Method and kit for detecting dephosphorylation activity of T4 polynucleotide kinase

Abstract

The present invention relates to the technical field of enzyme activity analysis, and specifically to a method and a kit for detecting the dephosphorylation activity of T4 polynucleotide kinase. The present invention designs a nucleic acid chain with both 5' and 3' ends modified with phosphate as a reaction substrate. Since the 5' end has been provided with a phosphate modification structure in advance, the 5' phosphorylation activity of the T4 polynucleotide kinase to be tested is prevented, so that only a 3' dephosphorylation reaction occurs during the entire detection process, and a nucleic acid chain reaction product with a phosphate modification at the 5' end and a hydroxyl at the 3' end is obtained. After a certain period of detection reaction, the content relationship between the reaction product and the reaction substrate in the reaction mixture is determined, and the conversion rate of the reaction substrate to the reaction product per unit time is used to define the dephosphorylation activity of the T4 polynucleotide kinase. The entire detection method is simple to operate and easy to control.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme activity analysis, and in particular to a method and a kit for detecting the dephosphorylation activity of T4 polynucleotide kinase. Background Art

[0002] T4 polynucleotide kinase, abbreviated as T4PNK, is a protein encoded by the pseT gene of T4 bacteriophage. It was originally isolated from Escherichia coli cells infected by T4 bacteriophage, so it is also called T4 polynucleotide kinase. The enzyme can catalyze the transfer of γ-phosphate of ATP to the 5' hydroxyl group of single-stranded or double-stranded DNA, RNA, oligonucleotide or mononucleotide with 3' phosphate group. The enzyme also has 3' phosphatase activity, which hydrolyzes the 3'-phosphate group from the 3' phosphate end of oligonucleotide, deoxy 3'-monophosphate nucleoside and deoxy 3'-diphosphonate nucleoside to form a 3'-hydroxyl structure.

[0003] T4PNK has a wide range of applications, such as labeling the 5' end of oligonucleotides, DNA or RNA, used as probes for Southern, Northern, EMSA, etc., markers for gel electrophoresis, DNA sequencing primers, PCR primers, etc.; phosphorylating the 5' end of oligonucleotides, DNA or RNA to ensure the smooth progress of subsequent ligation reactions; catalyzing the 5' phosphorylation of 3' phosphorylated mononucleotides so that the mononucleotide can be connected to the 3' end of DNA or RNA; removing the phosphate group at the 3' end.

[0004] During the production of T4PNK, it is usually necessary to evaluate and judge the activity of T4PNK, including the 5' end phosphorylation activity and the 3' end dephosphorylation activity. At present, there are some methods to analyze and detect the 5' end phosphorylation activity of T4PNK, such as the [γ-33P]ATP incorporation method using radioactive isotopes; Patent CN104293929A discloses a method of detecting the 5' phosphorylation activity of T4PNK by a luciferase reporter group system that can produce chemiluminescence by oxidizing ATP to produce oxygenated luciferin; Patent CN108823280A discloses a method for detecting the activity of T4 polynucleotide kinase based on a silver nanocluster fluorescent probe (by using a label-free, "turn-off" type fluorescent probe).

[0005] The above-mentioned T4PNK enzyme activity detection methods all detect the activity of T4PNK 5' end phosphorylation, and lack a simple, efficient and accurate analytical method for detecting the dephosphorylation activity of T4PNK 3' end. Summary of the invention

[0006] One of the purposes of the present invention is to provide a method for detecting the dephosphorylation activity of T4 polynucleotide kinase. The method comprises designing a primer probe with phosphate groups at both the 5' and 3' ends as a substrate, adding the T4 polynucleotide kinase to be tested to react, obtaining a nucleic acid chain product with a phosphate group at the 5' end and a hydroxyl group at the 3' end, and determining the content relationship between the substrate and the product to further deduce the dephosphorylation activity of the T4 polynucleotide kinase. The method is simple to operate and easy to control.

[0007] At the same time, the present invention also aims to provide a kit for detecting the dephosphorylation activity of T4 polynucleotide kinase.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A method for detecting the dephosphorylation activity of T4 polynucleotide kinase, comprising sequentially adding a reaction substrate and a T4 polynucleotide kinase to be detected into a reaction buffer, detecting the content relationship between the reaction substrate and the reaction product in the reaction mixture after the reaction is completed, and deriving the dephosphorylation activity of the T4 polynucleotide kinase;

[0010] The reaction substrate is a nucleic acid chain with both the 5' end and the 3' end modified with phosphate; the reaction product is a nucleic acid chain with the 5' end modified with phosphate and the 3' end being a hydroxyl group.

[0011] Furthermore, the above method for detecting the content relationship between the reaction substrate and the reaction product in the reaction mixture is: using HPLC detection, the reaction substrate and the reaction product have different retention times, and the dephosphorylation activity of T4 polynucleotide kinase is deduced based on the ratio of the peak areas corresponding to the reaction substrate and the reaction product.

[0012] In order to further improve the accuracy of the detection result, the total content of nucleotide A and nucleotide T in the reaction substrate sequence is 70%, and the content ratio of nucleotide A to nucleotide T is 1:1; further, the reaction substrate sequence does not contain nucleotide G.

[0013] Furthermore, the reaction substrate sequence consists of: 5'-P-CATTATTATCACCCATAAC-P-3'; and the reaction product sequence consists of: 5'-P-CATTATTATCACCCATAAC -3'.

[0014] Optionally, the above HPLC analysis conditions are: using a Poroshell 5μm 300SB-C18 2.1*75mm chromatographic column; mobile phase: A: 0.1M TEAB; B: 30% acetonitrile solution by volume in 0.1M TEAB.

[0015] Furthermore, the flow rate of the mobile phase was 0.5 ml / min; the mobile phase elution was gradient elution: 0-15 min: 90% A, 10% B; 15-20 min: 40% A, 60% B; 20-25 min: 90% A, 10% B.

[0016] Furthermore, the HPLC analysis conditions were: wavelength 260 nm; column temperature 30°C.

[0017] Furthermore, the HPLC analysis conditions are: using DAD detector, 220-400nm full-band scanning mode

[0018] Optionally, the above detection method further comprises centrifuging the reaction mixture and taking the supernatant to detect the content relationship between the reaction substrate and the reaction product.

[0019] Furthermore, the centrifugation is performed at 12000 rpm for 20 min.

[0020] Furthermore, the completion of the reaction includes reacting at 37° C. for 20 to 30 minutes and terminating the reaction.

[0021] Optionally, the method for terminating the reaction includes adding EDTA to terminate the reaction or heat-treating to inactivate the phosphorylation activity of T4 polynucleotide kinase.

[0022] Optionally, the heating temperature of the heat treatment is 65° C. and the heat treatment time is 20 min.

[0023] Optionally, the reaction buffer in the above detection method is 70mM Tris-HCl, 10mM MgCl2, 5mM DTTpH7.6@25

[0024] There are two common methods for enzyme activity determination: endpoint method and kinetic method. The kinetic method is to define the activity of the enzyme by measuring the amount of chemical reaction in a certain period of time. The above-mentioned method for detecting the dephosphorylation activity of T4 polynucleotide kinase is to use the kinetic method. T4 polynucleotide kinase has a variety of active effects, such as catalyzing the transfer of γ-phosphate of ATP to the 5' hydroxyl of single-stranded or double-stranded DNA, RNA, oligonucleotide or mononucleotide with 3' phosphate group, which is called 5' phosphorylation activity. The enzyme also has 3' phosphatase activity, which hydrolyzes the 3'-phosphate group from the 3' phosphate end of the oligonucleotide, deoxy 3'-monophosphate nucleoside and deoxy 3'-diphosphonate nucleoside to form a 3'-hydroxyl structure, which is called 3' dephosphorylation activity.

[0025] Therefore, in the process of determining the dephosphorylation activity of T4 polynucleotide kinase, the first technical problem to be solved is to prevent T4 polynucleotide kinase from exerting 5' phosphorylation activity. Then, the present invention designs a nucleic acid chain with both 5' and 3' ends modified with phosphate as a reaction substrate. Since the phosphate modification structure is set in advance at the 5' end, only 3' dephosphorylation reaction occurs during the entire detection process, and a nucleic acid chain reaction product with phosphate modification at the 5' end and hydroxyl at the 3' end is obtained. After a certain reaction time, the content relationship between the reaction product and the reaction substrate in the reaction mixture is detected, and the dephosphorylation activity of T4 polynucleotide kinase is defined as the conversion rate of the reaction product substrate into the reaction product per unit time.

[0026] Furthermore, the key to detecting the content relationship between the reaction product and the reaction substrate in the reaction mixture is to identify the reaction product and the reaction substrate respectively, and then quantify the content relationship between the reaction product and the reaction substrate. The present invention adopts HPLC as a detection means, distinguishes the reaction product and the reaction substrate by retention time, determines the ratio of the reaction product to the reaction substrate by the ratio of the peak area, and then determines the conversion rate of the reaction substrate per unit time, and defines the dephosphorylation activity of the T4 polynucleotide kinase to be tested. The entire analysis method is simple to operate and easy to control.

[0027] Furthermore, when HPLC is selected as the analytical method, in order to improve the accuracy and sensitivity of the detection results, it is necessary to further ensure that the reaction products and reaction substrates have clearly distinguishable retention times. Therefore, it is necessary to select a suitable reaction substrate chemical structure and select suitable chromatographic columns, mobile phases, flow rates, elution gradients and other analytical conditions according to the reaction substrate chemical structure. In view of this, the present invention designs a reaction substrate sequence composition of 5'-P-CATTATTATCACCCATAAC-P-3' through creative labor, adopts a Poroshell 5um 300SB-C18 2.1*75 chromatographic column, uses A: 0.1MTEAB; B: 30% acetonitrile solution with a volume content of 0.1MTEAB as a mobile phase, and selects a gradient elution program of 0-15min, 90%A, 10%B; 15-20min, 40%A, 60%B; 20-25min, 90%A, 10%B, so as to enhance the distinguishability of the retention time of the reaction substrate and the reaction product, reduce the impurity peaks, narrow the peak broadening, and improve the accuracy and sensitivity of detection.

[0028] Furthermore, the present invention also provides a kit for detecting the dephosphorylation activity of T4 polynucleotide kinase, comprising a reaction substrate reagent; the reaction substrate is a nucleic acid chain with both the 5' end and the 3' end modified with phosphate.

[0029] Furthermore, the concentration of the above reaction substrate reagent is 2 μM.

[0030] Furthermore, the kit also includes an enzyme reaction buffer. Preferably, the enzyme reaction buffer is 70mM Tris-HCl, 10mM MgCl2, 5mM DTT pH7.6@25. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a liquid chromatogram of the reaction substrate in Example 2;

[0032] Figure 2 is a liquid chromatogram of the reaction mixture after T4 polynucleotide kinase was added to complete the reaction in Example 2;

[0033] Figure 3 is a liquid chromatogram of the reaction mixture after T4 polynucleotide kinase was added to complete the reaction in Comparative Example 1;

[0034] Figure 4 is a liquid chromatogram of the reaction mixture after T4 polynucleotide kinase was added to complete the reaction in Comparative Example 2;

[0035] Figure 5 is a liquid chromatogram of the reaction mixture after T4 polynucleotide kinase was added to complete the reaction in Comparative Example 3;

[0036] Figure 6 It is a liquid chromatogram of the reaction mixture after T4 polynucleotide kinase was added to complete the reaction in Comparative Example 4. DETAILED DESCRIPTION

[0037] The present invention designs a nucleic acid chain with both 5' and 3' ends modified with phosphate as a reaction substrate. Since the 5' end is provided with a phosphate modification structure in advance, the 5' phosphorylation activity of the T4 polynucleotide kinase to be tested is prevented, so that only a 3' dephosphorylation reaction occurs during the entire detection process, and a nucleic acid chain reaction product with 5' end modified with phosphate and 3' end as a hydroxyl group is obtained. After a certain reaction time, the content relationship between the reaction product and the reaction substrate in the reaction mixture is detected, and the dephosphorylation activity of the T4 polynucleotide kinase is defined by the conversion rate of the reaction substrate to the reaction product per unit time. HPLC is further selected to detect the content relationship between the reaction product and the reaction substrate in the reaction mixture, and the accuracy and sensitivity of the detection are further improved by selecting a suitable reaction substrate sequence structure and chromatographic analysis conditions.

[0038] Below, in conjunction with specific embodiments, the present invention is further described, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0039] The reagents used in the following examples include: 10* reaction buffer: 700 mM Tris-HCl 100 mM MgCL 250 mM DTT pH 7.6@25; reaction substrate: 100 μM 5'-P-CATTATTATCACCCATAAC -P-3'.

[0040] Example 1

[0041] A method for detecting the dephosphorylation activity of T4 polynucleotide kinase comprises the following steps:

[0042] 1) Take 5 μL of reaction buffer, add 1 μL of reaction substrate, 1uL of T4PNK (New England Biolabs, catalog number #M0201), and then add DDH2O to make up to 50 μL. After reacting at 37°C for 10 minutes, heat to 65°C for inactivation treatment for 20 minutes, and then add 50 μL of DDH2O to mix evenly. Centrifuge at 4°C at 12000 rpm for 20 minutes, and take the supernatant for HPLC analysis;

[0043] 2) The injection volume of each HPLC analysis was 10uL, and the analysis conditions were: Agilent Poroshell 5μm300SB-C18 2.1*75 chromatographic column; wavelength 260nm; column temperature 30℃; mobile phase: A: 0.1M TEAB; B: 30% acetonitrile solution in 0.1MTEAB; mobile phase flow rate 0.5ml / min; mobile phase elution was gradient elution: 0~15min, 90%A, 10%B; 15~20min, 40%A, 60%B; 20~25min, 90%A, 10%B; chromatographic analysis spectrum was obtained;

[0044] 3) According to the chromatographic analysis spectrum obtained in step 2), the ratio of the peak areas corresponding to the reaction substrate and the reaction product is calculated, and the conversion rate of the reaction substrate into the reaction product under the action of T4 polynucleotide kinase per unit time is calculated, which is defined as the dephosphorylation activity of the T4 polynucleotide kinase to be tested.

[0045] Example 2

[0046] NEB T4 polynucleotide kinase (New England Biolabs, catalog number #M0201) was used as described in Example 1. Before adding T4 polynucleotide kinase to the reaction, the chromatographic analysis of the reaction substrate was as shown in the figure below. Figure 1 As shown; the chromatographic analysis after adding T4 polynucleotide kinase reaction is shown Figure 2As shown, the substrate conversion rate per unit enzyme amount per unit time is: 2uM*conversion rate / 10min / enzyme amount 1uL= 0.048 uM / (min*uL)=48nM / (min*uL)

[0047] Comparative Example 1

[0048] This comparative example provides a method for detecting the dephosphorylation activity of T4 polynucleotide kinase, which is different from the detection method provided in Example 1 in that the reaction substrate is different. The reaction substrate used in this comparative example is 5'-P-CCCATCTACCCACCCCCAA-P-3', and other reaction and detection conditions are the same as in Example 1.

[0049] The detection method provided in this comparative example was used to detect the dephosphorylation activity of the same batch of T4 polynucleotide kinase prepared in Example 2. The chromatographic analysis pattern of the detection was as follows: Figure 3 As shown in the figure, there are many impurity peaks between the reaction substrate and the reaction product, which affect the accuracy of the final calculation results.

[0050] Comparative Example 2

[0051] This comparative example provides a method for detecting the dephosphorylation activity of T4 polynucleotide kinase, which is different from the detection method provided in Example 1 in that the reaction substrate is different. The reaction substrate used in this comparative example is 5'-P-AATTATTATAAAAAATAAA-P-3', and other reaction and detection conditions are the same as in Example 1.

[0052] The detection method provided in this comparative example was used to detect the dephosphorylation activity of the same batch of T4 polynucleotide kinase prepared in Example 2. The chromatographic analysis pattern of the detection was as follows: Figure 4 As shown, the adsorption and elution processes of the reaction substrate and reaction product are unstable, and the peak stability of the reaction substrate and reaction product is poor, which affects the accuracy of the final calculation results.

[0053] Comparative Example 3

[0054] This comparative example provides a method for detecting the dephosphorylation activity of T4 polynucleotide kinase, which is different from the detection method provided in Example 1 in that the reaction substrate is different. The reaction substrate used in this comparative example is 5'-P-CAAAATTAACACCCATAAC-P-3', and other reaction and detection conditions are the same as in Example 1.

[0055] The detection method provided in this comparative example was used to detect the dephosphorylation activity of the same batch of T4 polynucleotide kinase prepared in Example 2. The chromatographic analysis pattern of the detection was as follows: Figure 5 As shown, the peak broadening of the reaction substrate and reaction product increases, affecting the accuracy of the final calculation results.

[0056] Comparative Example 4

[0057] This comparative example provides a method for detecting the dephosphorylation activity of T4 polynucleotide kinase, which is different from the detection method provided in Example 1 in that the reaction substrate is different. The reaction substrate used in this comparative example is 5'-P-GATTATTATGAGGGATAAG-P-3', and other reaction and detection conditions are the same as in Example 1.

[0058] The detection method provided in this comparative example was used to detect the dephosphorylation activity of the same batch of T4 polynucleotide kinase in Example 2. The chromatographic analysis pattern of the detection was as follows: Figure 6 As shown, after multiple verifications, the reaction substrate and reaction product could not be separated at all, the reaction product conversion rate could not be calculated, and the enzyme activity could not be determined.

[0059] By comparing the chromatograms of the above embodiment and the comparative example, it can be seen that the present invention, through the creative design of the 5'-end and 3'-end phosphate-modified nucleotide chains as substrates, can calculate the dephosphorylation activity of T4 polynucleotide kinase by detecting the content ratio of the final reaction substrate and the reaction product. Further, by creatively selecting a suitable AT content, AT ratio, and the selection and use of nucleotides C and G, the performance of the substrate nucleotide chain is optimized, the accuracy of the detection of the reaction substrate and the reaction product content is improved, and the accuracy of the final enzyme activity calculation result is thereby improved.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the dephosphorylation activity of T4 polynucleotide kinase, characterized in that: The method comprises sequentially adding a reaction substrate and a T4 polynucleotide kinase to be tested into a reaction buffer, detecting the content relationship between the reaction substrate and the reaction product in the reaction mixture after the reaction is completed, and deriving the dephosphorylation activity of the T4 polynucleotide kinase; The reaction substrate is a nucleic acid chain with both the 5' and 3' ends modified with phosphate; the reaction product is a nucleic acid chain with the 5' end modified with phosphate and the 3' end with a hydroxyl group; The method for detecting the content relationship between the reaction substrate and the reaction product in the reaction mixture is: using HPLC detection, the reaction substrate and the reaction product have different retention times, and the dephosphorylation activity of T4 polynucleotide kinase is deduced according to the ratio of the peak areas corresponding to the reaction substrate and the reaction product; The reaction substrate sequence consists of: 5'-P-CATTATTATCACCCATAAC-P-3'; the reaction product sequence consists of: 5'-P-CATTATTATCACCCATAAC -3'.

2. The method for detecting the dephosphorylation activity of T4 polynucleotide kinase according to claim 1, characterized in that: HPLC analysis conditions: Poroshell 5um 300SB-C18 2.1*75 column; mobile phase: A: 0.1M TEAB; B: 30% acetonitrile solution in 0.1M TEAB; The flow rate of the mobile phase was 0.5 ml / min; the mobile phase elution was gradient elution: 0-15 min: 90% A, 10% B; 15-20 min: 40% A, 60% B; 20-25 min: 90% A, 10% B; The HPLC analysis conditions were as follows: wavelength 260 nm; column temperature 30°C; DAD detector, 220-400 nm full-band scanning mode.

3. The method for detecting the dephosphorylation activity of T4 polynucleotide kinase according to any one of claims 1 to 2, characterized in that: The method also includes centrifuging the reaction mixture and taking the supernatant to detect the content relationship between the reaction substrate and the reaction product; the centrifugation is performed at 12000 rpm for 20 minutes.

4. The method for detecting the dephosphorylation activity of T4 polynucleotide kinase according to claim 3, characterized in that: The reaction is completed by reacting at 37° C. for 20 to 30 minutes, and then terminating the reaction; The method for terminating the reaction includes adding EDTA to terminate the reaction or heat treatment to inactivate the phosphorylation activity of T4 polynucleotide kinase; the heating temperature of the heat treatment is 65°C and the heat treatment time is 20 minutes; The reaction buffer was 70 mM Tris-HCl, 10 mM MgCl2, 5 mM DTT.

5. A kit for detecting the dephosphorylation activity of T4 polynucleotide kinase, characterized in that: The invention comprises a reaction substrate reagent; the reaction substrate is a nucleic acid chain with 5' end and 3' end both modified by phosphoric acid; the sequence composition of the reaction substrate is 5'-P-CATTATTATCACCCATAAC -P-3'.

6. The kit for detecting the dephosphorylation activity of T4 polynucleotide kinase according to claim 5, characterized in that: The final concentration of the reaction substrate reagent was 2 μM.

7. The kit for detecting the dephosphorylation activity of T4 polynucleotide kinase according to any one of claims 5 to 6, characterized in that: It also includes an enzyme reaction buffer; the enzyme reaction buffer is 70mM Tris-HCl, 10mM MgCl2, 5mM DTT, pH7.6.

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