CN118879647B

CN118879647B - Mutant T4 DNA ligase and kit

Info

Publication number: CN118879647B
Application number: CN202411041714.7A
Authority: CN
Inventors: 刘想; 商曰朋; 朱思思; 余玺玺
Original assignee: Yisheng Biotechnology Shanghai Co ltd
Current assignee: Yisheng Biotechnology Shanghai Co ltd
Priority date: 2024-07-31
Filing date: 2024-07-31
Publication date: 2025-05-23
Anticipated expiration: 2044-07-31
Also published as: CN118879647A

Abstract

The invention provides a mutant T4DNA ligase which is formed by mutation on the basis of the T4DNA ligase with the amino acid sequence shown as SEQ ID No. 1. The mutant of the present invention includes improvement of one or at least two of the following characteristics, such as improved thermostability, improved specific activity, reduced linker-linker self-ligation ratio, improved DNA library production, etc., as compared with the wild-type T4DNA ligase.

Description

Mutant T4 DNA ligase and kit

Technical Field

The invention relates to a mutant T4 DNA ligase and a kit, belonging to the technical field of biology.

Background

DNA ligase can block phosphodiester bonds of nucleic acid cleavage, is an enzyme necessary in organisms and is an indispensable tool enzyme in life science research. T4 DNA ligase is one of the most commonly used. The T4 DNA ligase can catalyze a diester bond between a 5 '-phosphate group and a 3' -hydroxyl group in double-helix DNA or RNA with the help of an ATP cofactor, so that a single-stranded gap in a double-helix DNA, RNA or DNA/RNA hybrid chain is repaired, and the application fields are very wide, such as connection of DNA fragments in molecular cloning, automatic cyclization of linear DNA, connection of DNA and a linker sequence in NGS sequencing and the like.

The wild type T4 DNA ligase is the gene encoding product of T4 phage, which is a single-chain polypeptide with molecular weight of about 55.23kDa, and consists of 487 amino acid residues, and the gene length is 1464bp. Although the overall performance of the T4 DNA ligase is better than that of other DNA ligases, the existing T4 DNA ligase has poor stability, and the activity of the T4 DNA ligase gradually decreases with time, so that the problems of reduced ligation products, reduced NGS library building yield and the like are caused during molecular cloning. In addition, in the NGS sequencing process, when DNA fragments are ligated to adaptors using T4 DNA ligase, adaptor-adaptor self-ligation, DNA fragment-fragment self-ligation, etc. often occur, thereby resulting in fewer correctly ligated DNA fragments to adaptors and eventually resulting in reduced NGS inventory production.

Disclosure of Invention

The object of the present invention is to provide a mutant T4 DNA ligase with improved performance compared to wild-type T4 DNA ligase.

The invention adopts the technical scheme that:

A mutant T4 DNA ligase which is a protein as described in any one of a1 to a 3:

a1, any mutation of one or more of 117, 118, 148, 160, 163, 255, 383, 391, 402, 403, 449, 458 occurs on the basis of the T4 DNA ligase having the amino acid sequence shown as SEQ ID No. 1;

a2, the protein with basically the same enzyme activity and performance is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in a1 except the mutation;

a3 is a protein which has at least 90% sequence identity with the protein of a1 and has substantially equivalent enzymatic activity and performance to the protein of a 1. Or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% sequence identity.

Preferably, any mutation comprising one or more of the following sites 40, 51, 207, 251, 332, 333, 334, 343, 371, 404, 405, 448, 487 occurs on the basis of the T4 DNA ligase having the amino acid sequence shown in SEQ ID No. 1.

Preferably, the T4 DNA ligase having the amino acid sequence shown in SEQ ID No.1 is one or more of the following mutations: a117S, S118T, K148E, A160Y, A163E, T255Y or T255W、R383S、G391C、K402V、V403G、N404K、A405V、G449D、L458A、S40D、P51S、I207V、A251P、K332E、V333K、I334F、L343H、D371C、D448G、L487R.

Preferably, it is a protein according to any one of the following b1-b 3:

b1. Protein obtained by mutating the T4 DNA ligase with the amino acid sequence shown in SEQ ID No.1 by any one of the following combinations:

(1)A117S;

(2)S118T;

(3)K148E;

(4)A160Y;

(5)A163E;

(6)T255Y;

(7)R383S;

(8)K402V/V403G/N404K/A405V;

(9)G449D;

(10)L458A;

(11)S118T/L487R;

(12)T255Y/L343H;

(13)R383S/K402V;

(14)R383S/D371C;

(15)R383S/D371C/S40D;

(16)K402V/V403G/N404K/A405V/G391C;

(17)K402V/V403G/N404K/A405V/G391C/L343H;

(18)K402V/V403G/N404K/A405V/G391C/D448G;

(19)K402V/V403G/N404K/A405V/P51S/A117S;

(20)K402V/V403G/N404K/A405V/K332E/V333K/I334F;

(21)K402V/V403G/N404K/A405V/R383S/D371C/S40D;

(22)L458A/I207V;

(23)L458A/I207V/A251P;

b2, the protein with basically the same enzyme activity and performance is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in b1 except the change;

b3-a protein having at least 90% sequence identity to the protein of b1 and having substantially equivalent enzymatic activity and properties to the protein of b 1.

Preferably, it has one or at least two of the following characteristics, improved thermal stability, improved specific activity, reduced linker-linker self-ligation ratio or improved DNA inventory production.

The coding gene of the mutant T4 DNA ligase.

Expression vectors or host bacteria of the above-described mutant T4 DNA ligase.

Use of the mutated T4 DNA ligase described above for DNA-DNA, DNA-RNA, RNA-RNA ligation.

A kit comprising the mutated T4 DNA ligase described above.

A sequencing library-building kit comprising the mutated T4 DNA ligase described above.

Herein, identity refers to identity of an amino acid sequence or a nucleotide sequence. Percent sequence identity may be calculated by any method known in the art, for example using BLOSUM62 matrix, see methods described by Henikoff et al in PNAS,89 (22): 10915-10919 (1992).

The description of the mutants of the present invention is recognized by those skilled in the art, and the A117S, exemplified by the sequence of one of the mutants, refers to the mutation of alanine (A) at position 117 of the amino acid sequence shown in SEQ ID NO. 1 to serine (S).

The mutant T4 DNA ligases provided herein also include amino acid sequences having at least 90% sequence identity, or at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% sequence identity, to the mutant sequence.

As used herein, "substantially identical" or "substantially equivalent" means that the variant T4 DNA ligase has no more than 20% variation in the test value of its enzymatic activity and performance under the same assay conditions. In some embodiments of the invention, the performance test includes testing thermal stability, testing specific activity, testing linker-linker self-coupling. T4 DNA ligase is an ATP-dependent ligase which has a DNA binding domain-DBD, a nucleotide transferase NTase domain and an OB folding domain from complex crystal structure analysis of the enzyme and DNA. FIG. 1 provides a basic route for T4 DNA ligase engineering. In the rational design, we analyzed the DNA substrate surroundings from the structureAmino acid residues within the range capable of reacting with DNA, and virtually screening and structure-activity analysis of the amino acids by means of calculation, and simultaneously, based on sequence conservation analysis, the ligase is also designed for sequence alignment, combined structure and force analysis, 14 key amino acids (117, 118, 148, 160, 163, 255, 383, 391, 402, 403, 404, 405, 449 and 458) are determined, as shown in figure 1, and site-directed saturation mutation libraries are constructed and screened for the sites. In the aspect of directed evolution, random mutation library construction and screening are performed on dominant mutants screened based on a fixed-point saturated library, the stability, activity and residual activity of the enzyme are subjected to parallel screening by using a MTPS method, screening data of all dimensions are synthesized, and the dominant mutants are purified and characterized. After fine purification of dominant mutants obtained in two aspects of rational design and directed evolution, deep analysis is carried out on multiple dimensions such as stability, specific activity, joint self-linking rate, fragment self-linking rate in library establishment, library establishment yield and the like of the mutants, so as to determine G1 generation dominant mutants. Based on the generation G1, DNA Shuffling screening and combined mutation CDM and other works are further carried out, and finally, the T4 DNA ligase mutant meeting various performance indexes is obtained.

The mutant of the present invention includes improvement of one or at least two of the following characteristics, such as improved thermostability, improved specific activity, reduced linker-linker self-ligation ratio, improved DNA library production, etc., as compared with the wild-type T4 DNA ligase.

Drawings

FIG. 1 is a schematic representation of the simulated spatial folding of a wild-type T4 DNA ligase.

FIG. 2 shows the thermal stability data of female parent V0 and mutants V1-V10.

FIG. 3 shows the thermal stability data of female parent V0 and mutant V11-V23.

Detailed Description

The following description of the embodiments of the invention is further illustrated in the accompanying drawings, but the description of the examples does not limit the scope of the invention in any way.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in this description of the invention are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention.

The materials or instruments used in the following examples, if not specifically described, were available from conventional commercial sources.

Example 1 engineering concept and acquisition of mutant T4 DNA ligase

Wild type T4 DNA ligase has the defects of mild action condition, sensitivity to temperature, easy inactivation, instability, storage intolerance, lower DNA yield in NGS library establishment application and the like. Therefore, we modified wild type T4 DNA ligase V0 (amino acid sequence shown as SEQ ID No. 1) protein, modified wild type T4 DNA ligase by adopting a method combining rational design and directed evolution, and screened dominant mutants from tens of thousands of sequences as shown in Table 1:

TABLE 1 mutant numbering and mutation types

EXAMPLE 2T 4 DNA ligase mutant thermal stability assay

Diluting the T4 DNA ligase mutant to 50U/. Mu.L according to the enzyme activity, incubating for 0 and 10min at 42 ℃, then adding the mutant into a following living detection reaction system,

Name of the name	Volume/. Mu.L
		FAM DNA(5μM)	1
BHQ-1DNA(5μM)	1
		10×ligase buffer	10
T4 DNA ligase	2
		ddH₂O	86

Fluorescence values of incubation for 0min and 10min were monitored and the T4 DNA ligase mutant thermostability was characterized by calculating the proportion of fluorescence decrease.

Mutant stability data are shown in fig. 2 and 3, which are residual viability values determined after comparing maternal V0 to mutants with simultaneous incubation at 42 ℃ for 10 minutes. Wherein V0 represents female parent, namely wild type T4 DNA ligase with the amino acid sequence shown as SEQ ID No.1, V1-V23 are mutant T4 DNA ligase obtained by modification on the basis of V0, and mutant numbers refer to a series of sequences in Table 1 respectively.

The thermal stability test at 42 ℃ is one of the methods commonly used by those skilled in the art to study T4DNA ligase mutations and their variants, and although the thermal stability test of the present application is performed at 42 ℃, this does not mean that the highest heat resistant temperature of the mutant of the present application is 42 ℃. The greater the residual activity number, the higher its thermal stability, the higher the reaction temperature and/or the longer the reaction time can be tolerated.

EXAMPLE 3 determination of the enzyme Activity of the T4 DNA ligase mutant

The principle of the method for detecting the enzyme activity of the T4 DNA ligase is that a section of DNA fragment (FAM-DNA) with a fluorescent group and a section of DNA fragment (BHQ-1 DNA) with a fluorescence quenching group are taken as substrates, and under the condition that the DNA ligase exists, the two DNA fragments are connected, and the fluorescence intensity is gradually reduced, namely, a certain relation exists between the fluorescence intensity and the DNA ligase. The enzyme activity of T4 DNA ligase was detected by measuring the intensity of fluorescence. The specific implementation mode is as follows:

(1) Substrate formulation

FAM-DNA was mixed by equal volumes of primers A (sequence: 5 '-TAG/i 6 FAMdT/ACACTGTCCTCATTG-3') and B (sequence: 5 '-CAATGAGGACAGTGT-3'), diluted with TE buffer to a final concentration of 5. Mu.M, 37℃for 5min, then left at room temperature for 10min, -20℃for storage for use. BHQ-1DNA was prepared by mixing primers C (sequence: 5 '-CTCCTCGTTCATCTAC-3') and D (sequence: 5 '-ACTAG/iBHQ dT/AGATGAACGAGGAG-3') in equal volume and preparing a 5. Mu.M final concentration solution according to FAM-DNA preparation method.

(2) Preparation of a reaction system

Name of the name	Volume/. Mu.L
		FAM DNA(5μM)	1
BHQ-1 DNA(5μM)	1
		10×ligase buffer	10
T4 DNA ligase	2
		ddH₂O	86

T4 DNA ligase with different concentrations is added into the reaction system, evenly mixed, incubated for 30min at 25 ℃, and fluorescence values at 485nm/535nm are read. The enzyme activity of the T4 DNA ligase mutant can be calculated by the relation between the T4 DNA ligase with known enzyme activity and the fluorescence value.

Table 2 provides a list of T4 DNA ligase mutants of specific sequences of particular relevant activities disclosed herein. Mutant numbers refer to the series of sequences in Table 1, respectively, and in the specific activity column, "-" indicates that the specific activity of the mutant protein is comparable to or lower than that of V0. The plus sign "+" indicates that the specific activity of the mutant protein is 120% -150% of the specific activity of V0, namely, the specific activity of the mutant/the specific activity of the wild type is less than or equal to 120%. The two plus signs "++" indicate that the specific activity of the mutant protein is 150% -200% of that of V0, namely that the specific activity of the mutant/the specific activity of the wild type is less than or equal to 150%. The three plus signs "++ + + +" indicate that the specific activity of the mutant protein is 200% -250% of that of V0, namely that the specific activity of the mutant/the specific activity of the wild type is less than or equal to 200%. The four plus signs "++ + ++" indicate that the specific activity of the mutant protein is 250% -400% of that of V0, namely that the specific activity of the mutant/the specific activity of the wild type is less than or equal to 250%. Wherein V0 represents a female parent, namely a wild type T4 DNA ligase with an amino acid sequence shown as SEQ ID No.1, and V1-V23 are mutants obtained by modification on the basis of V0.

TABLE 2 specific Activity of mutants

EXAMPLE 4T 4 DNA ligase mutant linker residual assay

In the embodiment, calf thymus DNA interrupted by ultrasound is used as a fragmentation template, and then YEASEN DNA library building kit (product number 12201) is used for building a library, and Qubit and Qsep are used for detecting yield and linker residues of the library building. The specific implementation mode is as follows:

(1) End repair/dA tail addition

The amplification procedure was as follows:

(2) Joint for connecting pipe

Temperature (thermal cover 105℃)	Time of
		30°C	30min
72°C	30min
		4°C	Hold

The amplification procedure was as follows:

Name of the name	Input amount
		dA-tailed DNA	60μL
12201-C	30μL
		T4 DNA ligase (300 ng/. Mu.L)	10μL
Adapter (0.5. Mu.M) (YEASEN cat No. 13519)	5μL
		ddH₂O	5μL

(3) Magnetic bead purification (1X)

1) Preparation work is toDNA Selection Beads (12601) the beads were removed from the refrigerator and equilibrated at room temperature for at least 30min. Preparing 80% ethanol.

2) Vortex or reverse the beads sufficiently to ensure adequate mixing.

Temperature (thermal cover off)	Time of
		20°C	15min
4°C	Hold

3) Suction 110. Mu.LDNA Selection Beads to Adapter Ligation products, vortexing or gently beating with a pipette until thoroughly mixed, incubating for 5min at room temperature.

4) The PCR tube was briefly centrifuged and placed in a magnetic rack to separate the beads from the liquid, after which the solution was clarified (about 5 min) and the supernatant carefully removed.

5) The PCR tube was kept always in a magnetic rack, the beads were rinsed with 200 μl of freshly prepared 80% ethanol, and after 30sec incubation at room temperature, the supernatant was carefully removed.

6) Step 5 was repeated for a total of two rinses. Finally, the residual liquid was sucked off using a 10. Mu.L small tip.

7) The PCR tube is kept to be always placed in the magnetic frame, and the magnetic beads are air-dried after being uncapped until cracks (not more than 5 min) just appear.

8) The PCR tube was removed from the magnetic rack, 21. Mu.L of ddH ₂ O was directly added, vortexed or gently beaten with a pipette until thoroughly mixed, and allowed to stand at room temperature for 5min. The PCR tube was briefly centrifuged and placed in a magnetic rack for standing, after the solution was clarified (about 5 min), 20. Mu.L of supernatant was carefully removed into the PCR tube without touching the magnetic beads.

(4) Library amplification system:

Name of the name	Input amount
		Adapter Ligated DNA	20μL
12201-E	25μL
		Primer(25μM)(12201-F)	5μL

Library amplification procedure:

Temperature (temperature)	Time of	Cycle number
			98°C	1min
98°C	10s	12
			60°C	30s
72°C	30s
			72°C	5min
4°C	Hold

(5) Magnetic bead purification (0.5X)

The same as the purification operation in the step (3). Using HieffDNA Selection Beads (0.5×, beads: dna=0.5:1) were purified of library amplification products. 30. Mu.L ddH ₂ O eluted.

(6) Concentration test

Before use, the components in the 1× DSDNA HS ASSAY KIT (YEASEN cat No. 12642) kit were returned to room temperature and mixed upside down. Qubit calibration was performed according to the kit instructions.

199. Mu.L of 1 XDsDNA detection solution was put into a 0.5mL thin-walled centrifuge tube, 1. Mu.L of the sample to be measured was added, and the mixture was gently vortexed and oscillated for 2-3sec to avoid air bubbles as much as possible. Readings were made using Qubit.

(7) Joint residual test

According to the concentration of the Qubit test, the library-building yield of different mutant T4 DNA ligases is diluted to 2 ng/. Mu.L, and then the percent data of the linker residue is obtained by using Qsep machine.

Table 3 provides the results of analysis of the ratio of the linker-linker self-ligation of T4 DNA ligase in the NGS library construction process, with lower linker-linker self-ligation ratios being more conducive to NGS library construction and increasing the yield of NGS library construction. In the following table, the mutant numbers refer to the series of sequences in Table 1, respectively, and in the linker-linker self-ligating ratio column, the negative "-" indicates that the linker-linker self-ligating ratio of the mutant protein is higher than that of V0. A plus sign "+" indicates that the linker-linker self-linking ratio of the mutant protein is equivalent to that of V0, and is within.+ -. 10% compared with the protein consisting of the amino acid sequence shown in SEQ ID No.1 of the sequence Listing. The two plus signs "++" indicate a 20% -50% decrease in the linker-linker self-ligating ratio of the mutant protein compared to the linker-linker self-ligating ratio of V0, i.e., a percentage decrease of <50% in the linker-linker self-ligating ratio of the mutant protein compared to the linker-linker self-ligating ratio of V0 of 20%. Three plus signs "+++" represent mutants linker-linker self-linking of proteins the ratio is reduced by 50% -100% compared with the ratio of the joint-joint self-connection of V0, that is, the percentage decrease of the linker-linker self-ligation ratio of the mutant protein to the linker-linker self-ligation ratio of V0 is less than 100%.

TABLE 3 self-ligation ratio of the mutants

The present invention is described in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e., it does not mean that the present invention must be practiced depending on the above detailed methods. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims

1. A mutant T4 DNA ligase, characterized by being a protein of the following:

a protein obtained by mutating any one of the following on the basis of T4 DNA ligase with the amino acid sequence shown in SEQ ID No. 1:

(7)R383S;

(13) R383S and K402V;

(14) R383S and D371C;

(15) R383S and D371C and S40D;

(21) K402V and V403G and N404K and A405V and R383S and D371C and S40D.

2. A mutated T4 DNA ligase encoding gene according to claim 1.

3. An expression vector or host bacterium for the mutated T4 DNA ligase of claim 1.

4. Use of the mutated T4 DNA ligase of claim 1 for DNA-DNA, DNA-RNA, RNA-RNA ligation.

5. A kit comprising the mutant T4 DNA ligase of claim 1.

6. A sequencing library kit comprising the mutated T4 DNA ligase of claim 1.