Mutated T4 DNA ligase and kit

Abstract

The present invention provides a mutant T4 DNA ligase, which is formed by mutation based on the T4 DNA ligase with an amino acid sequence as shown in SEQ ID No. 1. Compared with the wild-type T4 DNA ligase, the mutant of the present invention includes improvements in one or at least two of the following properties: improved thermal stability, improved specific activity, reduced linker-linker self-ligation ratio, improved DNA library construction yield, etc.

Description

Technical Field

[0001] The patent of this invention relates to a mutant T4 DNA ligase and a kit, belonging to the field of biotechnology. Background Art

[0002] DNA ligase can seal the phosphodiester bonds of broken nucleic acids. It is an essential enzyme in organisms and an indispensable tool enzyme in life science research. T4 DNA ligase is the most commonly used DNA ligase. With the help of ATP cofactor, T4 DNA ligase can catalyze the formation of diester bonds between the 5'-phosphate group and the 3'-hydroxyl group in double-helix DNA or RNA, thereby repairing single-stranded gaps in double-helix DNA, RNA or DNA / RNA hybrid chains. It has a wide range of application scenarios, such as the connection of DNA fragments in molecular cloning, automatic circularization of linear DNA, and the connection of DNA and adapter sequences in NGS sequencing.

[0003] Wild-type T4 DNA ligase is a gene-encoded product of T4 bacteriophage, which is a single-chain polypeptide with a molecular weight of about 55.23 kDa, composed of 487 amino acid residues, and a gene length of 1464 bp. Although T4 DNA ligase has better overall performance than other DNA ligases, the existing T4 DNA ligase has poor stability, and its activity gradually decreases over time, leading to problems such as reduced ligation products during molecular cloning and reduced NGS library production. In addition, during NGS sequencing, when T4 DNA ligase is used to connect DNA fragments to adapters, adapter-adapter self-ligation and DNA fragment-fragment self-ligation often occur, resulting in fewer products of correct connection between DNA fragments and adapters, and ultimately reducing the NGS library production. Summary of the invention

[0004] The purpose of the present invention is to provide a mutant T4 DNA ligase, which has improved performance compared to the wild-type T4 DNA ligase.

[0005] The technical solution adopted by the present invention is:

[0006] A mutant T4 DNA ligase, which is a protein described in any one of the following a1-a3:

[0007] a1: Based on the amino acid sequence of T4 DNA ligase shown in SEQ ID No. 1, any mutation occurs in one or more of the following sites: 117, 118, 148, 160, 163, 255, 383, 391, 402, 403, 449, 458;

[0008] a2: a protein having substantially the same enzyme activity and performance obtained by replacing and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutation in the amino acid sequence shown in a1;

[0009] a3: A protein having at least 90% sequence identity with the protein of a1 and having enzyme activity and performance substantially equivalent to the protein of a1, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% sequence identity.

[0010] Preferably, based on the amino acid sequence of T4 DNA ligase as shown in SEQ ID No. 1, any mutation occurs at one or more of the following sites: 40, 51, 207, 251, 332, 333, 334, 343, 371, 404, 405, 448, 487.

[0011] Preferably, one or more of the following mutations occur on the basis of the amino acid sequence of T4 DNA ligase as shown in SEQ ID No. 1: 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.

[0012] Preferably, it is a protein described in any one of the following b1-b3:

[0013] b1: A protein obtained by subjecting the amino acid sequence of T4 DNA ligase shown in SEQ ID No. 1 to any of the following mutations:

[0014] (1)A117S;

[0015] (2) S118T;

[0016] (3) K148E;

[0017] (4) A160Y;

[0018] (5)A163E;

[0019] (6) T255Y;

[0020] (7) R383S;

[0021] (8)K402V / V403G / N404K / A405V;

[0022] (9)G449D;

[0023] (10) L458A;

[0024] (11)S118T / L487R;

[0025] (12)T255Y / L343H;

[0026] (13) R383S / K402V;

[0027] (14) R383S / D371C;

[0028] (15) R383S / D371C / S40D;

[0029] (16)K402V / V403G / N404K / A405V / G391C;

[0030] (17)K402V / V403G / N404K / A405V / G391C / L343H;

[0031] (18)K402V / V403G / N404K / A405V / G391C / D448G;

[0032] (19)K402V / V403G / N404K / A405V / P51S / A117S;

[0033] (20)K402V / V403G / N404K / A405V / K332E / V333K / I334F;

[0034] (21)K402V / V403G / N404K / A405V / R383S / D371C / S40D;

[0035] (22) L458A / I207V;

[0036] (23) L458A / I207V / A251P;

[0037] b2: a protein having substantially the same enzyme activity and performance obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in b1 except for the aforementioned changes;

[0038] b3: A protein that has at least 90% sequence identity with the protein of b1 and has enzyme activity and performance that are substantially equivalent to those of the protein of b1.

[0039] 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 library construction yield.

[0040] The gene encoding the mutant T4 DNA ligase described above.

[0041] The expression vector or host bacteria of the above-mentioned mutant T4 DNA ligase.

[0042] The mutant T4 DNA ligase is used for DNA-DNA, DNA-RNA and RNA-RNA ligation.

[0043] A kit comprising the mutant T4 DNA ligase described above.

[0044] A sequencing library construction kit contains the mutant T4 DNA ligase.

[0045] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. Percent sequence identity can be calculated by any method known in the art, for example using the BLOSUM62 matrix, with reference to the method described by Henikoff et al. in PNAS, 89(22):10915-10919 (1992).

[0046] The description of mutants in the present invention is a description recognized by those skilled in the art, and a sequence example of a mutant, A117S, refers to a mutation of alanine (A) at position 117 of the amino acid sequence shown in SEQ ID NO: 1 to serine (S).

[0047] The mutant T4 DNA ligase provided herein also includes an amino acid sequence having at least 90% sequence identity to the mutant sequence, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% sequence identity.

[0048] Herein, "substantially the same" or "substantially equivalent" means that under the same test conditions, the deviation of the test values ​​of the enzyme activity and performance of the variant T4 DNA ligase does not exceed 20%. In some embodiments of the present invention, the performance test includes testing thermal stability, testing specific activity, and testing adapter-adapter self-ligation. T4 DNA ligase is an ATP-dependent ligase. From the crystal structure analysis of the complex of the enzyme and DNA, it has a DNA binding domain-DBD, a nucleotidyl transferase NTase domain and an OB fold domain. Figure 1 This paper provides a basic route for the modification of T4 DNA ligase. In terms of rational design, we start from the structure and analyze the surrounding DNA substrate. We identified amino acid residues in the range that can interact with DNA, and used computational methods to perform virtual screening and structure-activity analysis on these amino acids. At the same time, based on sequence conservation analysis, we also compared the sequences of such ligases and designed them in combination with structure and force analysis, and identified 14 key amino acids (117, 118, 148, 160, 163, 255, 383, 391, 402, 403, 404, 405, 449, 458), such as Figure 1 As shown, we constructed and screened the site-saturation mutation library for these sites. In terms of directed evolution, we constructed and screened the random mutation library based on the dominant mutants screened by the site-saturation library. We used the MTPS method to screen the stability, activity and residual activity of the enzyme in parallel. We purified and characterized the dominant mutants based on the screening data of various dimensions. After fine purification of the dominant mutants obtained from both rational design and directed evolution, we conducted in-depth analysis of the mutants’ stability, specific activity, linker self-ligation rate, fragment self-ligation rate during library construction, library construction yield and other dimensions to determine the dominant mutants of the G1 generation. On the basis of the G1 generation, we further carried out DNA Shuffling screening and combined mutation CDM, and finally obtained T4 DNA ligase mutants that met various performance indicators.

[0049] Compared with the wild-type T4 DNA ligase, the mutant of the present invention has improved one or at least two of the following properties: improved thermal stability, improved specific activity, reduced linker-linker self-ligation ratio, improved DNA library construction yield, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Schematic diagram of the simulated spatial folding of wild-type T4 DNA ligase.

[0051] Figure 2 The thermal stability data of the parent V0 and mutants V1-V10.

[0052] Figure 3 The thermal stability data of the parent V0 and mutants V11-V23. DETAILED DESCRIPTION

[0053] The specific implementation modes of the present invention are further described below in conjunction with the accompanying drawings, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0055] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.

[0056] Example 1: Transformation ideas and acquisition of mutant T4 DNA ligase

[0057] Wild-type T4 DNA ligase has defects such as mild reaction conditions, temperature sensitivity, easy inactivation, instability, poor storage resistance, and low DNA yield in NGS library construction applications. Therefore, we modified the wild-type T4 DNA ligase V0 (amino acid sequence as shown in SEQ ID No.1). Based on the wild-type T4 DNA ligase, we adopted a combination of rational design and directed evolution to modify it, and screened out mutants with advantages from tens of thousands of sequences, as shown in Table 1:

[0058] Table 1 Mutant numbers and mutation types

[0059]

[0060]

[0061] Example 2: Thermal stability assay of T4 DNA ligase mutants

[0062] The T4 DNA ligase mutant was diluted to 50 U / μL according to the enzyme activity, incubated at 42°C for 0 and 10 min, and then added to the following activity test reaction system:

[0063] name Volume / μL FAM DNA (5 μM) 1 BHQ-1 DNA (5 μM) 1 10×ligase buffer 10 T4 DNA ligase 2 <![CDATA[ddH 2 The]]> 86

[0064] The fluorescence values ​​at 0 min and 10 min of incubation were monitored, and the thermal stability of the T4 DNA ligase mutant was characterized by calculating the ratio of the fluorescence decrease.

[0065] Mutant stability data are shown in Figure 2 and Figure 3 , which is the residual activity value measured after comparing the parent V0 and the mutants at 42°C for 10 minutes. V0 represents the parent, i.e. the wild-type T4 DNA ligase with the amino acid sequence shown in SEQ ID No. 1, V1-V23 are mutant T4 DNA ligases obtained by modification based on V0; the mutant numbers refer to a series of sequences in Table 1.

[0066] Conducting a thermal stability test at 42°C is one of the common methods used by those skilled in the art to study the thermal stability of T4 DNA ligase mutants and their variants. Although the thermal stability test of the present application is conducted at 42°C, this does not mean that the highest heat-resistant temperature of the mutant of the present application is 42°C. The larger the residual activity value, the higher the thermal stability, and the higher the reaction temperature and / or the longer the reaction time can be tolerated.

[0067] Example 3: Determination of enzyme activity of T4 DNA ligase mutants

[0068] The principle of the method for detecting the activity of T4 DNA ligase is to use a DNA fragment with a fluorescent group (FAM-DNA) and a DNA fragment with a fluorescent quenching group (BHQ-1DNA) as substrates. In the presence of DNA ligase, the two DNA fragments are connected and the fluorescence intensity gradually decreases, that is, there is a certain relationship between the fluorescence intensity and the DNA ligase. The activity of T4 DNA ligase is detected by measuring the intensity of fluorescence. The specific implementation method is as follows:

[0069] (1) Substrate preparation

[0070] FAM-DNA was prepared by mixing primers A (sequence: 5`-TAG / i6FAMdT / ACACTGTCCTCATTG-3`) and B (sequence: 5`-CAATGAGGACAGTGT-3`) in equal volumes, and diluted with TE buffer to a final concentration of 5 μM, incubated at 37°C for 5 min, and then placed at room temperature for 10 min and stored at -20°C for later use. BHQ-1DNA was prepared by mixing primers C (sequence: 5`-CTCCTCGTTCATCTAC-3`) and D (sequence: 5`-ACTAG / iBHQ1dT / AGATGAACGAGGAG-3`) in equal volumes, and prepared into a solution with a final concentration of 5 μM according to the FAM-DNA preparation method for later use.

[0071] (2) Reaction system preparation

[0072] name Volume / μL FAM DNA (5 μM) 1 BHQ-1 DNA (5 μM) 1 10×ligase buffer 10 T4 DNA ligase 2 <![CDATA[ddH 2 The]]> 86

[0073] Add different concentrations of T4 DNA ligase to the above reaction system, mix well, incubate at 25°C for 30 minutes, and read the fluorescence value at 485nm / 535nm. The enzyme activity of the T4 DNA ligase mutant can be measured by calculation based on the relationship between the T4 DNA ligase with known enzyme activity and the fluorescence value.

[0074] Table 2 provides a list of T4 DNA ligase mutants of specific sequences with specific relevant activities disclosed in the present invention. The mutant numbers refer to a series of sequences in Table 1 respectively; the mutant numbers refer to a series of sequences in Table 1 respectively; in the specific activity column, "-" indicates that the specific activity of the mutant protein is equivalent to or reduced compared with the specific activity of V0. One plus sign "+" indicates that the specific activity of the mutant protein is 120% to 150% of the specific activity of V0, that is, 120% ≤ mutant specific activity / wild-type specific activity <150%. Two plus signs "++" indicate that the specific activity of the mutant protein is 150% to 200% of the specific activity of V0, that is, 150% ≤ mutant specific activity / wild-type specific activity <200%. Three plus signs "+++" indicate that the specific activity of the mutant protein is 200% to 250% of the specific activity of V0, that is, 200% ≤ mutant specific activity / wild-type specific activity <250%. Four plus signs "++++" indicate that the specific activity of the mutant protein is 250% to 400% of the specific activity of V0, i.e., 250% ≤ mutant specific activity / wild-type specific activity < 400%. V0 represents the parent, i.e., the wild-type T4 DNA ligase whose amino acid sequence is shown in SEQ ID No. 1, and V1-V23 are mutants obtained by modification based on V0.

[0075] Table 2 Specific activity of mutants

[0076]

[0077]

[0078] Example 4: T4 DNA ligase mutant linker residue determination

[0079] In this implementation case, ultrasonically sheared calf thymus DNA was used as a fragmentation template, and then the YEASEN DNA library construction kit (Cat. No. 12201) was used to build the library, and then Qubit and Qsep were used to detect the yield of the library and the residual adapter. The specific implementation method is as follows:

[0080] (1) End repair / dA tail addition

[0081] The amplification procedure is as follows:

[0082]

[0083] (2) Add connector

[0084] Temperature (heat cover 105℃) time 30℃ 30min 72℃ 30min 4℃ Hold

[0085] The amplification procedure is as follows:

[0086] name Input dA-tailed DNA 60μL 12201-C 30μL T4 DNA ligase (300 ng / μL) 10μL adapter (0.5μM) (YEASEN Product No. 13519) 5μL <![CDATA[ddH 2 The]]> 5μL

[0087] (3) Magnetic bead purification (1×)

[0088] 1) Preparation: Take the DNA Selection Beads (12601) magnetic beads out of the refrigerator and equilibrate them at room temperature for at least 30 minutes. Prepare 80% ethanol.

[0089] 2) Vortex or invert the beads thoroughly to ensure thorough mixing.

[0090] Temperature (heat cover off) time 20℃ 15min 4℃ Hold

[0091] 3) Pipette 110 μL Add DNA Selection Beads to Adapter Ligation product, vortex or pipette gently to mix thoroughly, and incubate at room temperature for 5 minutes.

[0092] 4) Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the magnetic beads and liquid. After the solution is clear (about 5 minutes), carefully remove the supernatant.

[0093] 5) Keep the PCR tube in the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0094] 6) Repeat step 5 for a total of two rinses. Finally, use a 10 μL pipette tip to remove the remaining liquid.

[0095] 7) Keep the PCR tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads until cracks just appear (no more than 5 minutes).

[0096] 8) Take the PCR tube out of the magnetic stand and add 21 μL ddH 2 O, vortex or use a pipette to gently blow until fully mixed, and let it stand at room temperature for 5 minutes. Centrifuge the PCR tube briefly and place it on a magnetic stand. After the solution is clear (about 5 minutes), carefully transfer 20 μL of the supernatant to the PCR tube without touching the magnetic beads.

[0097] (4) Library amplification system:

[0098] name Input Adapter Ligated DNA 20μL 12201-E 25μL Primer(25μM)(12201-F) 5μL

[0099] Library amplification procedure:

[0100] temperature time Number of cycles 98℃ 1min 98℃ 10s 12 60℃ 30s 72℃ 30s 72℃ 5min 4℃ Hold

[0101] (5) Magnetic bead purification (0.5×)

[0102] The purification steps are the same as those in step (3). DNA Selection Beads (0.5×, Beads:DNA=0.5:1) were used to purify the library amplification products. 30μL ddH 2 O elution.

[0103] (6) Concentration test

[0104] Before use, return all components of the 1×dsDNA HS Assay Kit (YEASEN Catalog No. 12642) to room temperature and mix by inverting. The Qubit calibration was performed according to the instructions of the kit.

[0105] Take 199 μL of 1×dsDNA detection solution into a 0.5 mL thin-walled centrifuge tube, add 1 μL of the sample to be tested, and gently vortex for 2-3 seconds to avoid bubbles. Use Qubit for reading.

[0106] (7) Joint residue test

[0107] According to the concentration tested by Qubit, the library production of different mutant T4 DNA ligases was diluted to 2 ng / μL, and then the percentage of the residual linker was obtained using Qsep.

[0108] Table 3 provides the analysis results of the linker-linker self-ligation ratio of T4 DNA ligase during NGS library construction. The lower the linker-linker self-ligation ratio, the more conducive it is to NGS library construction and improve the yield of NGS library construction. In the following table, the mutant numbers refer to a series of sequences in Table 1; in the linker-linker self-ligation ratio column, the minus sign "-" indicates that the linker-linker self-ligation ratio of the mutant protein is higher than the linker-linker self-ligation ratio of V0. A plus sign "+" indicates that the linker-linker self-ligation ratio of the mutant protein is equivalent to the linker-linker self-ligation ratio of V0, and is within ±10% compared with the protein composed of the amino acid sequence shown in SEQ ID No.1 in the sequence table. Two plus signs "++" indicate that the linker-linker self-ligation ratio of the mutant protein is reduced by 20%-50% compared with the linker-linker self-ligation ratio of V0, that is, 20%≤The linker-linker self-ligation ratio of the mutant protein is reduced by less than 50% compared with the linker-linker self-ligation ratio of V0. Three plus signs “+++” indicate that the linker-linker self-ligation ratio of the mutant protein is reduced by 50%-100% compared with the linker-linker self-ligation ratio of V0, that is, 50%≤the linker-linker self-ligation ratio of the mutant protein is reduced by <100% compared with the linker-linker self-ligation ratio of V0.

[0109] Table 3 The proportion of self-ligation of mutants

[0110]

[0111] The present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A mutant T4 DNA ligase, characterized in that The protein is as follows: A protein obtained by subjecting the amino acid sequence of T4 DNA ligase shown in SEQ ID No. 1 to any of the following mutations: (7) R383S; (13) R383S and K402V; (14) R383S and D371C; (15) R383S, D371C and S40D; (21)K402V and V403G and N404K and A405V and R383S and D371C and S40D.

2. The gene encoding the mutant T4 DNA ligase according to claim 1.

3. The expression vector or host bacteria of the mutant T4 DNA ligase according to claim 1.

4. Use of the mutant T4 DNA ligase according to claim 1 in ligating DNA-DNA, DNA-RNA, and RNA-RNA.

5. A kit, characterized in that A T4 DNA ligase comprising the mutation according to claim 1.

6. A sequencing library construction kit, characterized in that A T4 DNA ligase comprising the mutation according to claim 1.

Images