direct detection without nucleic acid amplification. This progressive modification from random mutation to rational design not only achieves quantitative improvement in performance, but also clarifies the regulatory mechanism of key residues on enzyme function, providing a clear molecular logic for the functional optimization of Ago proteins.

[0034] (2) The high salt tolerance of the PfAgo mutant protein provided by this invention offers a key advantage for the detection of complex samples. M2A4 at 200-400 mmol•L -1NaCl maintains a relative activity of >97% over a wide ionic strength range, significantly superior to the wild type's sensitivity to salt concentration. This characteristic allows for direct application in the detection of samples from high-salt environments (such as seawater and saline lake microbial samples) or clinical specimens containing high-ionic-strength matrices (such as serum and urine), eliminating the need for complex desalination pretreatment steps, greatly simplifying the detection process, shortening pretreatment time, and reducing operational costs in practical applications. Simultaneously, combined with its enhanced catalytic activity, the mutant can rapidly recognize and cleave target nucleic acids in complex matrices, effectively resisting environmental interference and improving the stability and reliability of detection, providing a practical enzyme tool for scenarios such as on-site point-of-care testing. This invention not only elucidates a universal strategy for the engineering modification of residue side chains and establishes a semi-rational design framework for Ago protein engineering, but also provides a theoretical basis and universal strategy for the design of high-performance nucleases in extreme environments and the development of detection tools for low-abundance nucleic acid biomarkers. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 The diagram shows the principle of directed evolution of this invention (A) and the structure and mutation site diagram of mutant M2A4 (B).

[0037] Figure 2 The image shows the SDS-PAGE detection and analysis results of the four unit point mutant proteins of this invention.

[0038] Figure 3 This is a comparison of the fluorescence signal intensities generated by the reactions of wild-type PfAgo, mutant M2A4, and four unit point mutant proteins of this invention.

[0039] Figure 4 This is a graph showing the SDS-PAGE analysis results of the saturated mutant protein at site 623 of this invention.

[0040] Figure 5 This is a diagram illustrating the activity verification of the saturated mutant protein at site 623 of this invention.

[0041] Figure 6 This is an optimization diagram showing the optimal NaCl concentration, optimal MnCl2 concentration, optimal reaction temperature, and optimal reaction pH for the wild-type PfAgo, mutant M2A4, and mutant M2A4-M of this invention.

[0042] Figure 7 Representative curves of the initial reaction rate versus time and Michaelis-Menten fitting plots of wild-type PfAgo, mutant M2A4, and mutant M2A4-M with different concentrations of substrate at 97°C.

[0043] Figure 8 This is a standard curve of fluorescence signal intensity versus single-stranded DNA when detecting single-stranded DNA using wild-type PfAgo, mutant M2A4, and mutant M2A4-M.

[0044] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0047] Example 1: Screening and preparation of PfAgo mutants 1. Constructing a PfAgo random mutant library based on directed evolution Constructing a PfAgo mutant library using error-prone PCR (the principle is as follows) Figure 1 (As shown), the specific operation steps are as follows: (1) Template preparation: The recombinant plasmid PfAgo-pET28a containing the wild-type PfAgo gene (containing the PfAgo gene fragment shown in SEQ ID NO.4) was used as the template; (2) Error-prone PCR amplification of the target gene: Using PfAgo-PET28a plasmid as a template, Error-F primer (5'-GGGAATTCCATATGAAGGCGAAAGTGGTT-3') and Error-R primer (5'-TCCGCTCGAGTTACACAAAGTACAGGAAG-3') were used as forward and reverse primers, respectively. PCR amplification was performed using low-fidelity Taq DNA polymerase. The PCR amplification reaction system is shown in Table 1. The reaction program was as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 58°C annealing for 30 s, 72°C extension for 2 min, for a total of 30 cycles; 72°C incubation for 5 min to obtain the PfAgo gene fragment containing random mutations. Table 1 Commonly Misunderstood PCR Reaction Systems Ingredients Volume (μL) PfAgo plasmid DNA 1 Error-F primer (10 μmol L -1 )]]> 1 Error-R primer (10 μmol L -1 )]]> 1 [dNTPs (2.5 mmol L -1 )]]> 4 [dCTP (25 mmol L -1 )]]> 1 [CAT] dTTP (25 mmol L -1 )]]> 1 10x Buffer 5 EasyTaq DNA polymerase 1 MnCl2(15 mmol L -1 )]]> 1 diH2O 34 (3) Enzyme digestion and ligation: The mutant PfAgo gene fragment and the vector pET-28a were double-digested with restriction endonucleases NdeI and XhoI, respectively, to produce complementary sticky ends. After purification, the digestion products were ligated with T4 DNA ligase to construct a plasmid containing the random mutant gene pET-28a-PfAgo. (4) Transformation and construction of random mutant libraries Wild-type PfAgo plasmid and its mutant plasmid were transformed into Escherichia coli BL21(DE3) competent cells to obtain a random mutant library.

[0048] The amino acid sequence of the wild-type PfAgo protein is shown in SEQ ID NO.1.

[0049] 2. Screening of PfAgo dominant mutants (1) Strain preparation and initial culture: Wild-type PfAgo plasmid and its mutant plasmid were transformed into Escherichia coli BL21(DE3) competent cells to obtain recombinant strains containing different PfAgo genes. Single clones were picked and inoculated into 200 µL of liquid LB medium (containing 50 μg / mL kanamycin). -1 Seed culture was obtained by shaking culture at 37°C and 180 r / min for 12 h in a 96-well deep plate.

[0050] (2) Induction of target protein expression: 20 µL of seed culture was transferred to each well to be pre-filled with 600 µL of LB medium (containing kanamycin to a concentration of 50 μg / mL). -1 The IPTG concentration was 0.2 mmol / L. -1Another 96-well culture plate was used. The 96-well culture plate was placed in a constant temperature shaker and induced at 28°C and 200 r / min for 6 h to fully express the target protein.

[0051] (3) Preparation of crude enzyme solution: Then add 20 µL of T7 phage to each well, transfer the 96-well culture plate to 22°C and 200 r / min for 2-3 h until the culture medium becomes clear, and then transfer the 96-well culture plate to 75°C water bath for 30 min to remove impurities and obtain crude enzyme solution.

[0052] (4) Enzyme activity detection and mutant screening: Take 10 µL of crude enzyme solution (the supernatant after lysis, containing the target protein) and add gDNA (1 μmol L). -1 ) 1 µL, ssDNA-FQ (2 µmol L -1 3 µL of PfAgo reaction buffer, 5 µL of DIwater, and 1 µL of DIwater were added, and the mixture was reacted at 95°C for 30 min. The fluorescence values ​​(Ex / Em: 485 / 520) were then read using a fluorescence microplate reader. Mutants with fluorescence values ​​greater than the control were selected for further screening and verification. The resulting positive mutant was sequenced and named M2A4 (amino acid sequence as shown in SEQ ID NO.2, mutation sites: V142A, Y413C, K417R, and I623F).

[0053] 3. Determination of the specific function of each mutation site To clarify the specific functions of each mutation site in M2A4 (containing four mutation sites: V142A, Y413C, K417R, and I623F), site-directed mutagenesis was performed on wild-type PfAgo using circular PCR, constructing four single-site mutants. Protein expression levels of the mutants were determined by Western blot, and nucleic acid cleavage activity was detected by fluorescence assay. The results are as follows: Figure 2 Figure 3 As shown, the key site affecting the catalytic efficiency and high salt tolerance of M2A4 is I623F, and the key site affecting the protein expression level is Y413C.

[0054] 4. Screening for dominant mutants using saturation mutations at site 623. Based on mutant M2A4, the folding free energy of the virtual saturation mutation at position 623 was calculated using YASARA-FoldX software. Subsequently, primers were designed using site-directed mutagenesis to construct a library of saturation mutants targeting position 623. The primer sequences are shown in Table 2. After screening, the dominant mutant M2A4-M was obtained (amino acid sequence shown in SEQ ID NO.3).

[0055] The nucleotide sequence encoding the mutant M2A4 protein is shown in SEQ ID NO.5; the nucleotide sequence encoding the mutant M2A4-M protein is shown in SEQ ID NO.6.

[0056] Table 2. Primer sequences for saturation mutations 5. Expression and purification of PfAgo wild-type and mutant proteins First, inoculate 1% of the wild-type and mutant *E. coli* BL21(DE3)-pET28a-PfAgo culture into a small shake flask containing 50 mL LB liquid medium (containing kanamycin) and incubate overnight at 37°C with a speed of 220 rpm. Then, transfer the overnight culture to a shake flask containing 1 L LB liquid medium (containing kanamycin) and incubate at 37°C with a speed of 220 rpm until OD (outcome limit) is reached. 600 When the value reaches 0.8-1.0, remove the shake flask from the shaker and place it on ice for 15 min, then add 0.2 mmol / L to the final concentration. -1 After inducing expression with IPTG at 18℃ and 220 r / min for 20 h, the bacterial cells were collected by centrifugation.

[0057] Use resuspension buffer (containing 20 mM Tris-HCl, pH approximately 8.0, 250 mmol / L) -1 The bacterial cells were resuspended in NaCl, then autoclaved, and the supernatant was obtained by centrifugation. The protein was purified using a Ni-NTA column affinity chromatography. The eluent was concentrated by ultrafiltration and desalted to obtain the purified protein. The purified protein was then validated for purity using SDS-PAGE gel chromatography. Figure 4 As shown, 10 mutants were successfully expressed, and the activity of 9 of these mutants was verified, yielding... Figure 5 ,Depend on Figure 5 It can be seen that, except for the three mutants M2A4-W623, M2A4-A623, and M2A4-Y623, whose enzyme activity catalytic efficiency was lower or basically the same as that of the wild type (WT), the remaining mutants were stronger than the wild type. The purified protein was stored in a solution containing 20 mmol / L... -1 The protein was prepared in Tris-HCl buffer and the concentration was determined by the Bradford method. The protein was then stored at -80°C for later use.

[0058] Example 2 Performance determination of PfAgo wild-type and mutant proteins 1. Determination of optimal reaction conditions for PfAgo wild-type and mutant proteins The optimal reaction conditions for PfAgo wild-type and screened mutant proteins were determined using gDNA and substrate ssDNA-FQ as shown in Table 3 for cleavage experiments. The optimal NaCl and MnCl2 concentrations, optimal reaction temperature, and optimal pH of the reaction buffer were determined by adjusting the concentrations and pH of the buffer components. The reaction system was placed in a real-time quantitative PCR instrument, and the FAM fluorescence signal intensity was collected every 30 seconds for a total of 30 collections before the program was terminated. Each substrate concentration gradient was performed in triplicate, and the results are shown below. Figure 6 As shown, compared to the wild-type PfAgo, mutants M2A4 and M2A4-M still exhibited higher activity across a wider range of NaCl and MnCl2 concentrations, indicating that mutants M2A4 and M2A4-M possess stronger salt tolerance. Further analysis showed that both also slightly outperformed the wild-type in terms of pH and temperature response characteristics.

[0059] Table 3 Sequences of gDNA and substrate ssDNA-FQ Name Sequence (5'-3') gDNA GAGGTCCTCGAGGTATG ssDNA-FQ 6FAM-CATACCTCGAGGACCTC-BHQ1 2. Determination of kinetic parameters of PfAgo wild-type and mutant proteases This example designed a final concentration gradient of the substrate ssDNA-FQ (0.10 μmol L). -1 0.20 μmol L -1 0.30 μmol L -1 0.50 μmol L -1 0.60 μmol L -1 0.80 μmol L -1 1.00 μmol L -1 1.20 μmol L -1 1.50 μmol L -1 1.80 μmol L -1 2.00 μmol L -1 2.40 μmol L -1 3.00 μmol L -1 The reaction system is shown in Table 4, where the reaction buffer represents the optimal reaction conditions for each mutant after assay. The following experimental conditions were used for the assay: the reaction system was placed in a real-time quantitative PCR instrument and incubated at 97°C. FAM fluorescence signal intensity was collected every 30 seconds for a total of 30 collections before the program terminated. Three replicates were performed for each substrate concentration gradient to ensure more accurate determination of the enzyme kinetic constant. Nonlinear fitting of the experimental data was performed using GraphPad Prism 8 software (e.g., ...). Figure 7The enzyme kinetic parameters were calculated using the Michaelis-Menten model: Michaelis constant (K... m ), maximum reaction rate (V max ) and catalytic constant (k cat The results are shown in Table 5.

[0060] Table 4 Reaction System Ingredients Volume (μL) Mutant protein (4.00 mg mL -1 ])]] 1.00 [CD AT] gDNA (2.00 pmol L -1 ])]] 0.50 ssDNA-FQ (0.10-3.00 μmol L -1 )]]> 2.00 2x Reaction Buffer 10.00 Deionized water 6.50 Table 5 Enzyme kinetic parameters Protein K m (M -1 )]]> k cat ( sec -1 )]]> k cat / K m ]]> WT 4.66 ± 2.04 x 10 -6 ]] 1892.60 ± 61.20 4.06 x 10 8 ]] [M2A4] 0.62 ± 0.54 x 10 -6 ]] 1463.00 ± 47.40 2.34 x 10 9 ]]> [M2A4-M] 0.48 ± 0.10 x 10 -6 ]] 1641.20 ± 14.00 3.42 x 10 9 ]] As shown in Table 5, compared with the wild type (WT), mutants M2A4 and M2A4-M increased their affinity for the substrate (reduced KL) by [the following method is needed]. m This significantly improved the overall catalytic efficiency (k). cat / K m Among them, M2A4-M, through further optimization, became a mutant with better catalytic performance.

[0061] 3. Determination of the sensitivity of wild-type PfAgo and mutant proteins to single-stranded DNA detection. In the detection system, the concentrations of wild-type PfAgo protein and mutant protein were set at the same level, and the concentration of the ssDNA-FQ probe was 0.6 μmol / L. -1 The 5'-P-gDNA was diluted to different concentration gradients, and the reactions were carried out according to the optimal reaction conditions for each protein as determined above. The results are as follows: Figure 8 As shown, the detection limit for wild-type PfAgo is calculated to be 129 pmol L / L. -1 The detection limit for the mutant protein M2A4 is 24 pmol / L. -1 The detection limit for the mutant protein M2A4-M is 13 pmol / L. -1 .

[0062] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A PfAgo mutant protein with enhanced target nucleic acid cleavage activity, characterized in that, The PfAgo mutant protein is obtained by mutating the wild-type PfAgo protein, whose amino acid sequence is shown in SEQ ID NO.1, by the following (1) or (2): (1) V142A, Y413C, K417R, I623F; (2) Based on (1), the 623rd amino acid is further mutated to methionine.

2. A polynucleotide, characterized in that, The polynucleotide encodes the PfAgo mutant protein as described in claim 1.

3. An expression cassette, vector, or transformant comprising the polynucleotide as described in claim 2.

4. A host cell, characterized in that, The host cell contains the polynucleotide as described in claim 2 or the expression cassette, vector, or transformant as described in claim 3.

5. A nucleic acid cleavage system, characterized in that, The nucleic acid cleavage system includes: (a) Single-stranded guide nucleic acid; (b) The PfAgo mutant protein of claim 1, wherein the PfAgo mutant protein is a programmable endonuclease.

6. The nucleic acid cleavage system according to claim 5, characterized in that, The single-stranded guide nucleic acid has a length of 12-64 nt and is 5'P-gDNA or 5'OH-gDNA.

7. The application of the nucleic acid cleavage system as described in claim 5 in the specific cleavage of target DNA, characterized in that, Target DNA is added to the nucleic acid cleavage system, and the target DNA is complementary to the single-stranded guide nucleic acid sequence. The PfAgo mutant protein specifically cleaves the target DNA under the guidance of the single-stranded guide nucleic acid. Specifically, complementary pairing refers to: The target DNA has a nucleotide sequence that is completely complementary to the single-stranded guide nucleic acid sequence; Alternatively, the target DNA may have a nucleotide sequence with a single, two, three, four, or five base mismatches with the single-stranded guide nucleic acid sequence.

8. The application according to claim 7, characterized in that, The nucleic acid cleavage system includes Mn 2+ Mg 2+ Co 2+ At least one divalent metal ion.

9. The application according to claim 8, characterized in that, In the nucleic acid cleavage system, Mn 2+ Mg 2+ Co 2+ The concentration is 0.25-10 mmol / L. -1 .

10. The use of the PfAgo mutant protein as described in claim 1 in the preparation of a nucleic acid detection kit.

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