T4 DNA ligase mutants and their applications

Abstract

The present invention provides a T4 DNA ligase mutant, 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 T4 DNA ligase mutant and its application, belonging to the field of biotechnology. Background Art

[0002] T4 DNA ligase can catalyze the formation of diester bonds between 5'-phosphate groups and 3'-hydroxyl groups in double-helix DNA or RNA with the help of ATP cofactor, thereby repairing single-strand 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, we found that the existing T4 DNA ligase has poor stability, and its activity gradually decreases over time, leading to problems such as reduced ligation products in molecular cloning and reduced NGS library production. In addition, during the NGS sequencing process, 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.

[0004] To solve the above common and urgent problems, we modified T4 DNA ligase and obtained mutants with better performance. Summary of the invention

[0005] The purpose of the present invention is to provide a T4 DNA ligase mutant with high thermal stability.

[0006] The technical solution adopted by the present invention is:

[0007] A T4 DNA ligase mutant, which is a protein described in any one of the following a1-a3:

[0008] a1: Based on the wild-type T4 DNA ligase with the amino acid sequence as shown in SEQ ID No. 1, any mutation is performed at one or more of the following sites: 243, 251, 296, 332, 334, 338, 343, 472, 486, 487;

[0009] 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;

[0010] a3: A protein that has at least 90% sequence identity with the protein of a1 and has enzyme activity and performance that are substantially equivalent to the protein of a1.

[0011] Preferably, it also includes any mutation of one or more of the following sites: 16, 19, 255, 333, 371, 383, 448, 470.

[0012] T4 DNA ligase is an ATP-dependent ligase. The crystal structure of the complex between the enzyme and DNA shows that it has a DNA binding domain (DBD), a nucleotidyl transferase (NTase) domain, and an OB fold domain. Figure 1 The basic route for the modification of T4 DNA ligase is provided. From the structural analysis of T4 DNA ligase, it can be seen that the active site is K159, the ATP binding sites are R164, R182, E217, R359 and K365, and the divalent metal ion binding sites are E217 and E344. Based on the structural analysis, we found that there is a Helix structure near the active region-Helix326-343 with obvious interaction, which is speculated to affect the performance of T4 DNA ligase by affecting the performance of the active site. Therefore, based on rational design, we used computational methods to conduct virtual screening and structure-activity analysis of these amino acids; at the same time, based on sequence conservation analysis, we also conducted sequence alignment of such ligases and combined structure and force analysis for design and screening, as shown in the figure. At the same time, in terms of directed evolution, we constructed more than 10,000 random mutation libraries based on dominant mutants, and used the MTPS method to screen the stability, activity and residual activity of the enzyme in parallel. The screening data of various dimensions were integrated to purify and characterize the dominant mutants. Further work was carried out on combined mutation of CDM, and finally a T4 DNA ligase mutant that met various performance indicators was obtained. After analysis, it was found that sites 251, 255, 296, 332, 333, 334, 338, 343, 371, 383, 448, 470, 472, 486, and 487 affected the performance of T4 ligase by affecting the structure of Helix326-343.

[0013] The mutant provided by the present invention can further improve the performance of T4 DNA ligase. Among them, 332, 334, 338, and 343 are located in the same α-helical secondary structure, and one or more mutations in these sites form more stable hydrogen bonds or other forces within the α-helical secondary structure or with other secondary structures, which can improve the thermal stability of the mutant.

[0014] Preferably, the mutation is any one of K16E, Q19E, K243E, A251P, T255Y, F296S, K332E, V333K, I334F, Y338A, L343A or L343F or L343H, D371C, R383S, D448G, K470R, N472D, G486V, L487R, or a combination of at least two thereof.

[0015] The description of the mutation in the present invention is a description of the mutation recognized by those skilled in the art. Taking the mutation of one of the sites as an example, K243E, it means that the lysine (K) at position 243 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to glutamic acid (E), that is, the lysine (K) at position 243 is replaced by glutamic acid (E).

[0016] 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).

[0017] The T4 DNA ligase mutants provided herein also include amino acid sequences having at least 90% sequence identity to the mutant sequence, or amino acid sequences having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% sequence identity.

[0018] 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 T4 DNA ligase mutant 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.

[0019] Preferably, it is a protein described in any one of the following b1-b3:

[0020] b1: A protein obtained by causing any combination of the following mutations to occur on the basis of the wild-type T4 DNA ligase with the amino acid sequence shown in SEQ ID No. 1:

[0021] 1)K332E / V333K / I334F

[0022] 2) Y338A

[0023] 3) L343A

[0024] 4) L343F

[0025] 5)L343H

[0026] 6)K332E / V333K / I334F / K243E

[0027] 7)K332E / V333K / I334F / Y338A

[0028] 8)K332E / V333K / I334F / R383S

[0029] 9)K332E / V333K / I334F / D448G

[0030] 10)Y338A / D448G

[0031] 11)L343H / T255Y

[0032] 12)L343H / Y338A

[0033] 13)L343H / D371C

[0034] 14)L343H / D448G

[0035] 15)L343H / D371C / L487R

[0036] 16)L343H / D371C / K16E / G486V

[0037] 17)L343H / D371C / Q19E / N472D

[0038] 18) L343H / D371C / F296S / K470R;

[0039] 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 other than the aforementioned mutation in the amino acid sequence shown in b1;

[0040] 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.

[0041] 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.

[0042] The gene encoding the aforementioned T4 DNA ligase mutant.

[0043] The expression vector or host bacteria of the aforementioned T4 DNA ligase mutant.

[0044] The aforementioned T4 DNA ligase mutant is used for the ligation of DNA-DNA, DNA-RNA and RNA-RNA.

[0045] The invention also discloses a kit containing the aforementioned T4 DNA ligase mutant.

[0046] The invention also discloses a sequencing library construction kit, which contains the aforementioned T4 DNA ligase mutant.

[0047] Compared with the prior art, the mutants of the present invention include 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Schematic diagram of the simulated spatial folding of wild-type T4 DNA ligase.

[0049] Figure 2 The thermal stability data of the parent V0 and each mutant. DETAILED DESCRIPTION

[0050] 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.

[0051] 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.

[0052] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.

[0053] Example 1: Transformation ideas and acquisition of mutant T4 DNA ligase

[0054] The wild-type T4 DNA ligase, denoted as parent V0, has an amino acid sequence as shown in SEQ ID NO.1. It has mild action conditions, is sensitive to temperature, is easily inactivated, unstable, not resistant to storage, and has low DNA yield in NGS library construction applications. Therefore, we modified the wild-type T4 DNA ligase protein. Based on the wild-type T4 DNA ligase, we adopted a combination of rational design and directed evolution to modify it, and screened out the numbers and amino acid mutation types of mutants with advantages from tens of thousands of sequences, as shown in Table 1 below:

[0055] Table 1 Numbers and amino acid mutation types of mutants

[0056]

[0057]

[0058] Example 2: Thermal stability assay of T4 DNA ligase mutants

[0059] 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 activity measurement reaction system as shown below.

[0060] 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

[0061] 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.

[0062] Figure 2 The mutant stability data in the table are residual activity values ​​measured after the parent V0 and each mutant were incubated at 42°C for 10 minutes. V0 represents the parent, i.e., the wild-type T4 DNA ligase with an amino acid sequence as shown in SEQ ID No. 1, V1-V18 are mutant T4 DNA ligases obtained by modification based on V0, and the mutant numbers refer to a series of sequences in Table 1.

[0063] 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.

[0064] Example 3: Determination of enzyme activity of T4 DNA ligase mutants

[0065] The principle of the T4 DNA ligase activity detection method 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 enzyme activity of T4 DNA ligase is detected by measuring the intensity of fluorescence. The specific detection method is as follows:

[0066] (1) Substrate preparation

[0067] 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, 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.

[0068] (2) Reaction system preparation

[0069]

[0070]

[0071] 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.

[0072] Table 2 provides the specific activity of the T4 DNA ligase mutants disclosed in the present invention, and 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 lower than 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, and V1-V18 are mutants obtained by transformation based on V0.

[0073] Table 2 Specific activity of mutants

[0074] Parent and mutant numbers Specific activity (compared to V0) V0 / V1 + V2 - V3 + V4 ++ V5 ++ V6 ++ V7 - V8 + V9 + V10 - V11 + V12 + V13 ++++ V14 + V15 ++++ V16 ++++ V17 ++++ V18 +++

[0075] Example 4: T4 DNA ligase mutant linker residue determination

[0076] 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:

[0077] (1) End repair / dA tail addition

[0078]

[0079] The amplification procedure is as follows:

[0080] Temperature (heat cover 105℃) time 30℃ 30min 72℃ 30min 4℃ Hold

[0081] (2) Adding joint system:

[0082]

[0083] The amplification procedure is as follows:

[0084] Temperature (heat cover off) time 20℃ 15min 4℃ Hold

[0085] (3) Magnetic bead purification (1×)

[0086] 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.

[0087] 2) Vortex or invert the beads thoroughly to ensure thorough mixing.

[0088] 3) Absorb 110 swing or charge Add DNA Selection Beads to Adapter Ligation product, vortex or pipette gently to mix thoroughly, and incubate at room temperature for 5 minutes.

[0089] 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.

[0090] 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.

[0091] 6) Repeat step 5 for a total of two rinses. Finally, use a 10 μL pipette tip to remove the remaining liquid.

[0092] 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).

[0093] 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.

[0094] (4) Library amplification system:

[0095] name Input Adapter Ligated DNA 20μL 12201-E 25μL Primer(25μM)(12201-F) 5μL

[0096] Library amplification procedure:

[0097] temperature time Number of cycles 98℃ 1min 98℃ 10s 12 60℃ 30s 72℃ 30s 72℃ 5min 4℃ Hold

[0098] (5) Magnetic bead purification (0.5×)

[0099] 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.

[0100] (6) Concentration test

[0101] 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.

[0102] Take 199 μL of the test solution in kit 11 into a 0.5 mL thin-walled centrifuge tube, add 1 μL of the sample to be tested, and vortex gently for 2-3 seconds to avoid bubbles. Use Qubit for reading.

[0103] (7) Joint residue test

[0104] 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.

[0105] Table 3 provides the results of the analysis of the adapter-to-adapter self-ligation ratio of the T4 DNA ligase mutants during the NGS library construction process. The lower the adapter-to-adapter self-ligation ratio, the more favorable it is for the NGS library construction yield. In the adapter-to-adapter self-ligation ratio column, the negative sign "-" indicates that the adapter-to-adapter self-ligation ratio of the mutant protein is higher than the adapter-to-adapter self-ligation ratio of V0. One plus sign "+" indicates that the adapter-to-adapter self-ligation ratio of the mutant protein is equivalent to the adapter-to-adapter self-ligation ratio of V0, within ±10% compared with V0. Two plus signs "++" indicate that the adapter-to-adapter self-ligation ratio of the mutant protein is reduced by 20%-50% compared with the adapter-to-adapter self-ligation ratio of V0, that is, 20%≤The percentage of reduction of the adapter-to-adapter self-ligation ratio of the mutant protein compared with the adapter-to-adapter self-ligation ratio of V0 is <50%. 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.

[0106] Table 3 Linker-linker self-ligation ratio of mutants

[0107]

[0108] The applicant declares that 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 T4 DNA ligase mutant, characterized in that The protein is as follows: A protein obtained by causing any of the following mutations to occur on the basis of the wild-type T4 DNA ligase with the amino acid sequence shown in SEQ ID No. 1: 3) L343A; 4) L343F; 5) L343H; 11) L343H / T255Y; 12) L343H / Y338A; 13) L343H / D371C; 14) L343H / D448G; 15) L343H / D371C / L487R; 16)L343H / D371C / K16E / G486V; 17) L343H / D371C / Q19E / N472D; or 18)L343H / D371C / F296S / K470R.

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

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

4. Use of the T4 DNA ligase mutant according to claim 1 in DNA-DNA ligation.

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

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

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