APOBEC3A deaminase activity determination method and kit

Abstract

The invention provides an APOBEC3A deaminase activity determination method and a kit. The determination method comprises the following steps: preparing U-probe-containing standard substance solutions with different concentrations, adding UDG, and carrying out enzymatic reaction and fluorescence test to obtain standard curves of different concentrations and fluorescence intensities of U-probe-containing standard substances; the method comprises the following steps: preparing APOBEC3A deaminase to-be-tested solutions with different dilution times and concentrations, adding a C-containing probe template, carrying out enzymatic reaction and fluorescence test, calculating the generation amount of ssDNA containing single U, drawing a linear relation curve between the generation amount of ssDNA containing single U and the enzyme input amount, and calculating the enzyme activity or specific activity of APOBEC3A deaminase. According to the method, the absolute enzyme activity of the APOBEC3A deaminase is calculated by deducing the linear relation between the generation amount of ssDNA containing single U and the input amount of the enzyme under the reaction time of 10 min, and the method is accurate in determination and good in repeatability.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and particularly relates to a method and kit for determining the deaminase activity of APOBEC3A. Background Art

[0002] Cytidine deaminase (APOBEC3A, A3A) is considered to be one of the main sources of APOBEC signature mutations in tumors. Therefore, detecting the activity of APOBEC3A is very important for understanding its relationship with malignant tumors. Summary of the Invention

[0003] Based on this, it is necessary to provide a method and kit for determining the deaminase activity of APOBEC3A, which can measure and obtain the absolute enzyme activity of APOBEC3A, and has good accuracy and repeatability.

[0004] The present invention adopts the following technical solutions:

[0005] The present invention provides a method for determining the deaminase activity of APOBEC3A, comprising the following steps:

[0006] Prepare standard solutions of U-containing probes with different concentrations, add the same amount of UDG, carry out an enzymatic reaction, and perform fluorescence testing to obtain a standard curve y = kx + b of different concentrations of the U-containing probe standard and its fluorescence intensity;

[0007] Prepare test solutions of APOBEC3A deaminase with different dilution multiples of concentrations, add the same amount of C-containing probe templates, carry out an enzymatic reaction, perform fluorescence testing, calculate the production amount of ssDNA containing a single U, and calculate and plot a linear relationship curve between the production amount of ssDNA containing a single U and the enzyme input amount:

[0008] The production amount of ssDNA containing a single U (pmol) x = (y - b) / k;

[0009] Calculate the enzyme activity or specific enzyme activity of APOBEC3A deaminase:

[0010] APOBEC3A enzyme activity (U) = (y - b) / (molar amount of c-containing probe * k);

[0011] APOBEC3A specific enzyme activity (U / μL) = (y - b) * APOBEC3A dilution multiple / (molar amount of c-containing probe * k).

[0012] In some embodiments, the sequence of the U-containing probe is:

[0013] 5'FAM-GAATGAAGUGTGAAAT-3'DABCYL;

[0014] In some of these embodiments, the sequence of the C probe template is:

[0015] 5'FAM-GAATGAAGCGTGAAAT-3'DABCYL.

[0016] In some of these embodiments, the composition of the PCR reaction system for the U probe standard is:

[0017] UDG (5U) / UDG (5U) dilution, 1 μL;

[0018] APOBEC Reaction Buffer, 5 μL;

[0019] U probe-containing, 5 μL;

[0020] BSA (20 mg / mL), 0.5 μL;

[0021] APOBEC Dilution Buffer, 5 μL;

[0022] Nuclease-free H2O, made up to 33.5 μL.

[0023] In some of these embodiments, the composition of the enzymatic reaction system of APOBEC3A deaminase is:

[0024] UDG (5U), 1 μL;

[0025] APOBEC Reaction Buffer, 5 μL;

[0026] C probe-containing, 5 μL;

[0027] BSA (20 mg / mL), 0.5 μL;

[0028] APOBEC3A deaminase, 5 μL;

[0029] Nuclease-free H2O, made up to 33.5 μL.

[0030] In some of these embodiments, the enzymatic reaction procedure is: set the PCR instrument to run at 37 °C for 10 min, hot lid temperature 105 °C, volume 50 μL; add 50 μL of 0.4 M NaOH to each tube of the reaction solution, react at 90 °C for 10 min, hot lid temperature 105 °C, volume 100 μL, and immediately take out and place on ice after the temperature drops to 45 °C; then add 10 μL of 3 M HAc to each tube of the reaction solution, and then add 80 μL of 0.5 M Tris-HCI (pH 8.0) to adjust the pH to 7.0.

[0031] In some of these embodiments, the preparation concentration range of the U-probe standard solution is 0.3 to 20 pmol.

[0032] In some of these embodiments, the dilution concentration range of the APOBEC3A deaminase solution to be measured is 2 to 200 ng / μL.

[0033] In some of these embodiments, the input amount of the C-probe template is 25 to 100 pmol.

[0034] The present invention can also provide a kit for measuring the activity of APOBEC3A deaminase, including a U-probe and a C-probe template; the sequence of the U-probe is:

[0035] 5'FAM-GAATGAAGUGTGAAAT-3'DABCYL;

[0036] The sequence of the C-probe template is:

[0037] 5'FAM-GAATGAAGCGTGAAAT-3'DABCYL.

[0038] Compared with the prior art, the core advantage of the present invention lies in:

[0039] The method for measuring the activity of APOBEC3A (A3A) deaminase of the present invention relies on specific U-probes and C-probe templates, combines enzymatic reactions and fluorescence detection, and can relatively accurately measure the activity of APOBEC3A deaminase. The system reacts quickly, the results are stable, the repeatability is good, the requirements for intermediate data are low, and the requirements for operators and the accuracy of detection instruments are reduced. Through rigorous formula derivation and calculation, the activity of APOBEC3A deaminase is determined, the template dosage is increased, and the fluorescence values showing a linear relationship in the amplification reflect the actual enzyme activity, improving the accuracy of the detection results. Description of the Drawings

[0040] Figure 1 It is the standard curve of the U-probe; wherein, the abscissa is the concentration of the U-probe, and the ordinate is the fluorescence value.

[0041] Figure 2 It is the corresponding relationship diagram between the input amounts of different APOBEC3A and the fluorescence values; wherein, the abscissa is the input amounts of different APOBEC3A, and the ordinate is the fluorescence value.

[0042] Figure 3 It is the relationship diagram between the input amount of APOBEC3A and the generation amount of ssDNA products containing U; wherein the abscissa is the different input amounts of APOBEC3A, and the ordinate is the generation amount of ssDNA products containing U. Detailed Embodiments

[0043] The technical concept of the present invention is to provide a method for measuring the activity of APOBEC3A (A3A) deaminase, which improves the operation efficiency and ensures the accuracy of test results.

[0044] The following further describes the present invention in detail with specific embodiments, so that those skilled in the art can understand the present invention more clearly. The following embodiments are only used to illustrate the present invention, but not to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative labor belong to the protection scope of the present invention. In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are all conventional means well known to those skilled in the art.

[0045] The probe sequences used in the experiments of the present invention were synthesized by Anhui General Bioscience Co., Ltd.; the other reagents are all conventional commercial products.

[0046] U-containing probe (standard product), its sequence is:

[0047] 5'FAM-GAATGAAGUGTGAAAT-3'DABCYL;

[0048] C-containing probe (template), its sequence is:

[0049] 5'FAM-GAATGAAGCGTGAAAT-3'DABCYL.

[0050] Prepare the U-containing probe standard solution: Dilute the above U-containing probe standard with 1×TE solution (pH 8.0), and the diluted concentrations are 10 pmol / μL, 5 pmol / μL, 2.5 pmol / μL, 1.2525 pmol / μL, and 0.3125 pmol / μL respectively.

[0051] Prepare the C-containing probe template solution: Dilute the C-containing probe template with 1×TE solution (pH 8.0) to a concentration of 10 pmol / μL, and a total of 50 pmol of C-containing probe is added.

[0052] Deaminase (APOBEC3A, A3A): sourced from ABclonal, product number: RM20542.

[0053] Uracil-DNA Glycosylase (UDG): sourced from ABclonal, product number: RM21505.

[0054] 1X UDG Diluent: 10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.4 @ 25 °C.

[0055] 10X APOBEC Reaction Buffer: 500 mM Bis-Tris (pH 6.0), 1% Triton X-100.

[0056] 1X APOBEC Dilution Buffer: 50 mM Tris-HCl, 50 mM NaCl, 0.5 mM DTT, 0.01% Tween-20, 50% glycerol, pH 8.0 @ 25 °C.

[0057] During the experiment of the present invention, the reaction system containing the U-probe standard is configured as follows in the table:

[0058] Reagent Reaction Control UDG 1 μL (5 U) / UDG Dilution Solution / 1 μL APOBEC Reaction Buffer 5 μL 5 μL Probe (containing U) 5 μL 5 μL BSA (20 mg / mL) 0.5 μL 0.5 μL APOBEC Dilution Buffer 5 μL 5 μL <![CDATA[Nuclease-free H2O]]> 33.5 μL 33.5 μL

[0059] All of the above experimental procedures are carried out under the condition of 4 °C.

[0060] During the experiment of the present invention, the reaction system of the APOBEC3A deaminase to be measured is configured as follows:

[0061] 1) Dilute the sample of the APOBEC3A deaminase (APOBEC3A, A3A) to be measured with 1×TE solution.

[0062] 2) Prepare the C-probe template solution: Dilute the C-probe template with 1×TE solution to a concentration of 10 pmol / μL, and a total of 50 pmol is added.

[0063] 3) Use 1X APOBEC Dilution Buffer (50 mM Tris-HCl, 50 mM NaCl, 0.5 mM DTT, 0.01% Tween-20, 50% glycerol, pH 8.0 @ 25 °C) to perform a 1.5-fold dilution on the APOBEC3A cytidine deaminase to be measured to obtain 7 different gradient diluents.

[0064] The activity test reaction system of the APOBEC3A deaminase is shown in the following table:

[0065]

[0066]

[0067] The PCR reaction procedure is as follows:

[0068] 1) Set the operating temperature of the PCR instrument to 37°C for 10 min, with the hot lid temperature at 105°C and a volume of 50 μL;

[0069] 2) Add 50 μL of 0.4 M NaOH to the reaction solution in each tube. Adjust the operating temperature of the PCR instrument to 90°C for 10 min, with the hot lid temperature at 105°C and a volume of 100 μL. Immediately remove the tubes and place them on ice after the temperature drops to 45°C.

[0070] 3) Add 10 μL of 3 M HAc, then add 80 μL of 0.5 M Tris-HCl (pH 8.0) to adjust the pH to 7.0.

[0071] Example 1

[0072] This example provides a method for obtaining a standard curve containing a U probe, which includes the following steps:

[0073] Add UDG (5 U) to the U-probe standard solutions with different dilution concentrations (10 pmol / μL, 5 pmol / μL, 2.5 pmol / μL, 1.2525 pmol / μL, and 0.3125 pmol / μL) respectively, and react using the U-probe standard reaction system. Record the fluorescence value a;

[0074] Add 1 μL of UDG dilution to the U-probe standard solutions with different dilution concentrations (10 pmol / μL, 5 pmol / μL, 2.5 pmol / μL, 1.2525 pmol / μL, and 0.3125 pmol / μL) respectively, and react using the U-probe standard reaction system. Record the fluorescence value b, and obtain:

[0075] Static fluorescence value A = fluorescence value a - fluorescence value b;

[0076] Use the reaction without adding the U-probe as a blank control, react using the U-probe standard reaction system, and obtain the fluorescence value C, and obtain:

[0077] Net fluorescence value B = static fluorescence value A - fluorescence value C;

[0078] Obtain the standard curve by plotting the concentration of the U-probe standard against the net fluorescence value B.

[0079] The results of the standard curve of the concentration of the U-probe standard against the net fluorescence value B are as Figure 1 shown, and the results show that:

[0080] After the reaction of the concentration of the U-probe standard with UDG, the fluorescence value is proportional to the input amount (pmol) of the U-probe, and the R 2 of the standard curve = 0.9997, meeting the requirements of a linear standard curve.

[0081] Example 2

[0082] This example provides a method for measuring the deaminase activity of APOBEC3A, which includes the following steps:

[0083] S1. Add the same C-containing probe template solution (50 pmol) to the blank control and the reaction solution as the substrate, and at the same time add the APOBEC3A deaminase samples with the same volume but different concentrations (diluted to the corresponding concentrations using APOBEC Dilution Buffer).

[0084] S2. Under the conditions of 10 min and the same input amount of the C-containing probe template (50 pmol), carry out the reaction using the test reaction system of APOBEC3A deaminase, record the net fluorescence mean values obtained by different input amounts of APOBEC3A deaminase at the same time point, and calculate and plot the curve of the input amount of APOBEC3A deaminase versus the net fluorescence value.

[0085] Among them, the net fluorescence value corresponding to the input amount of APOBEC3A deaminase at each gradient = the average fluorescence intensity value of 2 replicate wells - the average fluorescence intensity value of 2 blank control replicate wells.

[0086] The statistical results are as Figure 2 , and the results show that: APOBEC3A proteins with different input amounts could not be completely converted. The fluorescence values of all concentrations of APOBEC3A were under the maximum fluorescence value, and the fluorescence values were proportional to the input amounts of different APOBEC3A, showing a good linear relationship. With an excessive 50 pmol of C-containing probe, they were all within the measurable range.

[0087] S3. According to the standard curve of Example 1, calculate the production amount of ssDNA containing a single U:

[0088] The production amount of ssDNA containing a single U = the consumption amount of ssDNA containing a single U = x = (y - b) / k

[0089] S4. Plot the linear relationship curve between the production amount of ssDNA containing a single U and the input amount of APOBEC3A deaminase at the same time point.

[0090] The statistical results are as Figure 3 shown; y = 0.3482x - 0.7463, where R 2 = 0.9945, indicating that:

[0091] The production amount of U-containing ssDNA is proportional to the input amount of APOBEC3A deaminase, and there is a good linear relationship between the two, so it can be used as a calculation scheme for enzyme activity.

[0092] S5. Calculate the APOBEC3A deaminase activity:

[0093] The activity C (U) of APOBEC3A enzyme = the production amount (pmol) of ssDNA containing a single U / 50 = (y - b) / 50k = (y - b) * dilution factor / 50k

[0094] In the formula, the formula for the specific activity C (U / μL) of the enzyme:

[0095] C = the input activity S of the enzyme / the input mass m of the enzyme.

[0096] Taking the average value of the specific activity values of the enzyme to be measured calculated with the enzyme input amounts at each gradient within each round of repeated experiments as the standard value of the specific activity of the enzyme to be measured, and a coefficient of variation ≤ 10% within each round of repeated experiments is considered qualified. Coefficient of variation = STDEV value / standard value.

[0097] Example 3

[0098] This example provides a method for measuring the APOBEC3A deaminase activity with only 2 input amounts, and a total of 7 repeated tests are carried out. The specific steps are as follows:

[0099] S1. According to Example 1, obtain the standard curve of the U-containing probe. After the reaction of the U-containing probe standard product with UDG, the fluorescence value is proportional to the input amount (pmol) of the U-containing probe, y = 1113.8x + 44.712, where R 2 = 0.9998, meeting the requirements of the linear standard curve.

[0100] S2. Add the same C-containing probe template solution (50 pmol) to the blank control and the reaction solution respectively as the substrate, and at the same time add APOBEC3A deaminase samples with the same volume but different input amounts (diluted to the corresponding concentrations with APOBEC Dilution Buffer).

[0101] S2. Under the conditions of 10 min and the same input amount (50 pmol) of the C-containing probe template, carry out the reaction using the test reaction system of APOBEC3A deaminase, and record the net fluorescence mean values obtained by APOBEC3A at 2 input amounts at the same time point.

[0102] Among them, the net fluorescence value corresponding to the input amount of APOBEC3A deaminase = the average fluorescence intensity value of 2 replicate wells - the average fluorescence intensity value of 2 blank control replicate wells.

[0103] S3. According to the standard curve in S1, calculate the production amount of ssDNA containing a single U:

[0104] The production amount of ssDNA containing a single U = the consumption amount of ssDNA containing a single U = x = (y - 44.712) / 1113.8

[0105] S4. Calculate the APOBEC3A deaminase activity:

[0106] The enzyme activity C (U) of APOBEC3A = the production amount of ssDNA containing a single U (pmol) / 50;

[0107] The formula for the specific enzyme activity C (U / μL):

[0108] C = the input enzyme activity S (U) / the input enzyme volume (uL).

[0109] Take the average value of the measured specific enzyme activity values calculated based on the enzyme input amount in each round of repeated experiments as the standard value of the measured specific enzyme activity. A coefficient of variation ≤ 10% in each round of repeated experiments is considered qualified.

[0110] Coefficient of variation = STDEV value / standard value.

[0111] The statistical results are as follows:

[0112]

[0113] The above results show that: the results of 7 rounds of tests are close, the coefficient of variation is only 0.02, the error is small, and the method is stable.

[0114] Example 4

[0115] This example can provide a kit for measuring APOBEC3A deaminase activity, including a U-containing probe and a C-containing probe template,

[0116] The sequence of the U-containing probe is:

[0117] 5'FAM-GAATGAAGUGTGAAAT-3'DABCYL;

[0118] The sequence of the C-containing probe template is:

[0119] 5'FAM-GAATGAAGCGTGAAAT-3'DABCYL.

[0120] Furthermore, the kit for measuring APOBEC3A deaminase activity may further include 1×TE solution, UDG, UDG dilution solution, APOBEC Reaction Buffer, APOBEC Dilution Buffer, BSA, etc.

[0121] It is necessary to point out here that the above embodiments are only limited to further elaborating and explaining the technical solutions of the present invention, rather than further restricting the technical solutions of the present invention. The method of the present invention is only a preferred implementation scheme and is not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for measuring the deaminase activity of APOBEC3A, characterized in that, It includes the following steps: Prepare standard solutions of U-containing probes with different concentrations, add the same amount of UDG, conduct an enzymatic reaction, perform fluorescence testing, and obtain a standard curve of different concentrations of the U-containing probe standard and its fluorescence intensity: y = kx + b; Prepare test solutions of APOBEC3A deaminase with different dilution multiples, add the same amount of C-containing probe templates, conduct an enzymatic reaction, perform fluorescence testing, calculate the generation amount of ssDNA containing a single U, and calculate and plot a linear relationship curve between the generation amount of ssDNA containing a single U and the enzyme input amount: The generation amount of ssDNA containing a single U (pmol) x = (y - b) / k; Calculate the enzyme activity or specific enzyme activity of APOBEC3A deaminase: APOBEC3A enzyme activity (U) = (y - b) / (molar amount of C-containing probe * k); APOBEC3A specific enzyme activity (U / μL) = (y - b) * APOBEC3A dilution multiple / (molar amount of C-containing probe * k).

2. The method for measuring the deaminase activity of APOBEC3A according to claim 1, characterized in that, The sequence of the U-containing probe is: 5'FAM-GAATGAAGUGTGAAAT-3'DABCYL.

3. The method for measuring the deaminase activity of APOBEC3A according to claim 1 or 2, characterized in that, The sequence of the C-containing probe template is: 5'FAM-GAATGAAGCGTGAAAT-3'DABCYL.

4. The method for measuring the deaminase activity of APOBEC3A according to claim 3, characterized in that, The composition of the reaction system for the enzymatic reaction of the U-containing probe standard is: UDG (5U) / UDG (5U) dilution solution, 1 μL; APOBEC Reaction Buffer, 5 μL; U-containing probe, 5 μL; BSA (20 mg / mL), 0.5 μL; APOBEC Dilution Buffer, 5 μL; Nuclease-free H2O, make up to 33.5 μL.

5. The method for measuring the deaminase activity of APOBEC3A according to claim 3, characterized in that, The composition of the reaction system for the enzymatic reaction of APOBEC3A deaminase is: UDG (5U), 1 μL; APOBEC Reaction Buffer, 5 μL; C-containing probe, 5 μL; BSA (20 mg / mL), 0.5 μL; APOBEC3A deaminase, 5 μL; Nuclease-free H2O, make up to 33.5 μL.

6. The method for measuring the deaminase activity of APOBEC3A according to claim 3, characterized in that, The reaction procedure is: Set the PCR instrument to run at 37 °C for 10 min, with a hot lid temperature of 105 °C and a volume of 50 μL; Add 50 μL of 0.4 M NaOH to each tube of the reaction solution, react at 90 °C for 10 min, with a hot lid temperature of 105 °C and a volume of 100 μL. Immediately take it out and place it on ice after the temperature drops to 45 °C; Then add 10 μL of 3 M HAc to each tube of the reaction solution, and then add 80 μL of 0.5 M Tris-HCI (pH 8.0) to adjust the pH to 7.

0.

7. The method for determining the APOBEC3A deaminase activity according to claim 3, wherein, The concentration range for preparing the U-containing probe standard solution is 0.3 - 20 pmol.

8. The method for determining the APOBEC3A deaminase activity according to claim 3, wherein, The dilution concentration range of the APOBEC3A deaminase test solution is 1 - 200 ng / μL.

9. The method for determining the APOBEC3A deaminase activity according to claim 3, wherein, The input amount of the C-containing probe template is 25 - 100 pmol.

10. An assay kit for determining the APOBEC3A deaminase activity, wherein, It includes a U-containing probe and a C-containing probe template; The sequence of the U-containing probe is: 5'FAM-GAATGAAGUGTGAAAT-3'DABCYL; The sequence of the C probe template is as follows: 5'FAM-GAATGAAGCGTGAAAT-3'DABCYL.

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