T4 DNA ligase mutant and use thereof

Abstract

Provided are a T4 DNA ligase mutant and the use thereof. The T4 DNA ligase mutant: a) has an amino acid sequence having at least 80% identity to SEQ ID NO: 1; b) comprises a substitution of an amino acid at the position corresponding to position 28 of SEQ ID NO: 1; and c) has a T4 DNA ligase activity. The T4 DNA ligase mutant has improved double-end linker connection efficiency and single-chain cyclization performance, and is compatible with different reaction systems. The T4 DNA ligase mutant can be widely used in library construction for various high-throughput sequencing, single-chain cyclization and ligation of DNA fragments in molecular cloning.

Description

T4 DNA ligase mutants and their applications Technical Field

[0001] The present invention relates to the field of biochemistry, and in particular to a T4 DNA ligase mutant and an application thereof. Background Art

[0002] DNA ligases play a vital role in biological processes, catalyzing the repair of single-strand breaks in double-stranded DNA. There are five common DNA ligases: T4 DNA ligase, T7 DNA ligase, T3 DNA ligase, E. coli DNA ligase, and Taq DNA ligase. Each ligase has distinct properties. For example, T7 DNA ligase and T3 DNA ligase are less efficient at ligating blunt-ended DNA, with virtually no ligation activity observed in the absence of PEG. E. coli DNA ligase and Taq DNA ligase are incapable of ligating blunt-ended DNA substrates. In comparison, T4 DNA ligase exhibits superior ligation performance with both sticky and blunt-ended substrates, catalyzing the ligation of sticky ends of DNA, oligonucleotides, and RNA-DNA hybrids, as well as blunt-end DNA. DNA ligases require biomolecules for energy to function. T4 DNA ligase utilizes ATP, while E. coli DNA ligase and Taq DNA ligase utilize NAD.

[0003] T4 DNA ligase is widely used in various fields of genetic engineering and diagnosis, including molecular cloning, high-throughput sequencing and high-throughput screening.

[0004] In conventional molecular cloning, T4 DNA ligase is primarily used for vector construction, ligation of sticky-ended or blunt-ended substrates, and fragment repair. Improvements in T4 DNA ligase ligation efficiency will facilitate its further application in conventional molecular cloning.

[0005] High-throughput sequencing technology is a popular technique in genetic diagnosis and a key component of DNA and RNA diagnostics. With the continuous advancement of life sciences, the demand for accurate base sequencing of DNA or RNA sequences has increased significantly, requiring rapid, multi-parallel sequencing, high sensitivity, and ease of use. Consequently, a variety of high-throughput sequencing technologies have emerged, including those based on the Illumina HiSeq X-10 and Nextseq 500 sequencing platforms, DNBSeq based on the MGI sequencing platform, ABI Solid sequencing technology, and nanopore sequencing. All sequencing technologies require library construction to convert the target DNA or RNA into a structure with a specific sequence that can be recognized by the sequencer. This library construction process requires end-repair and adapter ligation, while the MGI sequencing platform's DNBSEQ technology also requires single-strand circularization. These processes all rely on T4 DNA ligase. Therefore, T4 DNA ligase plays a key role in library construction kits for various platforms.

[0006] It's well known that the efficiency and quality of ligation between target sequencing genes and adapters significantly impacts the quality of subsequent library construction and sequencing data. As the application of high-throughput sequencing technologies continues to expand and deepen, the enzymes used for library construction are evolving, placing higher demands on their performance. For example, in cfDNA sequencing, FFPE sample sequencing, and single-cell sequencing, these samples feature low template content, damaged DNA, and a wide range of lengths. Traditional T4 DNA ligases are prone to mismatches, low ligation efficiency, low substrate conversion rates, substrate bias, and the formation of adapter dimers, significantly impacting subsequent library screening and sequencing quality. Therefore, a T4 DNA ligase with superior performance is needed. Furthermore, high-throughput sequencing is characterized by multi-parallelism, rapid speed, and high throughput. Minimizing library construction and sequencing time is a trend. To ensure compatibility with other library construction steps, this also places higher demands on the compatibility of ligase reaction systems.

[0007] To address these technical requirements, currently available library construction kits optimize buffer components or utilize engineered T4 DNA ligase to improve performance, such as enzyme ligation efficiency and low-template ligation. Optimizing buffer components (e.g., adding compounds like PEG and 1,2-propylene glycol) can improve ligation efficiency, but also places higher demands on the compatibility of upstream and downstream systems. Products that achieve superior mutants through engineering are relatively rare, and these still largely fail to meet market performance requirements.

[0008] Summary of the Invention

[0009] The main purpose of the present invention is to provide a T4 DNA ligase mutant and its application, so as to solve the problem of low ligation performance of T4 DNA ligase in the prior art.

[0010] To achieve the above object, according to one aspect of the present invention, a T4 DNA ligase mutant is provided, which: a) has an amino acid sequence that is at least 80% identical to SEQ ID NO: 1; b) includes an amino acid substitution, and the position of the substituted amino acid corresponds to position 28 of SEQ ID NO: 1; and c) has T4 DNA ligase activity.

[0011] Furthermore, the amino acid substitution is any one of the following groups: Group 1) N at position 28 is substituted with G, I, Y, T or W; Group 2) N at position 28 is substituted with L or D; Group 3) N at position 28 is substituted with S, V, P, K, H, E, C or M.

[0012] Furthermore, the T4 DNA ligase mutant has the above-mentioned substitution at amino acid position 28 of SEQ ID NO: 1.

[0013] According to the second aspect of the present application, a nucleic acid molecule is provided, which encodes the aforementioned T4 DNA ligase mutant of the present invention.

[0014] According to the third aspect of the present application, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the aforementioned nucleic acid molecule of the present invention.

[0015] According to the fourth aspect of the present application, a host cell is provided, wherein the host cell contains the aforementioned nucleic acid molecule or recombinant plasmid of the present invention.

[0016] Furthermore, the host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell includes Escherichia coli.

[0017] According to a fifth aspect of the present application, a method for connecting DNA fragments is provided, which comprises: using the aforementioned T4 DNA ligase mutant of the present invention to connect the DNA fragments to be connected.

[0018] Furthermore, the ligation is performed by any of the following methods: 1) sticky end ligation; 2) blunt end ligation; 3) single-stranded circularization with the help of a circularization auxiliary sequence.

[0019] Furthermore, 1) sticky end ligation includes: the 3' end of the first DNA fragment and the 5' end of the second DNA fragment both have sticky ends, and the 3' end of the first DNA fragment is ligated to the 5' end of the second DNA fragment using the aforementioned T4 DNA ligase mutant; or the 3' protruding end of the first DNA fragment is A, and the 5' protruding end of the second DNA fragment is T, and the 3' protruding end A of the first DNA fragment is ligated to the 5' protruding end T of the second DNA fragment using the aforementioned T4 DNA ligase mutant; preferably, the first DNA fragment and the second DNA fragment having sticky ends are obtained by the action of a nuclease.

[0020] Furthermore, 2) blunt-end ligation includes: the ends of the first DNA fragment and the ends of the second DNA fragment are both blunt ends, the 3' end of the first DNA fragment has a hydroxyl group, and the 5' end of the second DNA fragment has a phosphorylated group, and the aforementioned T4 DNA ligase mutant is used to catalyze the formation of a phosphodiester bond between the hydroxyl group at the 3' end of the first DNA fragment and the phosphorylated group at the 5' end of the second DNA fragment, thereby completing the ligation of the first DNA fragment and the second DNA fragment.

[0021] Furthermore, 3) single-stranded cyclization with the help of a cyclization auxiliary sequence includes: incubating a single-stranded DNA fragment, a cyclization auxiliary sequence, and the aforementioned T4 DNA ligase mutant, wherein the single-stranded DNA fragment has a phosphorylated group at the 5' end and a hydroxyl group at the 3' end, the cyclization auxiliary sequence is completely complementary to the nucleotide sequences at both ends of the gap formed by head-to-tail cyclization of the 5' and 3' ends of the single-stranded DNA fragment, and the T4 DNA ligase mutant catalyzes the formation of phosphodiester bonds between the phosphorylated groups at both ends of the gap and the hydroxyl groups, thereby completing the cyclization of the single-stranded DNA fragment.

[0022] According to the sixth aspect of the present application, a kit is provided, which contains the aforementioned T4 DNA ligase mutant.

[0023] Furthermore, the kit also includes a 10× ligation buffer, which includes: 300–700 mM Tris-HCl, 100 mM Mg 2+ , 5-50 mM DTT, pH 7–8 or 300–700 mM Tris-acetate, 100 mM Mg 2+ , 5-50 mM DTT, pH 7–8.

[0024] Furthermore, the kit further comprises at least one of the following: ATP or a cyclization auxiliary sequence.

[0025] According to a seventh aspect of the present application, provided is the use of the aforementioned T4 DNA ligase, the aforementioned method for ligating DNA fragments, or the aforementioned kit in ligating DNA fragments.

[0026] Furthermore, the application includes at least one of sequencing library construction, single-strand circularization and molecular cloning.

[0027] The T4 DNA ligase mutant provided by the present invention has significantly improved double-end adapter ligation efficiency and single-strand circularization performance, and is highly compatible with different reaction systems. It can be widely used in various high-throughput sequencing library construction (specifically, for the connection of sequencing adapters to target fragments) and single-strand circularization (such as single-strand circularization in the MGI sequencing platform), which can improve the quality of sequencing libraries and sequencing. It can also be used for the connection between conventional molecular cloning DNA fragments. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0029] FIG1 shows the results of SDS-PAGE purification of wild-type and mutant T4 DNA ligase according to Example 2 of the present application;

[0030] FIG2 shows a schematic diagram of the connection principle of a double-ended connector in Example 3 of the present application;

[0031] FIG3 shows an example peak diagram of the detection of double-end adapter ligation products of wild-type T4 DNA ligase in Example 3 of the present application;

[0032] FIG4 shows a schematic flow chart of the single-chain cyclization assay method in Example 4 of the present application. DETAILED DESCRIPTION

[0033] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0034] As mentioned in the background, existing T4 DNA ligases have low ligation performance and are unable to meet the diverse ligation reaction systems currently available. To improve this situation, the inventors of this application attempted to mutate existing T4 DNA ligase to screen for improved ligation performance. Furthermore, they sought T4 DNA ligase mutants with high compatibility with various ligation reaction systems. The inventors first randomly mutated existing T4 DNA ligase and performed activity tests. They discovered that mutations at position 28 significantly impacted activity, so they conducted a saturation mutagenesis screen at this position. The general steps for this screening were as follows: 1) Utilizing the NNN degenerate primers, saturation mutagenesis was performed at position 28 of the T4 DNA ligase and an expression vector was constructed. 2) The constructed expression vector was transformed into competent Escherichia coli DH5α cells, cultured, and induced for protein expression. 3) The wild-type and mutant T4 DNA ligase proteins were purified using nickel-filled affinity chromatography. 4) The wild-type and mutant T4 DNA ligases were tested for double-end adaptor ligation efficiency and single-strand circularization performance. Based on the screening results, a series of T4 DNA ligase mutants were obtained. These mutants significantly improved their double-end adapter ligation efficiency and single-strand circularization performance. They also exhibited high compatibility with various reaction systems. Based on this, the applicants proposed a series of protection schemes for this application.

[0035] In a typical embodiment, a T4 DNA ligase mutant is provided, which: a) has an amino acid sequence that is at least 80% identical to SEQ ID NO: 1; b) includes an amino acid substitution, the position of the substituted amino acid corresponding to position 28 of SEQ ID NO: 1; and c) has T4 DNA ligase activity.

[0036] The protein with the amino acid sequence set forth in SEQ ID NO:1 is a wild-type T4 DNA ligase. Mutations in this amino acid sequence yield variants that share at least 80% identity with SEQ ID NO:1 and contain an amino acid substitution corresponding to position 28 of SEQ ID NO:1, while retaining T4 DNA ligase activity. These mutants, which conform to the sequence structure and functional activity, not only enhance the ligation performance of the wild-type T4 DNA ligase set forth in SEQ ID NO:1 but also exhibit varying degrees of improved compatibility with various ligation reaction systems. These mutants can be widely used in library construction (ligating adapter sequences to target fragments) for second- and third-generation high-throughput sequencing, as well as in single-strand circularization (e.g., single-strand circularization on the MGI sequencing platform), improving both sequencing library and sequencing quality. They can also be used for ligating DNA fragments in conventional molecular cloning.

[0037] Through screening, the present application found that when the amino acid N at position 28 corresponding to SEQ ID NO: 1 is substituted with the following amino acid types, the following aspects of the connection performance are improved: Group 1) when substituted with G (e.g., Mut 1 in the embodiment), I (e.g., Mut 9 in the embodiment), Y (e.g., Mut 11 in the embodiment), T (e.g., Mut 12 in the embodiment) or W (e.g., Mut 14 in the embodiment), the double-ended adapter connection activity and the single-chain cyclization activity are greatly improved compared with the wild type; Group 2) when substituted with L (e.g., Mut 4 in the embodiment) or D (e.g., Mut 10 in the embodiment), the double-ended adapter connection activity and the single-chain cyclization activity are slightly improved compared with the wild type; Group 3) when substituted with S (e.g., Mut 6 in the embodiment), V (e.g., Mut 7 in the embodiment), P (e.g., Mut 8 in the embodiment), K (e.g., Mut 13 in the embodiment), H (e.g., Mut 15 in the embodiment), E (e.g., Mut 16 in the embodiment), When Mut 16), C (e.g., Mut 17 in the examples) or M (e.g., Mut 18 in the examples) is used, the single-chain circularization efficiency is improved compared to the wild type.

[0038] In a preferred embodiment, the mutants are obtained by mutating N at position 28 of SEQ ID NO: 1. As mentioned above, this site was selected because random mutagenesis and activity screening revealed that mutations at this site have a greater impact on T4 DNA ligase activity than mutations at other sites. Therefore, saturation mutagenesis at this site facilitates the screening of T4 DNA ligase mutants with significantly improved ligation performance. Furthermore, these mutants are compatible with different ligation reaction systems, enabling their application in diverse ligation applications.

[0039] Those skilled in the art can flexibly add commonly used elements in the prior art (including but not limited to promoters for regulating transcription and translation, molecular tags for protein purification, signal peptides for protein localization, and other known protein sequences in the prior art) to the aforementioned T4 DNA ligase or mutants according to actual application needs. Such elements do not affect the activity of the T4 DNA ligase.

[0040] Homology in this application refers to the "sequence identity" between two amino acid sequences, i.e., the percentage of identical amino acids between the sequences. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, it can be determined using the BLAST program of the NCBI database. For determination of sequence identity, see, for example, Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, and Primers for Sequence Analysis, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0041] Proteins with 80%, 85%, 90%, 95%, 99% or more (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) identity and the same function, whose active sites, active pockets, active mechanisms, protein structures, etc. are most likely the same as the proteins provided in a).

[0042] Amino acid residues can be represented by the standard three-letter or one-letter amino acid code commonly known and agreed upon in the art. Herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0043] Conservative amino acid substitutions or replacements are well known in the art. For example, conservative amino acid substitutions preferably involve replacing one amino acid residue from the following groups (1)-(5) with another amino acid from the same group: (1) smaller aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (2) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (3) polar positively charged residues: His, Arg, and Lys; (4) larger aliphatic non-polar residues: Met, Leu, Ile, Val, and Cys; and (5) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative amino acid substitutions are as follows: Ala is substituted by Gly or Ser; Arg is substituted by Lys; Asn is substituted by Gln or His; Asp is substituted by Glu; Cys is substituted by Ser; Gln is substituted by Asn; Glu is substituted by Asp; Gly is substituted by Ala or Pro; His is substituted by Asn or Gln; Ile is substituted by Leu or Val; Leu is substituted by Ile or Val; Lys is substituted by Arg, Gln or Glu; Met is substituted by Leu, Tyr or Ile; Phe is substituted by Met, Leu or Tyr; Ser is substituted by Thr; Thr is substituted by Ser; Trp is substituted by Tyr; Tyr is substituted by Trp or Phe; and Val is substituted by Ile or Leu.

[0044] Those skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0045] Therefore, variant sequences of the above SEQ ID NOs: 2 obtained by replacing the above conventional conservative amino acids are also within the protection scope of the present application.

[0046] In a second typical embodiment of the present application, a nucleic acid molecule is provided, which encodes the aforementioned T4 DNA ligase mutant.

[0047] Nucleic acids are typically RNA or DNA. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. DNA nucleic acids are preferably used when incorporated into a vector.

[0048] It should be noted that, due to the principle of codon degeneracy, the nucleotide sequence encoding the amino acid sequence is not a unique constant sequence, and any nucleotide sequence that can encode the amino acid sequence of the above-mentioned T4 DNA ligase is within the scope of protection of this application.

[0049] In a third typical embodiment of the present application, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the aforementioned nucleic acid molecule.

[0050] The term "recombinant expression vector," also referred to as a recombinant vector, refers to a nucleic acid delivery vehicle into which a nucleic acid molecule can be inserted. When a vector is capable of expressing the protein encoded by the inserted nucleic acid molecule, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, or animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). In some embodiments, the vectors in the present application contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0051] In a fourth exemplary embodiment of the present application, a host cell is provided, wherein the host cell contains the aforementioned nucleic acid molecule or recombinant plasmid. Preferably, the host cell comprises a prokaryotic cell or a eukaryotic cell; more preferably, the prokaryotic cell comprises Escherichia coli.

[0052] In a fifth typical embodiment of the present application, a method for connecting DNA fragments is provided. The method comprises: using the aforementioned T4 DNA ligase mutant to connect the DNA fragments to be connected.

[0053] It should be noted that the T4 DNA ligase mutant is used to ligate the DNA fragments to be ligated, and the ligation methods include any of the following: 1) sticky end ligation; 2) blunt end ligation; 3) single-stranded circularization with the help of a circularization auxiliary sequence.

[0054] Specifically, the sticky end ligation step includes: the 3' end of the first DNA fragment and the 5' end of the second DNA fragment both have sticky ends, and the 3' end of the first DNA fragment is ligated to the 5' end of the second DNA fragment using the aforementioned T4 DNA ligase mutant. Preferably, the first DNA fragment and the second DNA fragment having sticky ends are obtained by the action of an endonuclease. Alternatively, the sticky end ligation step includes: the 3' overhanging end of the first DNA fragment is A, and the 5' overhanging end of the second DNA fragment is T, and the 3' overhanging end A of the first DNA fragment is ligated to the 5' overhanging end T of the second DNA fragment using the aforementioned T4 DNA ligase mutant. This ligation method is suitable for enzyme digestion and ligation library construction, such as double-end adapter ligation of DNA fragments with sticky ends at both 5' and 3' ends for high-throughput sequencing library construction.

[0055] The blunt-end ligation step includes: the ends of the first DNA fragment and the second DNA fragment are both blunt-end, the 3' end of the first DNA fragment has a hydroxyl group, and the 5' end of the second DNA fragment has a phosphorylated group. The T4 DNA ligase mutant is used to catalyze the formation of a phosphodiester bond between the hydroxyl group at the 3' end of the first DNA fragment and the phosphorylated group at the 5' end of the second DNA fragment, thereby completing the ligation of the first DNA fragment and the second DNA fragment. This ligation method is suitable for ligating two DNA fragments with blunt ends.

[0056] The step of cyclizing a single-stranded DNA fragment with the help of a cyclization auxiliary sequence includes incubating a single-stranded DNA fragment, the cyclization auxiliary sequence, and the T4 DNA ligase mutant, wherein the single-stranded DNA fragment has a phosphorylated group at its 5' end and a hydroxyl group at its 3' end, the cyclization auxiliary sequence is completely complementary to the nucleotide sequences at both ends of the gap formed by end-to-end cyclization of the 5' and 3' ends of the single-stranded DNA fragment, and the T4 DNA ligase mutant catalyzes the formation of phosphodiester bonds between the phosphorylated groups and the hydroxyl groups at both ends of the gap, thereby completing the cyclization of the single-stranded DNA fragment. This ligation method is suitable for cyclization of single strands.

[0057] In a sixth exemplary embodiment of the present application, a kit is provided, which contains the aforementioned T4 DNA ligase mutant. The kit containing the T4 DNA ligase mutant of the present application has higher ligation performance and is suitable for more ligation application scenarios.

[0058] Depending on the structure of the target fragment to be connected or the specific application scenario, the kit may also contain other components to facilitate the connection. In some preferred embodiments, the kit also includes 10× connection buffer, which includes: 300-700mM Tris-HCl, 100mM Mg 2+ , 5-50 mM DTT, pH 7-8 or 300-700 mM Tris-acetate, 100 mM Mg 2+ , 5-50 mM DTT, pH 7-8. In other preferred embodiments, the kit further comprises at least one of the following: ATP or a cyclization auxiliary sequence.

[0059] In a seventh typical embodiment of the present application, the use of the aforementioned T4 DNA ligase, the aforementioned method for ligating DNA fragments, or the aforementioned kit in DNA ligation is provided, and the specific applications include at least one of sequencing library construction (for example, linker ligation of the target fragment), single-stranded circularization, and molecular cloning.

[0060] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0061] Example 1: T4 DNA ligase vector construction

[0062] The gene encoding wild-type T4 DNA ligase (WT, SEQ ID NO: 1) (SEQ ID NO: 2) was synthesized by Changzhou New Life Science Technology Co., Ltd. and subcloned into the E. coli expression vector pCA24N. The constructed expression vector, pCA24N / T4-WT, adds a 7-histidine tag to the N-terminus of the wild-type T4 DNA ligase.

[0063] Wild-type T4 DNA ligase has the amino acid sequence shown in SEQ ID NO: 1:

[0064] Wild-type T4 DNA ligase has the nucleotide sequence shown in SEQ ID NO: 2:

[0065] First, random mutations were performed on the wild-type T4 DNA ligase and activity was tested. It was found that mutations at position 28 significantly affected activity, so saturation mutagenesis was performed at this site.

[0066] Secondly, using the pCA24N / T4-WT plasmid as a template, the primer pair N28-F and N28-R with the NNN sequence at position 28 were designed (see Table 1), and saturation mutagenesis was performed at the N28 site by rapid PCR amplification.

[0067] The rapid PCR reaction system consisted of 5 μL of 10× Reaction Buffer (containing MgSO₄, Promega), 1 μL of dNTP Mix (10 mM each), 1.5 μL of forward primer N28-F (10 μM), 1.5 μL of reverse primer N28-R (10 μM), 50 ng of pCA24N / T4-WT plasmid, 0.5 μL of Pfu DNA polymerase (3 U / μL, Promega, Cat. No. M7741), and nuclease-free water to a total volume of 50 μL. The rapid PCR reaction program was as follows: initial denaturation at 95°C for 2 min; 16 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 6 min; a final extension at 72°C for 5 min, and storage at 4°C.

[0068] Add 1 μL of DpnI (20 U / μL, NEB, Cat. No. R0176V) to the PCR product and incubate at 37°C in a metal bath for 2 hours to degrade the template. The reaction product was then transformed into E. coli DH5α competent cells purchased from Tiangen (Cat. No. CB101). The transformation steps were as follows: add 10 μL of the reaction product to 100 μL of DH5α competent cells, gently flick to mix, and place on ice for 30 minutes; heat shock at 42°C for 1 minute, then place on ice again for 10 minutes; add 400 μL of resistance-free LB medium and shake at 37°C at 220 rpm / min for 1 hour. After centrifugation at 3000 g for 3 minutes, discard some of the supernatant, retaining approximately 100 μL, resuspend and mix, then spread on a chloramphenicol-resistant LB plate and incubate at 37°C for 12-16 hours.

[0069] Several single colonies were selected and cultured overnight at 37°C in a shaker. Glycerol culture was then used for plasmid DNA miniprep. Plasmids were sent to Beijing Liuhe BGI Genomics Co., Ltd. for Sanger sequencing to verify the correct introduction of the mutation site. Glycerol culture strains corresponding to plasmids with correct sequencing results were recombinant E. coli strains expressing 19 T4 DNA ligase mutants (see Table 2, Mut1-Mut19) as well as the wild-type protein.

[0070] Table 1: Primer sequences for mutant construction

[0071] Note: Bold italic bases are introduced mutation sites

[0072] Table 2: List of wild-type and mutant T4 DNA ligase

[0073] Example 2: Fermentation expression and purification of T4 DNA ligase mutants

[0074] 1. Fermentation expression: The glycerol strain obtained in Example 1 was inoculated into 3 mL of liquid LB medium containing chloramphenicol at a ratio of 1:200, and cultured at 37°C with shaking at 220 rpm until the OD 600 =0.6, add IPTG to a final concentration of 0.4 mM and incubate at 16°C, 220 rpm, to induce expression overnight (16 h). Centrifuge the culture at 6000 rpm for 8 min, and collect the bacterial precipitate.

[0075] 2. Fermentation cell treatment: The collected cells were resuspended in a resuspension solution (20 mM Tris-HCl, 500 mM NaCl, 5% glycerol, pH 7.5), and ultrasonically disrupted. Centrifuged at 12,000 rpm for 20 minutes, and the supernatant was collected.

[0076] 3. Protein Purification: Purify T4 DNA ligase using a His SpinTrap column (purchased from Cytiva, Cat. No. 28401353). Add 500 μL of the supernatant to the His SpinTrap column, centrifuge at 100 g for 30 s, and discard the filtrate. Add wash buffer (20 mM Tris-HCl, 500 mM NaCl, 5% glycerol, 30 mM imidazole, pH 7.5) to elute contaminants. Centrifuge at 100 g for 30 s, discarding the filtrate until the total elution volume reaches twice the volume of the supernatant. Elute the target protein twice with 200 μL of elution buffer (20 mM Tris-HCl, 500 mM NaCl, 5% glycerol, 500 mM imidazole, pH 7.5). Collect the eluate. The collected eluates were combined and concentrated using a 0.5 mL 30 kDa ultrafiltration tube (Millipore, Cat. No. UFC503096). The protein was finally stored in storage buffer (20 mM Tris-HCl, 50 mM KCl, 50% glycerol, 0.1 mM EDTA, 1 mM DTT, pH 7.5) at a concentration of 0.2 mg / mL and stored at -20°C for subsequent performance testing.

[0077] The protein purity of the purified wild-type and mutant T4 DNA ligase samples was determined by SDS-PAGE electrophoresis analysis, as shown in Figure 1. The results showed that the wild-type and mutant T4 DNA ligase were successfully obtained, and the purity of the remaining proteins, except Mut8, was similar.

[0078] Example 3: Testing the Dual-End Adapter Ligation Performance of T4 DNA Ligase Mutants

[0079] During high-throughput sequencing library construction, sequencing adapters are attached to both ends of the sequencing fragment using T4 DNA ligase. For example, using MGI sequencing library construction as an example, a 200bp DNA fragment is ligated with double-ended Ad153 adapters. Figure 2 shows a schematic diagram of the double-ended Ad153 adapter ligation process.

[0080] In Figure 2, the sequence of the double-ended Ad153 linker includes: Ad153_5T_1-index, whose sequence is:

[0081] 5'phos-AGTCGGAGGCCAAGCGGTCTTAGGAAGACAATGTCATAAATCAACTCCTTGGCTCACA-3' (SEQ ID NO: 5); and Ad153Ω_Bottom_2, the sequence of which is:

[0082] 5'-TTGTCTTCCTAAGGAACGACATGGCTACGATCCGACTT-3' (SEQ ID NO: 6).

[0083] A 200-bp DNA substrate (i.e., the sequence shown in Figure 2, phosphorylated at the 5' end, with 7 N residues on the left, omitted in the middle, 8 N residues on the right, and an A residue at the end) was prepared as follows: E. coli gDNA was PCR amplified using the forward primer 5'Phos-GACGGGTGAGTAATGTCTG-3' (SEQ ID NO: 7) and the reverse primer 5'Phos-GTCATCCTCTCAGACCAGC-3' (SEQ ID NO: 8). The reaction system was prepared as follows: 10 μL 10x rTaq Buffer, 8 μL dNTPs Mix (2.5 mM each), 2 μL forward primer, 2 μL reverse primer, 10 ng E. coli gDNA, 0.5 μL rTaq DNA polymerase (2 U / μL, Changzhou New Life Technology Co., Ltd., Cat. No. LS-EZ-E-00001O), and nuclease-free water to a total volume of 100 μL. After the reaction is set up, perform PCR amplification under the following conditions: initial denaturation at 95°C for 5 minutes, followed by 30 cycles of 95°C for 20 seconds, 61°C for 30 seconds, and 72°C for 20 seconds; and a final extension at 72°C for 5 minutes, followed by storage at 4°C. Upon completion of the reaction, a 200-bp DNA substrate with an A tail was obtained.

[0084] Ad153 adapter annealing pretreatment: Mix equal volumes of Ad153_5T_1-index and Ad153Ω_Bottom_2 at a concentration of 100 μM each. Anneal at 95°C for 2 min, 75°C for 2 min, 60°C for 2 min, 50°C for 2 min, 40°C for 2 min, and 25°C for 20 min, with a cooling rate of 0.1°C / s per step. After the reaction, dilute the Ad153 adapter with TE buffer to a final concentration of 10 μM and store at -20°C.

[0085] Detection method: The above-obtained DNA with an A tail length of 200bp was used as a substrate, and Ad153 adapters were ligated on both sides of the substrate (the 3' end of the DNA substrate is A, and the 3' end of the Ad153 adapter has a single base protruding end with a T, and the two are connected through AT pairing). The reaction system shown in Table 3 was prepared and reacted at 23°C for 30 minutes. After the reaction, the enzyme ligation product was detected using a High Sensitivity DNA Detection Kit (AGILENT, Cat. No. 5067-4626). The conversion rate of the double-ended adapter product was calculated based on the molar concentration corresponding to each peak given in the test report. The conversion rate of the double-ended adapter product = molar concentration (double-ended ligation product) / molar concentration (double-ended product + single-ended product + substrate).

[0086] Table 3 Reaction system for determination of double-end adapter ligation efficiency

[0087] 10×Reaction Buffer: 700mM Tris-HCl, 100mM MgCl2, 50mM DTT, pH7.6@25℃.

[0088] Ligation products of wild-type and mutant T4 DNA ligase were detected using the High Sensitivity DNA Kit. With the exception of Mut19, all mutants and wild-type T4 DNA ligase successfully produced ligation products. Figure 3 shows the peak peaks of wild-type T4 DNA ligase. The conversion rates of double-ended adapter ligation products of wild-type and mutant T4 DNA ligase were calculated based on the peak peaks, and the results are shown in Table 4. The results show that the conversion rates of double-ended adapter ligation products of some mutants were significantly improved compared to the wild-type. No ligation products were produced with Mut19, suggesting that its ligation activity is significantly impaired after the mutation to Mut19.

[0089] Table 4 Conversion rate of double-ended connector ligation products

[0090] Example 4: Single-strand circularization performance test of T4 DNA ligase mutants

[0091] The principle of the detection method is shown in Figure 4. The auxiliary sequence Splint Oligo (SEQ ID NO: 9: 5'-GCCATGTCGTTCTGTGAGCCAAGG-3') is fully complementary to the nucleotide sequence at both ends of the substrate gap. T4 DNA ligase catalyzes the formation of a phosphodiester bond between the phosphate and hydroxyl groups at both ends of the substrate, completing the cyclization process.

[0092] The specific reaction steps are as follows:

[0093] a. Substrate PCR Amplification: 50 μL 2X Kapa HiFi HotStart Readymix (Roche, Cat. No. KK2602), 3 μL PCR forward primer (SEQ ID NO: 10: 5'-Phos-GAACGACATGGCTACGA-3'), 2 μL PCR reverse primer (SEQ ID NO: 11: 5'-TGTGAGCCAAGGAGTTG-3'), 1 ng pMD19T plasmid, and nuclease-free water to a total volume of 100 μL. After reaction preparation, PCR amplification was performed under the following conditions: initial denaturation at 95°C for 3 min; 20 cycles of 98°C for 20 s, 60°C for 15 s, and 72°C for 30 s; a final extension at 72°C for 5 min, and storage at 4°C. A 300-bp PCR product was obtained.

[0094] b. Prepare single-stranded circularization substrate: Dissolve 150 ng of the 300 bp PCR product in TE buffer and dilute to 23 μL. Preheat the PCR instrument to 95°C, with the heated lid at 105°C. Place the dissolved and diluted PCR product in the preheated PCR instrument and incubate at 95°C for 3 minutes. Immediately remove the sample and place on ice to obtain the single-stranded product.

[0095] c. Single-strand cyclization: Prepare the reaction system according to Table 5 and incubate at 37°C for 30 min. 10×TA buffer contains 330 mM Tris-Acetate, 660 mM Potassium Acetate, 100 mM Magnesium Acetate, and 5 mM DTT, pH 7.5.

[0096] d. Enzymatic digestion of uncircularized single-stranded substrates: After the single-stranded cyclization reaction is completed, directly add 2U of nuclease I and 65U of nuclease III and continue the reaction at 37°C for 30 minutes to digest the uncircularized single-stranded substrate or double-stranded substrate.

[0097] e. Reaction termination and quantification: After the reaction is completed, add 3.3 μL 0.5M EDTA directly to the reaction tube. TM The concentration of single-stranded products in each reaction system was determined using an ssDNA detection kit (Invitrogen, catalog number Q10212).

[0098] During the reaction, a negative control group was set up, and the enzyme storage solution was used instead of the wild-type or mutant T4 DNA ligase. The volume and reaction procedures were the same. The concentration of the single-stranded circularization product in each group was measured by ssDNA calibration. The single-stranded circularization efficiency = ΔssDNA concentration / theoretical ssDNA concentration of the system. The relative single-stranded circularization efficiency of the mutant relative to the wild-type was calculated as: relative single-stranded circularization efficiency = single-stranded circularization efficiency (mutant) / single-stranded circularization efficiency (wild-type), that is, the relative single-stranded circularization efficiency of the wild-type T4 DNA ligase was set as 1. The results are shown in Table 6. With the exception of Mut2, Mut3, Mut5, and Mut19, the single-stranded circularization efficiency of the remaining mutants was improved to varying degrees compared to the wild-type. The relative single-stranded circularization efficiency of Mut19 was close to that of the blank control (using a reaction system without ligase as a blank control), suggesting that its ligation activity was significantly impaired after the mutation to Mut19.

[0099] Table 5 Single-chain cyclization system preparation

[0100] Table 6 Single chain cyclization results

[0101] As can be seen from the foregoing description, the embodiments of the present invention achieve the following technical effects: the present invention provides a series of T4 DNA ligase mutants with N28 mutations, exhibiting enhanced performance. These mutants exhibit improved double-end adapter ligation efficiency and single-strand circularization efficiency compared to the wild-type, enabling both sticky-end and blunt-end ligation. They have potential applications in conventional molecular cloning, library construction for high-throughput sequencing (ligating adapter sequences to target fragments), and single-strand circularization (e.g., single-strand circularization on the MGI sequencing platform).

[0102] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A T4 DNA ligase mutant, characterized in that, The T4 DNA ligase mutant: a) has an amino acid sequence with at least 80% identity to SEQ ID NO: 1; b) includes a substitution of one amino acid, and the position of the substituted amino acid corresponds to the 28th position of SEQ ID NO: 1; and c) has T4 DNA ligase activity.

2. The T4 DNA ligase mutant according to claim 1, wherein The substitution of the amino acid is any one of any of the following groups: Group 1) N at the 28th position is substituted with G, I, Y, T or W; Group 2) N at the 28th position is substituted with L or D; Group 3) N at the 28th position is substituted with S, V, P, K, H, E, C or M.

3. The T4 DNA ligase mutant according to claim 1 or 2, characterized in that, The T4 DNA ligase mutant is the substitution of the amino acid at the 28th position of SEQ ID NO:

1.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the T4 DNA ligase mutant according to any one of claims 1-3.

5. A recombinant plasmid, characterized in that, The recombinant plasmid is ligated with the nucleic acid molecule according to claim 4.

6. A host cell, characterized in that, The host cell contains the nucleic acid molecule according to claim 4 or the recombinant plasmid according to claim 5.

7. The host cell according to claim 6, characterized in that, The host cell includes a prokaryotic cell or a eukaryotic cell; Preferably, the prokaryotic cell includes Escherichia coli.

8. A method for ligating DNA fragments, characterized in that, The ligation method includes: using the T4 DNA ligase mutant according to any one of claims 1-3 to ligate the DNA fragments to be ligated.

9. The connection method according to claim 8, characterized in that, The ligation is carried out by any one of the following methods: 1) Sticky-end ligation; 2) Blunt-end ligation; 3) Single-strand circularization assisted by a circularization assisting sequence.

10. The connection method according to claim 9, wherein 1) The sticky-end ligation includes: Both the 3' end of the first DNA fragment and the 5' end of the second DNA fragment have sticky ends, and the 3' end of the first DNA fragment is ligated to the 5' end of the second DNA fragment using the T4 DNA ligase mutant; preferably, the first DNA fragment and the second DNA fragment with sticky ends are obtained by the action of an endonuclease. 1) The sticky-end ligation includes:

11. The connection method according to claim 9, characterized in that, The 3' overhanging end of the first DNA fragment is A, and the 5' overhanging end of the second DNA fragment is T. The 3' overhanging end A of the first DNA fragment is ligated to the 5' overhanging end T of the second DNA fragment using the T4 DNA ligase mutant. 2) The blunt-end ligation includes:

12. The connection method according to claim 9, wherein, Both the end of the first DNA fragment and the end of the second DNA fragment are blunt ends, and the 3' end of the first DNA fragment has a hydroxyl group, and the 5' end of the second DNA fragment has a phosphorylated group. The T4 DNA ligase mutant is used to catalyze the formation of a phosphodiester bond between the hydroxyl group at the 3' end of the first DNA fragment and the phosphorylated group at the 5' end of the second DNA fragment, thereby completing the ligation of the first DNA fragment and the second DNA fragment. 3) The single-strand circularization assisted by a circularization assisting sequence includes:

13. The connection method according to claim 9, characterized in that, ​ Co-incubate the single-stranded DNA fragment, the cyclization assisting sequence and the T4 DNA ligase mutant, wherein the 5'-end of the single-stranded DNA fragment has a phosphoryl group and the 3'-end has a hydroxyl group, the cyclization assisting sequence is completely complementary to the nucleotide sequences at both ends of the nick formed by head-to-tail cyclization of the 5'-end and 3'-end of the single-stranded DNA fragment, and the T4 DNA ligase mutant is used to catalyze the formation of a phosphodiester bond between the phosphoryl group and the hydroxyl group at both ends of the nick, thereby completing the cyclization of the single-stranded DNA fragment.

14. A kit, characterized in that, The kit contains the T4 DNA ligase mutant according to any one of claims 1-3.

15. The kit according to claim 14, wherein The kit further includes a 10× ligation buffer, and the 10× ligation buffer includes: 300 - 700 mM Tris-HCl, 100 mM Mg 2+ , 5 - 50 mM DTT, pH 7 - 8 or 300 - 700 mM Tris-acetate, 100 mM Mg 2+ , 5 - 50 mM DTT, pH 7 - 8.

16. The kit according to claim 14, characterized in that, The kit further includes at least one of the following: ATP or a cyclization assisting sequence.

17. Use of the T4 DNA ligase according to any one of claims 1-3, the method for ligating the DNA fragment according to any one of claims 8 to 13, or the kit according to any one of claims 14-16 in DNA fragment ligation.

18. The application according to claim 17, wherein The application includes at least one of sequencing library construction, single-stranded cyclization and molecular cloning.

Images