Highly active T4 DNA ligase mutants, their preparation methods and applications

Abstract

This invention discloses a highly active T4 DNA ligase mutant, its preparation method, and its applications. In this invention, through crystal structure and bioinformatics analysis combined with site-directed mutagenesis, saturation mutagenesis, and combinatorial mutagenesis, as well as rigorous experimental analysis, several T4 DNA ligase mutants with significantly improved performance were screened, including single mutants and combinatorial mutants. The improved performance includes: enzyme activity, thermal stability, and / or ligation efficiency.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and enzymology. More specifically, this invention relates to a highly active T4 DNA ligase mutant, its preparation method, and its application. Background Technology

[0002] T4 DNA ligase (T4Lig) is a key enzyme in the DNA ligation process. It catalyzes the chemical reaction between the 3'-hydroxyl group (3'-OH) and the 5'-phosphate group (5'-PO4) of the DNA strands between two double-stranded DNA strands, forming a stable 3'-5'-phosphodiester bond, thus achieving precise ligation of the DNA strands.

[0003] In molecular cloning, T4 DNA ligase ligates vector DNA and insert fragments to construct recombinant DNA molecules, a fundamental step in genetic engineering. In DNA repair and recombination, T4 DNA ligase participates in the DNA repair process within cells and can be used in vitro to ligate PCR products and synthesize adapters. In sequencing library construction, such as in next-generation sequencing (NGS), T4 DNA ligase is used to ligate DNA fragments to sequencing adapters.

[0004] T4 DNA ligase can ligate sticky ends (high efficiency) and blunt ends (low efficiency, usually requiring auxiliary conditions such as the addition of PEG or increased enzyme concentration), while E. coli DNA ligase mainly ligates sticky ends; the catalytic reaction of T4 DNA ligase requires ATP as an energy donor, while E. coli DNA ligase requires NAD+. + As an energy donor.

[0005] Currently, researchers' studies on the ligation mechanism of T4 DNA ligase mainly focus on nick DNA (nDNA), while the understanding of the ligation process of linear DNA (L-DNA) with blunt or sticky ends is still insufficient.

[0006] Although T4 DNA ligase is widely used in laboratories both domestically and internationally for DNA ligation experiments, in-depth research on T4 DNA ligase is relatively limited. The general consensus is that T4 DNA ligase catalyzes the formation of 3'-5' phosphodiester bonds in blunt-ended L-DNA, achieving complete double-strand ligation and constructing a covalently closed circular DNA structure. However, in practical applications, especially during intramolecular ligation using blunt-ended L-DNA to construct vectors, base deletions at the ligation ends are frequently observed. This indicates that there are still unresolved details in its ligation mechanism, potentially limiting ligation efficiency. Nevertheless, in the field of high-throughput sequencing, efficient DNA ligation is a core step in library construction, directly impacting sequencing data quality and experimental efficiency.

[0007] With advancements in medical technology and the development of precision medicine, personalized treatment is gradually becoming dominant in diseases such as cancer. The high-throughput and low-cost characteristics of high-throughput sequencing technology provide a solid foundation for personalized medicine. By analyzing patient sample DNA libraries, gene mutation information can be rapidly obtained to formulate precise treatment plans. Therefore, modifying and optimizing the activity of T4 DNA ligase to meet the performance requirements of different application scenarios has become a continuously attracting research hotspot in this field.

[0008] Therefore, modifying the activity of T4 DNA ligase to improve its ligation efficiency has become the key to promoting the development of high-throughput sequencing technology and is also the top priority of current research. Summary of the Invention

[0009] The purpose of this invention is to provide a highly active T4 DNA ligase mutant, its preparation method, and its application.

[0010] In a first aspect of the present invention, a method for enhancing the performance of T4 DNA ligase is provided, comprising: mutating T4 DNA ligase to form a T4 DNA ligase mutant, wherein the modification comprises: mutating amino acid residue D at positions 112, 371 and / or 448 corresponding to the amino acid sequence shown in SEQ ID NO: 1; wherein the performance comprises: enzyme activity, thermal stability and / or ligation efficiency.

[0011] In one or more embodiments, the "thermal stability" is characterized by retaining enzyme activity after incubation at a temperature greater than 40°C for 30 minutes; preferably, it retains more than 40% of its activity.

[0012] In one or more embodiments, the method includes performing the following mutations corresponding to the amino acid sequence shown in SEQ ID NO: 1: A. Mutations selected from (a)-(c) or combinations thereof (unless otherwise stated, hereinafter referred to as protein A): (a) The D at position 112 is mutated to I (M51), C (M49), V (M52), A (M12), or G (M50); (b) The 371st position is mutated from D to S (M10) or N (M9); (c) The 448th position is mutated from D to A (M53) or Q (M45); B. A derivative protein that has the function of the protein in item A, formed by substituting, deleting, or adding one or more (e.g., 1-20; preferably 1-15; more preferably 1-10, such as 8, 6, 5, 4, 2) amino acid residues, but whose corresponding sites defined in (a)-(c) of item A are conserved (the amino acid sequence remains unchanged). C. A derivative protein that has 80% or more (preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, such as 98% or more, 99% or more) homology with the amino acid sequence of the protein in item A and has the function of the protein in item A, but the sites corresponding to (a)-(c) in item A are conserved.

[0013] In one or more embodiments, in item A, the combination includes: The 112th D position mutates to I, the 448th D position mutates to A, and the 371st D position mutates to S; The 112th D position mutates to I, the 448th D position mutates to A, and the 371st D position mutates to N; or The 112th D position mutates to I, and the 448th D position mutates to A.

[0014] In another aspect of the invention, a T4 DNA ligase mutant is provided, corresponding to the amino acid sequence shown in SEQ ID NO: 1, wherein amino acid residue D at positions 112, 371 and / or 448 is mutated; it has enhanced enzyme activity, thermostability and / or ligation efficiency.

[0015] In one or more embodiments, the "enhanced enzyme activity, thermal stability, and / or ligation efficiency" refers to enzyme activity, thermal stability, and / or ligation efficiency that are significantly higher than those of wild-type T4 DNA ligase (SEQ ID NO: 1).

[0016] In one or more embodiments, in the T4 DNA ligase mutant, position 371 is not mutated alone; preferably, position 371 is mutated simultaneously with position 112 or with position 448.

[0017] In one or more embodiments, in the T4 DNA ligase mutant, position 448 is not mutated alone; preferably, position 448 is mutated simultaneously with position 112 or with position 371.

[0018] In one or more embodiments, the T4 DNA ligase mutant includes the amino acid sequence corresponding to SEQ ID NO:1, which undergoes the following mutations: A. Mutations selected from (a)-(c) or combinations thereof: (a) The D at position 112 is mutated to I (M51), C (M49), V (M52), A (M12), or G (M50); (b) The 371st position is mutated from D to S (M10) or N (M9); (c) The 448th position is mutated from D to A (M53) or Q (M45); B. A derivative protein that has the function of the protein in item A, formed by substituting, deleting, or adding one or more (e.g., 1-20; preferably 1-15; more preferably 1-10, such as 8, 6, 5, 4, 2) amino acid residues, but whose corresponding sites defined in (a)-(c) of item A are conserved (the amino acid sequence remains unchanged). C. A derivative protein that has 80% or more (preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, such as 98% or more, 99% or more) homology with the amino acid sequence of the protein in item A and has the function of the protein in item A, but the sites corresponding to (a)-(c) in item A are conserved.

[0019] In one or more embodiments, in item A, the combination includes: The 112th D position mutates to I, the 448th D position mutates to A, and the 371st D position mutates to S; The 112th D position mutates to I, the 448th D position mutates to A, and the 371st D position mutates to N; or The 112th D position mutates to I, and the 448th D position mutates to A.

[0020] In another aspect of the invention, a polynucleotide is provided, said polynucleotide encoding said T4 DNA ligase mutant.

[0021] In another aspect of the invention, a vector or a genetically engineered host cell containing the vector is provided, wherein the vector contains the polynucleotide; or, the host cell contains the vector or the polynucleotide integrated into its genome.

[0022] In another aspect of the invention, a method for producing a mutant of the T4 DNA ligase is provided, comprising the steps of: (i) Culturing the host cells; (ii) Collect cultures containing the T4 DNA ligase mutant described above; (iii) Isolate the T4 DNA ligase mutant from the culture.

[0023] In another aspect of the invention, the use of the T4 DNA ligase mutant, a host cell expressing the mutant, or a lysis product thereof is provided for the ligation of DNA strands.

[0024] In one or more embodiments, the T4 DNA ligase mutant catalyzes a chemical reaction between the 3'-hydroxyl group (3'-OH) and the 5'-phosphate group (5'-PO4) of the DNA strands between two double-stranded DNA (fragments) to form a stable 3'-5'-phosphodiester bond, thereby achieving precise ligation of the DNA strands.

[0025] In one or more embodiments, the double-stranded DNA (fragment) may include (but is not limited to): a vector fragment or plasmid fragment, or a DNA insert fragment.

[0026] In one or more embodiments, the double-stranded DNA (fragment) may be (but is not limited to): two or more short-stranded DNA or long-stranded DNA.

[0027] In one or more embodiments, the DNA fragment is double-stranded.

[0028] In another aspect of the present invention, a method for catalyzing the ligation of DNA chains is provided, comprising: using the T4 DNA ligase mutant, a host cell expressing the mutant, or its lysis product for catalysis, to cause the DNA chains (DNA fragments) to ligate.

[0029] In another aspect of the invention, a kit for performing DNA ligation is provided, the kit comprising: the T4 DNA ligase mutant; or, an expression vector or host cell expressing the T4 DNA ligase mutant.

[0030] In one or more embodiments, the kit further includes reagents selected from the group consisting of: T4 DNA ligase buffer, adenosine triphosphate (ATP), nuclease-free water, and DNA template (optional).

[0031] In one or more embodiments, the T4 DNA ligase buffer provides a suitable pH environment and ionic strength.

[0032] In one or more embodiments, the adenosine triphosphate serves as an energy source to drive the linkage reaction.

[0033] In one or more embodiments, the nuclease-free water is used to dissolve reagents and adjust reaction volume to avoid nuclease contamination.

[0034] In one or more embodiments, the application of the DNA template includes: in certain ligation experiments, adding a small amount of uncut vector DNA as a positive control to verify the effectiveness of the ligation reaction.

[0035] In another aspect of the invention, a DNA ligation system is provided, comprising the T4 DNA ligase mutant described above; preferably, the DNA ligation system further comprises: T4 DNA ligase buffer, adenosine triphosphate (ATP), nuclease-free water, and / or a DNA template (optional).

[0036] In another aspect of the present invention, a library of T4 DNA ligase mutants or polynucleotides encoding them is provided, including two or more of the aforementioned T4 DNA ligase mutants, such as 3, 4, 5, 6, 7, 8, 9, etc.; preferably including all of the aforementioned T4 DNA ligase mutants.

[0037] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Attached Figure Description

[0038] Figure 1 A bar chart comparing the activity of wild-type T4 DNA ligase and enzymes from various single-point mutants.

[0039] Figure 2 Thermostability study of wild-type T4 DNA ligase and D371 site mutant.

[0040] Figure 3A bar chart comparing the activities of wild-type T4 DNA ligase and various saturated mutant enzymes; the horizontal axis represents the type of residues after mutation. The difference was statistically significant (p < 0.0001).

[0041] Figure 4 Bar chart comparing the relative activities of wild-type T4 DNA ligase and various combined mutants.

[0042] Figure 5 Comparison of enzyme activity and thermostability between wild-type T4 DNA ligase and triplet mutant. The difference was statistically significant (p < 0.0001).

[0043] Figure 6 The ligation efficiency was determined by gray-scale quantification after electrophoresis of the ligation products catalyzed by wild-type T4 DNA ligase and mutants. Detailed Implementation

[0044] In this invention, through crystal structure and bioinformatics analysis combined with site-directed mutagenesis, saturation mutagenesis, and combinatorial mutagenesis, as well as rigorous experimental analysis, several T4 DNA ligase mutants with significantly improved performance were screened, including single mutants and combinatorial mutants. The improved performance includes enzyme activity, thermal stability, and / or ligation efficiency.

[0045] the term

[0046] As used herein, unless otherwise stated, the terms "T4 DNA ligase mutant" and "mutant T4 DNA ligase" are used interchangeably. They refer to an enzyme (peptide / protein) formed by mutation at certain sites related to the enzyme's activity / thermal stability as determined by the inventors, corresponding to the original T4 DNA ligase. Preferably, the mutation corresponds to the amino acid sequence shown in SEQ ID NO: 1. The mutation is selected from the following sites (amino acid residue D site) or combinations thereof: position 112, position 371, or position 448.

[0047] If a representation of the pre-mutant T4 DNA ligase is required, it can be an enzyme with an amino acid sequence such as SEQ ID NO: 1. Unless otherwise stated, the mutation sites of the mutants in this invention are based on the sequence shown in SEQ ID NO: 1.

[0048] In this invention, unless otherwise stated, the T4 DNA ligase mutant is identified by “the amino acid that was replaced at the original amino acid position” to indicate the mutated amino acid in the T4 DNA ligase mutant, such as D112C, which means that the amino acid at position 112 is replaced by C from the amino acid residue D of the starting enzyme.

[0049] As used herein, "isolated T4 DNA ligase" refers to a T4 DNA ligase mutant that is substantially free of other naturally occurring proteins, lipids, carbohydrates, or other substances associated with it. Those skilled in the art can purify the T4 DNA ligase mutant using standard protein purification techniques. A substantially pure protein will produce a single master band on a non-reducing polyacrylamide gel.

[0050] As used in this article, "enhanced / improved enzyme activity or thermostability" refers to a statistically significant enhancement / improvement, or a marked enhancement / improvement, of the enzyme activity or thermostability of the mutated T4 DNA ligase compared to the original T4 DNA ligase starting peptide. For example, under the same reaction conditions / environment, after a certain reaction time, the mutant T4 DNA ligase with enhanced enzyme activity or thermostability shows a significant increase of more than 5%, 10%, 20%, 30%, 50%, 70%, 80%, 100%, or 150% in enzyme activity or thermostability compared to the original enzyme.

[0051] As used herein, "a library of T4 DNA ligase mutants or polynucleotides encoding them" refers to a collection of polypeptides or polynucleotides containing a series of mutant T4 DNA ligases provided by this invention. The assembly of multiple T4 DNA ligase mutants or polynucleotides encoding them with different enzyme activities or thermostabilities into a library facilitates the selection of a suitable T4 DNA ligase or its encoded nucleic acid by those skilled in the art based on their required reaction conditions.

[0052] As used herein, the terms “containing” or “comprising” include “comprising,” “substantially constitutes,” and “consisting of.” The term “substantially constitutes” means that, in addition to containing the essential components or essential parts, the composition / reaction system / kit may contain small amounts of minor components and / or impurities that do not affect the active ingredient.

[0053] As used herein, the term "effective amount" refers to the amount that produces the functional or enzymatic activity of the reaction (DNA ligation reaction) of interest in this invention, achieving the desired effect (accurate detection results).

[0054] T4 DNA ligase mutant and its construct

[0055] To improve the substrate recognition, binding, and ligation efficiency of T4 DNA ligase, i.e., to significantly improve enzyme activity and achieve efficient production of highly active T4 DNA ligase for better application in various related technologies, the inventors, based on their research experience with previously developed T4 DNA ligase reporter gene system products, analyzed the three-dimensional spatial results of wild-type T4 DNA ligase and substrate complexes, combined with EVcoupling bioinformatics, and performed homologous sequence alignment analysis of T4 DNA ligase. They calculated the correlation between amino acid residues, mapped the generated protein co-evolutionary network onto reference sequences with high similarity, selected key amino acid residues with co-evolutionary relationships, and optimized the molecular structure and improved enzyme activity of wild-type T4 DNA ligase using a combination of site-directed mutagenesis, saturation mutagenesis, and synergistic mutagenesis. Through in-depth experimental analysis, they obtained a class of T4 DNA ligase mutants with significantly improved performance, including improved enzyme activity, thermostability, and / or ligation efficiency.

[0056] After confirmation by sequencing, the mutant plasmid was simultaneously transformed into competent *E. coli* cells along with the wild-type plasmid. Following culture and induction, *E. coli* bacterial cultures expressing T4 DNA ligase were obtained. T4 DNA ligase was released by cell lysis, and the T4 DNA ligase proteins were purified. The enzyme activities of each mutant were then detected and compared. Using this strategy, T4 DNA ligase mutants with higher activity and better thermostability were successfully screened. The D112 site mutant was found to significantly increase enzyme activity, as was the mutation of the D448 site to A or Q, which also significantly enhanced enzyme activity. Furthermore, an appropriate mutant of the D371 site was found to improve thermostability. More importantly, the D112 site can combine with the D448 and / or D371 sites to form mutants with even higher enzyme activity and better stability.

[0057] The T4 DNA ligase mutant of the present invention can be a chemically synthesized product or produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology.

[0058] This invention also includes fragments, derivatives, and analogs of the T4 DNA ligase mutant. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to proteins that substantially retain the same biological function or enzymatic activity as the native T4 DNA ligase mutant of this invention. The protein fragments, derivatives, or analogs of this invention may be (i) proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by fusing additional amino acid sequences to this protein sequence (such as leader sequences, secretory sequences, sequences used to purify this protein, or proteomic sequences, or fusion proteins). These fragments, derivatives, and analogs are within the scope of knowledge known to those skilled in the art as defined herein. However, a condition must be met: the amino acid sequence of the T4 DNA ligase mutant and its fragments, derivatives, and analogs must necessarily contain at least one mutation specifically pointed out above in this invention.

[0059] In this invention, the term "T4 DNA ligase mutant" also includes (but is not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-20, more preferably 1-10, and even more preferably 1-8, 1-5, 1-3, or 1-2), and the addition or deletion of one or more amino acids (typically up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition or deletion of one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein. The term also includes enzyme-active fragments and enzyme-active derivatives of the T4 DNA ligase mutant. However, at least one mutation described above in this invention is certainly present in these variant forms.

[0060] In this invention, the term "T4 DNA ligase mutant" also includes (but is not limited to): derived proteins that retain the protease activity of the T4 DNA ligase mutant and have at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, such as at least 98% or 99%, sequence identity with the amino acid sequence of the T4 DNA ligase mutant. Similarly, these derived proteins certainly contain at least one mutation as described above in this invention.

[0061] The present invention also provides analogs of the T4 DNA ligase mutant. These analogs may differ from the T4 DNA ligase mutant in that they may differ in amino acid sequence, in the form of modification that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to a mutagen, site-directed mutagenesis, or other known molecular biology techniques. Analogs also include those having residues different from naturally occurring L-amino acids (e.g., D-amino acids), and those having non-naturally occurring or synthetic amino acids (e.g., β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above. Modifications (generally without altering the primary structure) include chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those polypeptides resulting from glycosylation modifications during or after polypeptide synthesis and processing. Such modifications can be accomplished by exposing the polypeptide to glycosylation enzymes (e.g., mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, and phosphotythreonine). They also include peptides modified to improve their resistance to proteolysis or optimize their solubility.

[0062] The present invention also provides a multinucleotide sequence encoding the T4 DNA ligase mutant of the present invention or a conserved variant thereof.

[0063] The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

[0064] The polynucleotide encoding the mature protein of the mutant includes: a coding sequence that encodes only the mature protein; a coding sequence for the mature protein and various additional coding sequences; a coding sequence for the mature protein (and optional additional coding sequences) and a non-coding sequence.

[0065] "A polynucleotide encoding a protein" can be a polynucleotide that includes the protein itself, or it can include polynucleotides that also include additional coding and / or non-coding sequences.

[0066] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered using the vectors of the present invention or T4 DNA ligase mutant coding sequences, and methods for generating the mutant enzymes of the present invention via recombination technology.

[0067] Recombinant T4 DNA ligase mutants can be expressed or produced using the polynucleotide sequence of the present invention via conventional recombinant DNA technology. The process includes the following steps: (1) transforming or transducing suitable host cells with the polynucleotide (or variant) encoding the T4 DNA ligase mutant of the present invention, or with a recombinant expression vector containing the polynucleotide; (2) culturing the host cells in a suitable culture medium; and (3) isolating and purifying the protein from the culture medium or cells.

[0068] In this invention, the T4 DNA ligase mutant polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, 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 remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0069] Methods well known to those skilled in the art can be used to construct expression vectors containing a T4 DNA ligase mutant encoding DNA sequence and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. The expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells.

[0070] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0071] In this invention, the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a mold cell or a yeast cell; or a higher eukaryotic cell, such as a plant cell.

[0072] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells. In a preferred embodiment of the present invention, the expression vector used is a pET vector; and the microbial host cells transformed by the expression vector are all *Escherichia coli*.

[0073] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0074] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their 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 precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0075] Application of T4 DNA ligase mutant

[0076] The present invention relates to a class of T4 DNA ligase mutants with significantly enhanced enzyme activity, wherein the D112 site mutant can significantly enhance enzyme activity, the D448 site mutation to A or Q can also significantly enhance enzyme activity, the appropriate D371 site mutant can significantly enhance enzyme thermostability, and more importantly, the D112 site can be combined with the D448 and / or D371 sites to form mutants with higher enzyme activity and better thermostability.

[0077] Having obtained the T4 DNA ligase mutant enzyme of the present invention, those skilled in the art can readily apply this enzyme to catalyze the ligation of DNA strands, as indicated by the present invention. In a preferred embodiment, the catalysis is carried out in a suitable reaction system, which may include: T4 DNA ligase buffer, adenosine triphosphate (ATP), nuclease-free water, and a DNA template (optional).

[0078] The T4 DNA ligase mutant of the present invention has wide applications in multiple fields of molecular biology, including in vitro nucleic acid analysis, vector preparation, DNA ligation, and ligation into complete genomic DNA during the synthesis of Okazaki fragments in vivo.

[0079] The applications of the T4 DNA ligase of this invention are not limited to the ligation of sticky-end and blunt-end double-stranded DNA, but also include the ligation of double-stranded oligonucleotide adapters to double-stranded DNA, the repair of gaps in double-stranded DNA and RNA or DNA-RNA complexes, ligase-mediated RNA detection, site-specific amplification, and analysis of amplified fragment length polymorphism.

[0080] In high-throughput sequencing library construction, the T4 DNA ligase of this invention significantly improves library construction efficiency and quality due to its high activity and thermostability. With advancements in medical technology and the development of precision medicine, personalized treatment is gradually becoming dominant in diseases such as cancer. The high-throughput and low-cost characteristics of high-throughput sequencing technology provide a solid foundation for personalized medicine. By analyzing patient sample DNA libraries, gene mutation information can be rapidly obtained to formulate precise treatment plans. The optimized T4 DNA ligase of this invention provides a superior approach for this application.

[0081] It should be understood that the T4 DNA ligase mutant of the present invention, which has higher enzyme activity and thermostability, is applicable to a wide variety of ligation reaction methods known or under development in the art and has broad applicability.

[0082] The present invention also provides a DNA ligation reaction system containing an effective amount of T4 DNA ligase mutant.

[0083] The present invention relates to the T4 DNA ligase mutant and other components required for detection. These components include (but are not limited to) components selected from the group consisting of: T4 DNA ligase buffer, adenosine triphosphate (ATP), nuclease-free water, and DNA template (optional).

[0084] This invention also provides a detection system comprising: the T4 DNA ligase mutant of this invention, T4 DNA ligase buffer, adenosine triphosphate (ATP), nuclease-free water, and / or a DNA template (optional). In this detection system, when the DNA (fragment) to be ligated is added, a ligation reaction can occur efficiently, yielding the assay results.

[0085] To facilitate expanded or commercial applications, this invention also provides a detection system or kit, comprising: the T4 DNA ligase mutant of this invention. Preferably, the kit may further comprise reagents selected from the group consisting of: T4 DNA ligase buffer, adenosine triphosphate (ATP), nuclease-free water, and DNA template (optional). The T4 DNA ligase mutant and other reagents are placed independently in different containers, or two or more of them are mixed in the same container.

[0086] As a preferred embodiment of the present invention, the detection kit may further include: a cell lysis reagent and / or an ATP extraction reagent.

[0087] As a preferred embodiment of the present invention, the test kit may also include an instruction manual to guide users in applying the test kit of the present invention in the correct manner.

[0088] The invention will be better understood from the following examples. However, those skilled in the art will understand that the specific methods and results are merely for illustrating the invention and not for limiting it. Experimental methods in the following examples that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0089] Materials and Methods

[0090] 1. Experimental materials, reagents and instruments

[0091] The materials and reagents used, including Escherichia coli DH5α, BL21(DE3), BeyoFusion™ PCR Master Mix (2X), colony direct PCR kit, restriction enzymes Nde I, Xho I, BamHI, EcoRI, LB medium-related reagents, antibiotics, inducers, plasmid mini-extraction kit, Ni affinity chromatography column packing, protein concentration assay reagents, and all-black 96-well plates, are all commercially available products from Shanghai Beyotime Biotechnology Co., Ltd.

[0092] The PCR instrument was purchased from Bio-Rad.

[0093] The ultrasonic cell disruptor was purchased from Ningbo Xinzhi Biotechnology Co., Ltd.

[0094] The high-pressure cell disruptor was purchased from Antos Nanotechnology (Suzhou) Co., Ltd.

[0095] The Clinx gel imaging system was purchased from Shanghai Qinxiang Scientific Instruments Co., Ltd.

[0096] The Varioskan LUX multi-functional microplate reader was purchased from ThermoFisher.

[0097] 2. EVcoupling analysis and screening of co-evolving amino acid residues

[0098] a. T4 DNA ligase is an ATP-dependent DNA ligase. T4 DNA ligase first generates an E-AMP complex powered by ATP. The E-AMP complex then recognizes the cleavage site of the double-stranded DNA and transfers AMP to the 5'-P group, forming a 5'-P-AMP complex. Finally, the 3'-OH nucleophilically attacks the 5'-P-AMP to form a phosphodiester bond, releasing AMP. Analysis of the spatial structure data of the T4 DNA ligase-substrate complex (PDB: 6DT1) currently available in the Protein Data Bank (PDB) database shows that T4 DNA ligase consists of three conserved domains: a DNA-binding domain (DBD, M1-L129), an adenylation domain (AdD, I130-E368), and an oligonucleotide-binding-fold domain (OBD, V369-E482). In the adenylation domain AdD, K159 is the adenylation site (key active site). ATP transfers AMP to the enzyme molecule K159, mediating the formation of intermediate products during double-stranded DNA ligation. Meanwhile, R164, R182, R359, and K365 are ATP binding sites, while E217 and E344 are divalent metal ion binding sites.

[0099] b. Based on the crystal structure of T4 DNA ligase, using the EVcoupling bioinformatics tool, based on statistical models and algorithms, long-range interactions between amino acid residues were predicted, and key amino acids that may have co-evolutionary effects were screened by parameters such as pseudo-likelihoodmaximization (PLM) and mean-field approximation.

[0100] c. Based on the virtual mutation prediction parameters Epistatic and Independent for each amino acid in the co-evolutionary analysis results, this experiment selected a total of 80 single-point mutation sites for subsequent experimental verification.

[0101] 3. Construction of T4 DNA ligase mutant library

[0102] a. Construction of wild-type T4 DNA ligase expression vector: A third party was commissioned to synthesize the wild-type T4 DNA ligase gene sequence (see attached sequence) using gene synthesis methods. The sequence was then cloned into the pET-N-His-Thrombin-C-His expression vector (Beyotime, D2908) using Nde I and Xho I restriction sites. A 6X His purification tag and a stop codon were added to the N-terminus and C-terminus of the protein, respectively, to obtain the pET-N-His-Thrombin-C-His-T4 DNA ligase (wild-type) plasmid.

[0103] b. Design and synthesis of mutant primers: Using pET-N-His-Thrombin-C-His-T4 DNA ligase (wild-type) plasmid as a template, gene site mutation PCR primers were designed and synthesized by a third party.

[0104] c. Site-directed mutagenesis: Site-directed mutagenesis PCR was performed using BeyoFusion™ PCR Master Mix (2X) (Beyotime, D7250). The 20 μl reaction mixture included: 7.9 μl Nuclease-free water, 10 μl 2X BeyoFusion™ PCR Master Mix, 0.5 μl pET-N-His-Thrombin-C-His-T4 DNA ligase plasmid (100 ng), and 1.6 μl a mixture of forward and reverse primers (10 μM).

[0105] d. PCR program: 92℃ pre-denaturation for 3 min, 92℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 7 min (15-60 sec / kb) (30 cycles in total), 72℃ further extension for 10 min, and storage at 4℃.

[0106] e. Template plasmid digestion: After the PCR reaction is completed, take an appropriate amount of the PCR reaction solution to be digested with enzymes, add 1 μl of restriction enzyme DpnI (Beyotime, D6257), mix well and incubate at 37°C for 1 hour to digest the template plasmid.

[0107] f. DNA Sample Transformation: Take 100 μl of DH5α supercompetent cells (Beyotime, D1031) and thaw them in an ice bath or ice-water bath. Add 10 μl of digested PCR product, gently tap the bottom of the tube about 2-3 times or gently shake it about 2-3 times to mix, and immediately incubate on ice for 30 min. Quickly place the centrifuge tube in a 42℃ water bath and heat shock it for 45 seconds. Then immediately transfer it to an ice-water bath and incubate for 2 minutes to rapidly cool it to near zero degrees. Add 900 μl of antibiotic-free LB medium and incubate at 37℃ with a shaker at about 150 rpm for 1 hour. Centrifuge at 5000g at room temperature for 1 min, aspirate about 900 μl of supernatant, leaving about 100 μl of supernatant. Gently pipette and resuspend the cells, then spread them onto LB agar plates containing kanamycin sulfate and incubate upside down at 37℃ overnight.

[0108] g. Colony PCR Detection: Single-clonal mutants were detected using a colony direct PCR kit (Beyotime, D7280). The 20 μl reaction mixture included: 8.4 μl Nuclease-free water, 1.6 μl forward and reverse primer mixture (10 μM), and 10 μl 2X E. coli Colony Direct PCR Mix. PCR program: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 45℃ annealing for 30 s, 72℃ extension for 2 min (15-60 sec / kb) (30 cycles), 72℃ further extension for 10 min, and storage at 4℃. 5 μl of the PCR reaction solution was validated on a 1% agarose gel (160V, 20 min). Single colonies with the correct target band size were picked and sequenced using universal primers T7 and T7-Terminator by a third party. The sequencing results were compared with wild-type T4 DNA ligase to identify the mutation site and mutation type.

[0109] h. Near-saturation mutation: For sites requiring near-saturation mutation, primers covering all amino acids at the site that are most likely to produce an optimization effect should be designed and synthesized, and subsequent operations should be performed using the same method as single-point mutation.

[0110] i. Combinatorial mutation: For mutants that require the introduction of multiple mutation sites, multiple rounds of site-directed mutagenesis are required, with one mutation introduced in each round. Each round must be verified by sequencing to ensure success before proceeding to the next round.

[0111] 4. Expression and purification of T4 DNA ligase mutant

[0112] a. The successfully constructed T4 DNA ligase mutant plasmid was transformed into BL21(DE3) strain supercompetent cells (Beyotime, D1013).

[0113] b. The following day, pick a single colony and transfer it to 10 mL of LB liquid medium containing kanamycin sulfate resistance. Incubate overnight at 37°C and 220 rpm with a shaker. Then, transfer 10 mL of the bacterial culture at a 1:100 ratio to 1000 mL of LB liquid medium containing kanamycin sulfate resistance and incubate at 37°C and 220 rpm with a shaker until OD (outcome limit) is reached. 600 The pH value was approximately between 0.6 and 0.8. The culture temperature was lowered to 16℃ and IPTG (Beyotime, ST098) was added to a final concentration of 0.1 mM. The culture was continued with shaking for 16 hours to induce protein expression. 500 μl of bacterial culture before and after induction were collected by centrifugation and resuspended in 500 μl of 1X PBS. Then, 50 μl of bacterial culture samples before and after induction were taken, and 10 μl of 6X SDS-PAGE protein loading buffer (Beyotime, P0015F) was added. The mixture was heated in a boiling water bath at 100℃ for 10 min, followed by centrifugation at 12000 rpm for 10 min. 20 μl of each sample was taken for SDS-PAGE electrophoresis to observe the expression of T4 DNA ligase protein in the induced bacterial cells.

[0114] c. Purification steps: Collect induced bacterial cells (1L), resuspend in 40mL buffer (50mM Tris, pH 7.4, 0.5M NaCl, 10% glycerol), homogenize, and after lysis, take 100μl sample for electrophoresis and use later. Centrifuge the remaining sample (12000rpm, 10min) and collect the supernatant. Purify the target protein using a BeyoGold™ His-tag Purification Resin (reduction-resistant chelate type) (BeyoGold, P2218) affinity purification column. Dialyze the purified T4 DNA ligase into storage buffer (36mM Tris, pH 7.5, 90mM KCl, 1.8mM DTT, 0.18mM EDTA, 10% glycerol).

[0115] d. Protein concentration was determined using the Bradford Protein Assay Kit (detergent compatible) (Beyotime, P0006) and detected by SDS-PAGE electrophoresis. Based on the standard protein BSA, the consistency of protein content in each T4 DNA ligase mutant sample was further determined for subsequent enzyme activity detection and comparison.

[0116] 5. Determination of enzyme activity between wild-type T4 DNA ligase and T4 DNA ligase mutant

[0117] The T4 DNA ligase activity assay system consists of one long DNA chain (P1 probe (DABCYL-DNA)) and two short DNA chains. The long chain has a fluorescent quencher group (DABCYL) at its 3' end; one short chain has a fluorescent group (TAMRA) at its 5' end (P3 probe (TAMRA-DNA)); and the other short chain has a phosphorylated 5' end (P2 probe).

[0118] P1 probe (DABCYL-DNA) sequence: 5'-CACGTCTACACGAAATTCATATGTGC-3' (SEQ ID NO:2)-DABCYL; P2 probe sequence: P-5'-ATGAATTTCGTGATGACGTG-3' (SEQ ID NO: 3); P3 probe (TAMRA-DNA) sequence: TAMRA-5'-GCACAT-'3.

[0119] Under the action of DNA ligase, a stable DNA double-stranded molecule is formed between two short DNA chains and a long DNA chain, causing TAMRA and DABCYL to approach each other, resulting in FRET. This causes a decrease in the fluorescence signal in the solution, so the ligation reaction process can be monitored in real time based on the change in fluorescence signal.

[0120] a. Substrate preparation

[0121] Dissolve the P1 probe (DABCYL-DNA), P2 probe, and P3 probe (TAMRA-DNA) separately in annealing buffer (10 mmol / L Tris-HCl, pH 7.5, 5 mmol / L MgCl2), mix them thoroughly at equimolar concentrations, and then dilute them to 200 nmol / L with hybridization buffer. Take 500 μl of the mixture and place it in a PCR instrument. Denature at 95°C for 2 min, then decrease the temperature by 0.1°C every 8 seconds until it reaches 25°C. Store at -20°C for later use.

[0122] b. The reaction system is prepared as shown in Table 1.

[0123] Table 1

[0124] c. Measurement of fluorescence signal

[0125] The maximum excitation wavelength of the fluorescently labeled TAMRA is 521 nm, and the maximum emission wavelength is 578 nm. Therefore, all fluorescence intensity measurements were performed using 521 nm excitation and 578 nm emission fluorescence signal detection. The entrance and emission slits of the fluorescence microplate reader were set to 5 nm and 10 nm, respectively. All samples were incubated at 25°C until the fluorescence signal stabilized, then different concentrations of T4 DNA ligase were added, rapidly vortexed to mix, and fluorescence signal changes were detected. By comparing the fluorescence curves of mutant and wild-type T4 DNA ligase at different dilutions, the relationship between T4 DNA ligase concentration and fluorescence value was obtained, thus determining the activity of the mutant enzyme relative to the wild-type enzyme. The activity test results of T4 DNA ligase wild-type and each single-point mutant are shown below. Figure 1 , saturated mutant activity test results as follows Figure 3 As shown, the activity test results of the combined mutants are as follows: Figure 4 and Figure 5 As shown in Figure A.

[0126] 6. Determination of the thermostability of wild-type T4 DNA ligase and T4 DNA ligase mutant

[0127] a. Dilute the wild-type T4 DNA ligase and each mutant enzyme to a concentration of 0.5 mg / mL.

[0128] b. Thermal stability screening test: The diluted enzyme solution was incubated at 30℃, 35℃, 40℃, 45℃, and 50℃ for 0 min and 30 min, and then added to the same ligation system as the "enzyme activity assay".

[0129] c. Monitor the fluorescence values ​​at 0 min and 30 min of incubation, and characterize the thermostability of the T4 DNA ligase mutant by calculating the proportion of fluorescence decrease. The results are as follows: Figure 2 and Figure 5 As shown in B.

[0130] 7. Determination of ligation efficiency between wild-type T4 DNA ligase and mutant T4 DNA ligase

[0131] In this embodiment, a 61 bp double-stranded DNA fragment with a prominent 3' end A was designed as a simulation fragment, along with a designed ligation adapter. Then, T4 DNA ligase mutant and wild-type T4 DNA ligase were used to perform the adapter ligation reaction in the same ligation buffer to simulate the DNA ligation process in the high-throughput sequencing sample preparation process.

[0132] a. Preparation of the simulated fragment: The simulated fragment was prepared by synthesizing two reverse complementary single-stranded primers (Jerui Biotechnology, HPLC purified, F1: 5'-CAATGAGGACAGTGTACTAGTAGATGAACGAGGAGCACGCCATGTCGAAATTCTTAAGTGA-3' (SEQ ID NO: 4); F2: 5'-CACTTAAGAATTTCGACATGGCGTGCTCCTCGTTCATCTACTAGTACACTG TCCTCATTGA-3' (SEQ ID NO: 5)). These two single-stranded primers were annealed in an annealing buffer (10 mmol / L Tris-HCl pH 7.5, 5 mmol / L MgCl2) to form a double-stranded DNA with a 3' end deoxyadenosine protrusion and a 5' end monophosphate group. This substrate model is similar to the fragment after end repair and addition of base A in library construction, and is used to reflect the corresponding ligation efficiency.

[0133] b. Preparation of ligation adapters: Two primers were synthesized separately: Adapter 1: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC TTCCGATCT-3' (SEQ ID NO: 6), and Adapter 2: 5'-GATCGGAAGAGCAC ACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 7). The two primers were annealed in an annealing buffer (10 mmol / L Tris-HCl pH 7.5, 5 mmol / L MgCl2) to form a Y-shaped adapter, which is consistent with the adapter type in library construction and can be used to monitor the ligation efficiency between fragments and adapters in high-throughput sequencing.

[0134] c. The reaction system and reaction conditions for the connector connection are shown in Table 2.

[0135] Table 2

[0136] When the initial sample size is less than 100 ng, the Adaptor should be diluted 10 times with deionized water to 2 μM before use.

[0137] d. Magnetic bead purification: After ligation, 2×BeyoMag™ DNA length sorting magnetic beads (Beyotime, D0038) were used to remove unligated adapters or adapter dimers and other invalid products. 20 μl of ddH2O was used for elution, and 10 μl was taken for agarose gel detection. Image J was used to quantify the target band and calculate the ligation efficiency of mutant and wild-type T4 DNA ligase for adapters and fragments.

[0138] Example 1: Construction of a T4 DNA ligase mutant library

[0139] 1. Wild-type T4 DNA ligase

[0140] The amino acid sequence (SEQ ID NO: 1) of the wild-type T4 DNA ligase is as follows: MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLTDMLDFIEFFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMR D LECGASVSIANKVWPGLIPEQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLKEELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPEN SKAKEFAEVAESRTASNGIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQVVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKEVI D VDLKIVGIYPHRKDPTKAGGFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKS D GRTDYVKLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL

[0141] 2. Mutant Design

[0142] As described in the preceding materials and methods, mutants were designed, expressed, analyzed, and measured. The T4 DNA ligase mutant PCR product was digested with Dpn I and transformed into E. coli DH5α. Three single clones were selected for small-scale culture and plasmid extraction.

[0143] Positive clones were obtained for the designed single-point mutants, near-saturation mutants, and combined mutants, and the plasmid sequences were all verified by sequencing to ensure their correctness.

[0144] Example 2: Enzyme activity analysis and thermal stability analysis of single-point mutants

[0145] As described above, the activity of wild-type T4 DNA ligase and enzymes of each single-point mutant was detected.

[0146] The bar chart comparing wild type and mutant is shown below. Figure 1 As shown, compared with wild-type T4 DNA ligase, the enzyme activity of the successfully constructed T4 DNA ligase single-point mutants M12 (D112A), M19 (T387N), M28 (F392V), M39 (E246M) and M45 (D448Q) was increased by more than 50%.

[0147] The thermostability of the two mutants, M9 (D371N) and M10 (D371S), was significantly improved compared to that of the wild-type T4 DNA ligase. Figure 2 As shown, M9(D371N) and M10(D371S) remained active after incubation at temperatures above 40°C for 30 minutes, while wild-type T4 DNA ligase completely lost its activity.

[0148] Based on the enzyme activity assay results of single-point mutants, five single-point mutation sites with significantly enhanced activity were subjected to near-saturation mutations to further screen for potential superior mutants. A bar chart comparing the activities of wild-type T4 DNA ligase and each near-saturation mutant is shown below. Figure 3 As shown in the figure. The results showed that at the D112 site, the activities of mutants M49 (D112C), M50 (D112G), M51 (D112I), and M52 (D112V) were significantly increased compared to wild-type T4 DNA ligase, with M51 (D112I) showing a particularly significant increase. At the D448 site, the activity of mutant M53 (D448A) was particularly significantly increased compared to wild-type T4 DNA ligase.

[0149] No mutants with better activity than the parental mutants were found among the near-saturation mutations at the M19(T387N), M28(F392V), and M39(E246M) sites.

[0150] Therefore, after in-depth analysis and experimentation, single mutants with improved performance were obtained, and the sequence information of each mutant is as follows: M9 (D371N) amino acid sequence: Based on SEQ ID NO: 1, position 371 is mutated from D to N.

[0151] M10 (D371S) amino acid sequence: Based on SEQ ID NO: 1, position 371 is mutated from D to S.

[0152] M12 (D112A) amino acid sequence: Based on SEQ ID NO: 1, position 112 is mutated from D to A.

[0153] M45 (D448Q) amino acid sequence: Based on SEQ ID NO: 1, position 448 is mutated from D to Q.

[0154] M49 (D112C) amino acid sequence: Based on SEQ ID NO: 1, position 112 is mutated from D to C.

[0155] M50 (D112G) amino acid sequence: Based on SEQ ID NO: 1, position 112 is mutated from D to G.

[0156] M51 (D112I) amino acid sequence: Based on SEQ ID NO: 1, position 112 is mutated from D to I.

[0157] M52 (D112V) amino acid sequence: Based on SEQ ID NO: 1, position 112 is mutated from D to V.

[0158] M53 (D448A) amino acid sequence: Based on SEQ ID NO: 1, position 448 is mutated from D to A.

[0159] Example 3: Enzyme activity analysis of combined mutants

[0160] A bar chart comparing the activities of wild-type T4 DNA ligase and various mutant combinations is shown below. Figure 4As shown: Based on the results of enzyme activity detection and comparison of single-point mutants and near-saturation mutants, D112C, D113G, D112I, D112V, D448A and D448Q mutants were selected for pairwise combination mutations to construct T4 DNA ligase multi-point superposition combination mutants in order to explore the possibility of further improving enzyme activity.

[0161] like Figure 4 As shown, most combined mutations did not exhibit synergistic effects. Only the combination of the D448A mutant and the D112I mutant showed a better increase in activity, with the superior mutant M60 (D112I / D448A) showing a particularly significant increase in activity compared to wild-type T4 DNA ligase. p < 0.0001).

[0162] Furthermore, two thermostability-enhancing mutation sites, M9 (D371N) and M10 (D371S), were introduced into the M60 mutant to construct multi-site combination mutants M64 (D112I / D448A / D371N) and M65 (D112I / D448A / D371S).

[0163] Activity results as follows Figure 5 As shown in Figure A, the activities of mutants M64 and M65 were significantly increased compared to the wild-type enzyme T4 DNA ligase, with the activity of mutant M65 (D112I / D448A / D371S) being more than 2.5 times higher than that of wild-type T4 DNA ligase.

[0164] The results of the thermal stability test are as follows Figure 5 As shown in B, the thermostability of the combined mutant with the D371 site was significantly improved compared to the wild-type T4 DNA ligase. After incubation at 40°C for 30 min, most of the activity remained, while the wild-type T4 DNA ligase almost completely lost its activity at 40°C.

[0165] Therefore, after in-depth analysis and experimentation, the following combined mutants with improved performance were obtained: M60 (D112I / D448A) amino acid sequence: Based on SEQ ID NO: 1, position 112 is mutated from D to I; position 448 is mutated from D to A.

[0166] M64(D112I / D448A / D371N) amino acid sequence: Based on SEQ ID NO: 1, position 112 is mutated from D to I; position 448 is mutated from D to A; and position 371 is mutated from D to N.

[0167] M65 (D112I / D448A / D371S) amino acid sequence: Based on SEQ ID NO: 1, position 112 is mutated from D to I; position 448 is mutated from D to A; and position 371 is mutated from D to S.

[0168] Example 4: Analysis of the ligation efficiency of mutants

[0169] As described above, the ligation efficiency of wild-type T4 DNA ligase and mutant T4 DNA ligase was determined.

[0170] A bar chart comparing the ligation efficiency of wild-type T4 DNA ligase and superior mutants and their parental mutants is shown below. Figure 6 As shown, mutants M51 (D112I) and M53 (D448A) significantly enhance the ligation efficiency of T4 DNA ligase for standard DNA fragments and adapters. The M60 (D112I / D448A) mutant, combining the M51 and M53 mutation sites, further improves the ligation efficiency of T4 DNA ligase. When the D371S site is introduced for mutation, the T4 DNA ligase mutants significantly enhance the ligation efficiency of standard DNA fragments and adapters, with the optimal mutant M65 (D112I / D448A / D371S) achieving a ligation efficiency of nearly 100%.

[0171] discuss

[0172] This invention analyzes and compares the sequence and structure of T4 DNA ligase by analyzing the three-dimensional spatial results of the wild-type T4 DNA ligase-substrate complex, combining EVcoupling bioinformatics analysis, and conducting in-depth experimental analysis, and screens for possible key mutation sites. A series of single-site, saturation, and combinatorial mutants of T4 DNA ligase were designed and constructed using site-directed mutagenesis.

[0173] Through in-depth analysis and experiments, compared with wild-type T4 DNA ligase, the enzyme activities of single-point mutants D112A, D112C, D112G, D112I, D112V, D448A, and D448Q were significantly improved, and the thermostability of mutants D371S and D371N was significantly improved.

[0174] After combined mutations, the superior double mutant M60 (D112I / D448A) showed a significantly higher enzyme activity compared to the wild-type T4 DNA ligase. The optimal multi-site mutant M65 (D112I / D448A / D371S) not only showed a 2.5-fold increase in activity compared to the wild-type T4 DNA ligase, but also improved thermostability. Furthermore, the optimal mutant was selected to simulate high-throughput sequencing fragments and ligate them with adapters. Compared to the wild-type enzyme, this mutant achieved a ligation efficiency of over 90%.

[0175] The development of efficient and convenient new high-throughput sequencing library preparation technologies using M65 mutants is highly suitable for high-throughput sequencing detection of clinical samples.

[0176] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method of enhancing the performance of T4 DNA ligase comprising: The T4 DNA ligase is mutated to form a T4 DNA ligase mutant. The modification includes mutating amino acid residues D at positions 112, 371, and / or 448, corresponding to the amino acid sequence shown in SEQ ID NO:

1. The performance characteristics include enzyme activity, thermostability, and / or ligation efficiency.

2. The method of claim 1, wherein, Including the amino acid sequence corresponding to SEQ ID NO: 1, the following mutations were performed: A. Mutations selected from (a)-(c) or combinations thereof: (a) The D at position 112 is mutated to I, C, V, A, or G; (b) The 371st position is mutated from D to S or N; (c) The 448th position is mutated from D to A or Q; B. A derivative protein formed by substituting, deleting, or adding one or more amino acid residues of the amino acid sequence of the protein in item A, and having the function of the protein in item A, but the sites corresponding to (a)-(c) in item A are conserved. C. A derivative protein that shares more than 80% homology with the amino acid sequence of the protein in item A and has the function of the protein in item A, but the sites corresponding to (a)-(c) in item A are conserved.

3. The method as described in claim 2, characterized in that, In A, the combination includes: The 112th D position mutates to I, the 448th D position mutates to A, and the 371st D position mutates to S; The 112th D position mutates to I, the 448th D position mutates to A, and the 371st D position mutates to N; or The 112th D position mutates to I, and the 448th D position mutates to A.

4. A T4 DNA ligase mutant, corresponding to the amino acid sequence shown in SEQ ID NO: 1, wherein amino acid residue D at positions 112, 371 and / or 448 is mutated; which has enhanced enzyme activity, thermostability and / or ligation efficiency.

5. The T4 DNA ligase mutant as described in claim 4, characterized in that, Including the amino acid sequence corresponding to SEQ ID NO: 1, the following mutations occurred: A. Mutations selected from (a)-(c) or combinations thereof: (a) The D at position 112 is mutated to I, C, V, A, or G; (b) The 371st position is mutated from D to S or N; (c) The 448th position is mutated from D to A or Q; B. A derivative protein formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence of the protein in item A, and having the function of the protein in item A, but the sites corresponding to (a)-(c) in item A are conserved. C. A derivative protein that shares more than 80% homology with the amino acid sequence of the protein in item A and has the function of the protein in item A, but the sites corresponding to (a)-(c) in item A are conserved.

6. The T4 DNA ligase mutant as described in claim 5, characterized in that, In option A, the combination includes: The 112th D position mutates to I, the 448th D position mutates to A, and the 371st D position mutates to S; The 112th D position mutates to I, the 448th D position mutates to A, and the 371st D position mutates to N; or The 112th D position mutates to I, and the 448th D position mutates to A.

7. Use of the T4 DNA ligase mutant of claim 4, 5 or 6, a host cell expressing the mutant or its lysis product, for the purpose of ligating DNA strands.

8. The use as described in claim 7, characterized in that, The T4 DNA ligase mutant catalyzes the chemical reaction between the 3' hydroxyl group and the 5' phosphate group of the DNA strands between two double-stranded DNA strands, forming a stable 3'-5'-phosphodiester bond, thus achieving precise ligation of the DNA strands.

9. A method for catalyzing the ligation of DNA strands, comprising: DNA strands are ligated by using the T4 DNA ligase mutant as described in claim 4, 5 or 6, a host cell expressing the mutant or its lysis product.

10. A kit for performing DNA ligation, the kit comprising: The T4 DNA ligase mutant as described in claim 4, 5 or 6; or An expression vector or host cell that expresses the T4 DNA ligase mutant as described in claim 4, 5 or 6.

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