Single-stranded DNA for detecting single-stranded DNA cytidine deaminase AID activity and complementary strand, kit and application thereof

Abstract

The invention relates to the technical field of biological fluorescence detection, in particular to a single-stranded DNA for detecting DNA cytidine deaminase AID activity, a complementary strand of the single-stranded DNA, a kit and application of the single-stranded DNA. The single-stranded DNA has a DNA cytidine deaminase AID recognition site, and the nucleotide sequences of the single-stranded DNA and the complementary strand of the single-stranded DNA are as shown in SEQ ID NO: 6 and SEQ ID NO: 3. The invention also provides a fluorescent kit and a method for detecting the activity of DNA cytidine deaminase AID or screening a DNA cytidine deaminase AID inhibitor, the activity of AID with different concentrations can be sensitively and stably detected in vitro by utilizing the kit and the method, the kit and the method have the advantages of convenience, rapidness, lower price, small raw material dosage and the like, the AID inhibitor can be screened at high throughput, and the application prospect is wide. The detection of a false positive inhibitor is avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological fluorescence detection, in particular to a single-stranded DNA and its complementary strand for detecting DNA cytidine deaminase AID activity, a kit and application. BACKGROUND

[0002] Cytidine deaminase (AID) is a cytidine deaminase that specifically acts on single-stranded DNA (ssDNA) and can convert cytidine (C) to uridine (U), a process known as C-to-U editing. AID is mainly expressed in activated B lymphocytes and plays a key role in the formation of antibody diversity, including somatic hypermutation (SHM) and class switch recombination (CSR). AID induces DNA mutations and double-strand breaks by targeting the variable region and switch region of immunoglobulin (Ig) genes, thereby promoting antibody affinity maturation and class switching.

[0003] However, abnormal expression or off-target activity of AID can lead to mutations in non-Ig genes, causing genomic instability and chromosomal translocation, and thus promoting the occurrence of various malignant tumors. For example, in Burkitt's lymphoma, AID-mediated translocation of the c-Myc gene and the IgH gene (t(8;14)) is a key driver of tumorigenesis. In addition, ectopic expression of AID is also closely related to B-cell malignancies such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL). In solid tumors, abnormal activation of AID can promote the occurrence of inflammation-related cancers such as gastric cancer, liver cancer, colorectal cancer, lung cancer, and cholangiocarcinoma, and the mechanism involves the accumulation of mutations in key genes such as TP53, KRAS, and BCL6.

[0004] In addition to tumors, dysregulation of AID is also associated with various immune diseases. In systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjogren's syndrome, excessive activation of AID can lead to abnormal production of autoantibodies. In high IgM syndrome (HIGM) and common variable immunodeficiency disease (CVID), the loss or abnormality of AID function can cause antibody class switching disorders, increasing the risk of infection.

[0005] Currently, the methods for detecting AID activity mainly include PCR, gene sequencing, Western Blot, immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) and the like. Although these methods can detect the expression or activity of AID, they generally have the shortcomings of complicated operation, long time consumption, high cost, and the need for special equipment. In addition, the existing AID activity detection techniques are mostly dependent on the complex environment in cells, and it is difficult to achieve efficient and specific detection in vitro. For example, although the cell line detection method based on fluorescent reporter gene can indirectly reflect the activity of AID, it is complicated to operate, has poor repeatability, and depends on expensive instruments and equipment.

[0006] Therefore, it is of great significance to develop a technical method capable of rapidly, sensitively and specifically detecting the activity of AID in vitro. Such a technology can not only be used for screening of AID inhibitors and providing potential drug targets for the treatment of tumors and autoimmune diseases, but also can be used for the detection of AID activity in clinical samples to assist in the diagnosis and treatment monitoring of diseases. By simulating the natural substrate of AID (such as G-quadruplex structure formed by single-stranded DNA rich in G repeats), a specific detection system is designed, which is expected to achieve efficient detection of AID activity and provide a powerful tool for related research and clinical application. SUMMARY

[0007] In order to overcome the deficiencies and shortcomings of the prior art, the first aspect of the present application aims to provide a single-stranded DNA and its complementary strand for detecting the activity of DNA cytosine deaminase AID or for screening DNA cytosine deaminase AID inhibitors and the like.

[0008] The second aspect of the present application aims to provide a fluorescence kit for detecting the activity of DNA cytosine deaminase AID or screening DNA cytosine deaminase AID inhibitors.

[0009] The third aspect of the present application aims to provide the application of the above-mentioned single-stranded DNA and its complementary strand and kit.

[0010] The fourth aspect of the present application aims to provide a method for detecting the activity of DNA cytosine deaminase AID.

[0011] The fifth aspect of the present application aims to provide a method for screening DNA cytosine deaminase AID inhibitors.

[0012] In order to achieve the above-mentioned purposes of the present application, the technical solutions adopted by the present application are as follows:

[0013] The single-stranded DNA and its complementary strand provided by the first aspect of the present application have a DNA cytosine deaminase AID recognition site, and the nucleic acid sequence of the single-stranded DNA is 5'-AAAAAAAAAAAAATATGTAGCTAGGATGAG-3' (SEQ ID NO: 6).

[0014] In some embodiments of the present application, the complementary strand of the single-stranded DNA is 5'-AAAAAAAACTCATCCTAACTACA-3' (SEQ ID NO: 3).

[0015] In some embodiments of the present application, the 5' end of the single-stranded DNA is preferably connected to a labeling group, which is used to detect whether the single-stranded DNA and its complementary strand form stable dsDNA under the action of a labeling reagent; and the 5' end of the complementary strand of the single-stranded DNA is preferably connected to a chemical modification group, which is used to immobilize the complementary strand of the single-stranded DNA.

[0016] In some embodiments of the present application, the labeling group is preferably biotin or digoxin; and the chemical modification group is preferably a carboxyl group.

[0017] The second aspect of the present application provides a fluorescent kit for detecting DNA cytosine deaminase AID activity or screening DNA cytosine deaminase AID inhibitors, which comprises the single-stranded DNA and its complementary strand according to the first aspect of the present application.

[0018] In some embodiments of the present application, the kit preferably further comprises at least one of the following components: a labeling reagent specifically combined with the labeling group at the 5' end of the single-stranded DNA, a color developing solution, a color developing termination solution, a buffer solution, and an eluent.

[0019] In some embodiments of the present application, the labeling reagent specifically combined with the labeling group at the 5' end of the single-stranded DNA is preferably SA-HRP (streptavidin-HRP) or a digoxin antibody.

[0020] In some embodiments of the present application, the color developing solution is used to develop color when the labeling group and the labeling reagent form a complex, thereby detecting the nucleic acid connected by the labeling group.

[0021] In some embodiments of the present application, the color developing solution is preferably a TMB color developing solution.

[0022] In some embodiments of the present application, the buffer solution is preferably a deamination buffer solution.

[0023] In some embodiments of the present application, the eluent is preferably a PBST solution; and the concentration is preferably 0.2-0.5x PBST solution.

[0024] In some embodiments of the present application, the kit further comprises DNA cytosine deaminase AID.

[0025] In a third aspect of the present application, there is provided use of the single-stranded DNA of the first aspect of the present application and its complementary strand, and the kit of the second aspect of the present application in the preparation of a product for detecting the activity of DNA cytosine deaminase AID or screening for inhibitors of DNA cytosine deaminase AID.

[0026] In a fourth aspect of the present application, there is provided a method for detecting the activity of DNA cytosine deaminase AID, comprising the following steps:

[0027] (1) uniformly mixing the complementary strand of the single-stranded DNA of the first aspect of the present application with EDCI in a PBS solution, and then incubating;

[0028] (2) adding the incubated mixture of step (1) to a BSA-coated enzyme-labeled plate for incubation, then washing the plate with PBST solution after the incubation, adding blocking solution for blocking, and then washing the plate with PBST solution after the blocking, to obtain an enzyme-labeled plate coupled with the complementary strand of the single-stranded DNA;

[0029] (3) adding a PBS solution containing the single-stranded DNA to the enzyme-labeled plate coupled with the complementary strand of the single-stranded DNA in step (2) for incubation, and then washing the plate with PBST solution after the incubation;

[0030] (4) mixing the DNA cytosine deaminase AID to be detected with deamination buffer for incubation, to obtain an incubated mixture;

[0031] (5) adding the incubated mixture of step (4) to the enzyme-labeled plate in step (3) for incubation, and then washing the plate with PBST solution after the incubation;

[0032] (6) adding SA-HRP (horseradish peroxidase-labeled streptavidin) or digoxin antibody to the enzyme-labeled plate, and then washing the plate with PBST solution after the incubation;

[0033] (7) adding a color developing solution to the enzyme-labeled plate, incubating in the dark, then adding a color developing termination solution, and then measuring the absorbance at a wavelength of 450 nm.

[0034] In some embodiments of the present application, the concentration of the complementary strand of the single-stranded DNA in the PBS solution in step (1) is preferably 2-50 nM.

[0035] In some embodiments of the present application, the amount of EDCI used in step (1) is preferably 2-50 times the amount of the complementary strand of the single-stranded DNA.

[0036] In some embodiments of the present application, the temperature of the incubation in step (1) is preferably 25°C, and the incubation time is preferably 10-60 min.

[0037] In some embodiments of the present application, the blocking solution in step (2) is preferably a 1% defatted milk solution or a 2% PEG-4000 solution.

[0038] In some embodiments of the present application, the blocking solution in step (2) is preferably prepared using a PBS solution.

[0039] In some embodiments of the present application, the BSA-coated enzyme-labeled plate in step (2) is prepared by the following method: a 1% BSA solution is prepared using a 50 mM carbonate buffer at pH 9.4, the 1% BSA solution is added to the enzyme-labeled plate, and the plate is coated overnight at 4°C, then washed 2-4 times with a PBST solution; the amount of 1% BSA solution added is preferably 200 μL.

[0040] In some embodiments of the present application, the amount of the mixed solution after incubation in step (2) is 80-120 μL, further 100 μL, and the amount of the blocking solution is preferably 150-250 μL, further 200 μL.

[0041] In some embodiments of the present application, the temperature of the incubation in step (2) is preferably 32-40°C, further 37°C, and the incubation time is preferably 1-3 h.

[0042] In some embodiments of the present application, the temperature of the blocking in step (2) is preferably 32-40°C, further 37°C, and the blocking time is preferably 1-3 h.

[0043] In some embodiments of the present application, the concentration of the single-stranded DNA in the PBS solution containing single-stranded DNA in step (3) is preferably 2-50 nM.

[0044] In some embodiments of the present application, the amount of the PBS solution containing single-stranded DNA in step (3) is preferably 80-120 μL, further 100 μL.

[0045] In some embodiments of the present application, the temperature of the incubation in step (3) is preferably 32-40°C, further 37°C, and the incubation time is preferably 1-3 h.

[0046] In some embodiments of the present application, the deamination buffer in step (4) comprises 0.1 mM MES and 0.1 mM TEA, pH = 8.5.

[0047] In some embodiments of the present application, the temperature of the incubation in step (4) is preferably 32-40℃, further preferably 37℃, and the incubation time is preferably 10-60 min.

[0048] In some embodiments of the present application, the amount of the mixed solution after incubation in step (5) is preferably 250-350 μL, further preferably 300 μL.

[0049] In some embodiments of the present application, the temperature of the incubation in step (5) is preferably 32-40℃, further preferably 37℃, and the incubation time is preferably 1-3 h.

[0050] In some embodiments of the present application, the SA-HRP used in step (6) is used according to the manufacturer's instructions.

[0051] In some embodiments of the present application, the temperature of the incubation in step (6) is 25℃, and the incubation time is 10-60 min.

[0052] In some embodiments of the present application, the temperature of the incubation in step (7) is preferably 25℃, and the incubation time is preferably 10-60 min.

[0053] In some embodiments of the present application, the color developing stop solution in step (7) is preferably 2M H2SO4.

[0054] In some embodiments of the present application, the absorbance in step (7) is preferably determined within 15 min after the color developing stop solution is added.

[0055] In some embodiments of the present application, the PBST solution in steps (2), (3), (5) and (6) is preferably a 0.5×PBST solution, and the pH value is pH 8.5.

[0056] In some embodiments of the present application, the number of times of washing the plate with the PBST solution in steps (2), (3), (5) and (6) is preferably 1-6 times.

[0057] In a fifth aspect of the present application, a method for screening a DNA cytidine deaminase AID inhibitor is provided, comprising the following steps:

[0058] S1) After the single-stranded DNA complementary strand is mixed with EDCI in a PBS solution, the mixture is incubated;

[0059] S2) adding the mixed solution after the incubation in step S1) to a BSA-coated enzyme-labeled plate for incubation, after the incubation, washing the plate with PBST solution, then adding a blocking solution for blocking, after the blocking, washing the plate with PBST solution, to obtain an enzyme-labeled plate coupled with a complementary strand of the single-stranded DNA;

[0060] S3) adding the single-stranded DNA to the enzyme-labeled plate in step S2) for incubation, after the incubation, washing the plate with PBST solution;

[0061] S4) mixing the compound to be tested and DNA cytosine deaminase AID in a deamination buffer for incubation;

[0062] S5) adding the mixed solution after the incubation in step S4) to the enzyme-labeled plate in step S3) for incubation, after the incubation, washing the plate with PBST solution;

[0063] S6) adding SA-HRP or digoxin antibody to the enzyme-labeled plate for incubation, after the incubation, washing the plate with PBST solution;

[0064] S7) adding a color developing solution to the enzyme-labeled plate, after incubation in the dark, adding a color developing termination solution, and measuring the absorbance at a wavelength of 450 nm.

[0065] In some embodiments of the present application, the temperature of the incubation in step S1) is preferably 32-40℃, further 37℃, and the incubation time is preferably 10-60 min.

[0066] In some embodiments of the present application, the temperature of the incubation in step S2) is preferably 32-40℃, further 37℃, and the incubation time is preferably 1-3 h.

[0067] In some embodiments of the present application, the temperature of the blocking in step S2) is preferably 32-40℃, further 37℃, and the blocking time is preferably 1-3 h.

[0068] In some embodiments of the present application, the temperature of the incubation in step S3) is preferably 32-40℃, further 37℃, and the incubation time is preferably 1-3 h.

[0069] In some embodiments of the present application, the temperature of the pre-incubation in step S4) is preferably 32-40℃, further 37℃, and the pre-incubation time is 10-60 min.

[0070] In some embodiments of the present application, the temperature of the incubation in step S5) is preferably 32-40℃, further 37℃, and the incubation time is preferably 1-3 h.

[0071] In some embodiments of the present application, the temperature of the incubation in step S6) is preferably 32-40℃, further 37℃, and the time of the incubation is preferably 10-60 min.

[0072] In some embodiments of the present application, the temperature of the incubation in step S7) is preferably 32-40℃, further 37℃, and the time of the incubation is preferably 10-60 min.

[0073] In some embodiments of the present application, the PBST solution in steps S2), S3), S4) and S5) is preferably a 0.5x PBST solution.

[0074] The technical principle of the present application is:

[0075] The present application designs a single-stranded DNA containing a DNA cytosine deaminase AID recognition site, and the 5' end of the single-stranded DNA is connected with biotin, and the 5' end of the complementary strand is modified with a carboxyl group; an AID recognition site - adenine A is designed in the single-stranded DNA sequence, and a mismatched base cytosine C is designed at the corresponding position on the DNA complementary strand. The complementary strand of the single-stranded DNA is added to an enzyme-labeled plate for incubation, and then the enzyme-labeled plate is washed with an eluent to form an enzyme-labeled plate coupled with the complementary strand of the single-stranded DNA. On this basis, the single-stranded DNA is added to the above enzyme-labeled plate to form double-stranded DNA with the complementary strand through base complementary pairing, and the double-stranded DNA forms a mismatch at the AID recognition site. AID recognizes adenine A in the single-stranded DNA sequence and mutates it into inosine I, and since adenine A is mutated into inosine I, it can be complementary to cytosine C at the corresponding position in the complementary strand, thereby repairing the base mismatch between the single-stranded DNA and its complementary strand. By utilizing the difference in binding capacity caused by the different number of mismatches between the single-stranded DNA and the complementary strand, the single-stranded DNA connected with biotin with few or no mismatches cannot be eluted, and at this time, SA-HRP is added for incubation and TMB color developing solution is used for color development, so that the absorbance can be detected.

[0076] The present application has the following advantages and effects compared with the prior art:

[0077] (1) The present application provides a single-stranded DNA and its complementary strand for detecting the activity of DNA cytosine deaminase AID, which can sensitively and stably detect the activity of AID of different concentrations in vitro, and more importantly, can be applied to screening inhibitors, and can effectively avoid the quenching interference of compounds on traditional bioluminescence and prevent the detection of false positive inhibitors by using TMB for color development.

[0078] (2) The application provides a fluorescent kit for detecting DNA cytosine deaminase AID activity or screening DNA cytosine deaminase AID inhibitors, the kit is simple in components, low in cost, and can be used for detecting DNA cytosine deaminase AID activity or screening DNA cytosine deaminase AID inhibitors.

[0079] (3) The application provides a method for detecting DNA cytosine deaminase AID activity or screening DNA cytosine deaminase AID inhibitors, the method can be used for detecting DNA cytosine deaminase AID activity or screening DNA cytosine deaminase AID inhibitors in vitro, is simple and convenient to operate, low in price, small in raw material consumption, and can be applied to high-throughput screening of AID inhibitors. BRIEF DESCRIPTION OF DRAWINGS

[0080] The application is further described below in combination with the drawings and examples, and wherein:

[0081] Figure 1 is a schematic diagram of single-stranded DNA and its complementary strand (SEQ ID NO: 1) for detecting DNA cytosine deaminase AID activity.

[0082] Figure 2 is a schematic diagram of single-stranded DNA and its complementary strand (SEQ ID NO: 2) for detecting DNA cytosine deaminase AID activity.

[0083] Figure 3 is a schematic diagram of single-stranded DNA and its complementary strand (SEQ ID NO: 3) for detecting DNA cytosine deaminase AID activity.

[0084] Figure 4 is a schematic diagram of single-stranded DNA and its complementary strand (SEQ ID NO: 4) for detecting DNA cytosine deaminase AID activity.

[0085] Figure 5 is a schematic diagram of single-stranded DNA and its complementary strand (SEQ ID NO: 5) for detecting DNA cytosine deaminase AID activity.

[0086] Figure 6 is an OD result analysis diagram of single-stranded DNA and its complementary strand (SEQ ID NO: 1) for detecting DNA cytosine deaminase AID activity. 450 Result analysis diagram.

[0087] Figure 7 is an OD result analysis diagram of single-stranded DNA and its complementary strand (SEQ ID NO: 2) for detecting DNA cytosine deaminase AID activity. 450 Result analysis diagram.

[0088] Figure 8OD450 results of detecting DNA cytosine deaminase AID activity by single-stranded DNA and its complementary strand (SEQ ID NO: 3) 450 Result analysis graph.

[0089] Figure 9 OD450 results of detecting DNA cytosine deaminase AID activity by single-stranded DNA and its complementary strand (SEQ ID NO: 4) 450 Result analysis graph.

[0090] Figure 10 OD450 results of detecting DNA cytosine deaminase AID activity by single-stranded DNA and its complementary strand (SEQ ID NO: 5)

[0091] Figure 11 Result analysis graph of the best PBST plate washing solution concentration screening in Example 3.

[0092] Figure 12 OD450 results of detecting DNA cytosine deaminase AID activity by 0.2x PBST plate washing in Example 3 450 Linear fitting graph of the results.

[0093] Figure 13 OD450 results of detecting DNA cytosine deaminase AID activity by 0.5x PBST plate washing in Example 3 450 Linear fitting graph of the results.

[0094] Figure 14 Chemical structure of AID inhibitor and result analysis graph of screening, wherein A: chemical structure, B: screening result, C: DNA cytosine deaminase concentration and OD450 value linear fitting graph, D: fitting calculation of AID inhibitor IC50. 450 DETAILED DESCRIPTION

[0095] The concept and technical effects of the present application will be described below in combination with examples to fully understand the purpose, features and effects of the present application. Obviously, the described examples are only a part of the examples of the present application, but not all the examples. Based on the examples of the present application, other examples obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0096] Example 1 Determination of ctDNA marker

[0097] ​The present application designs and screens 1 single-stranded DNA and 5 single-stranded DNA complementary chains with different sequences, and the nucleic acid sequences are shown in Table 1. The 5' end of the single-stranded DNA sequence is labeled with biotin, and the 5' end of the single-stranded DNA complementary chain is modified with a carboxyl group. The synthesis of the primer, the labeling of biotin and the modification of the carboxyl group are completed by Shengong Bioengineering (Shanghai) Co., Ltd.

[0098] Table 1 Nucleic acid sequences of single-stranded DNA and its complementary chain

[0099]

[0100] Example 2 Screening of the optimal single-stranded DNA sequence

[0101] (1) Add EDCI to the PBS solution (pH=7.4) containing a final concentration of 5nM single-stranded DNA complementary chain (any one of SEQ ID NO: 1-5, Table 1), mix thoroughly, and then incubate at room temperature 25℃ for 20min to obtain the incubated mixed solution; wherein the amount of EDCI is 50 times the equivalent of the single-stranded DNA complementary chain, which activates the carboxyl group at the 5' end of the single-stranded DNA complementary chain to react with the amino group in the BSA protein, thereby stably fixing the single-stranded DNA complementary chain on the enzyme-labeled plate 96-well plate.

[0102] (2) Prepare a 1% BSA solution with 50mM carbonate buffer as the solvent; add 200μL of 1% BSA solution to each well of the enzyme-labeled plate 96-well plate, and then stand at 4℃ for overnight coating, then wash the plate 3 times with 0.5×PBST solution to obtain the BSA-coated enzyme-labeled plate for standby; take the incubated mixed solution in step (1) and add it to each well of the BSA-coated enzyme-labeled plate, 100μL per well, and incubate at 37℃ for 2h, then wash the plate 1 time with 0.5×PBST solution (300μL) after incubation; add 200μL of 1% skimmed milk powder (prepared with PBS solution) to each well, and then block at 37℃ for 1h, then wash the plate 3 times with 0.5×PBST solution, 300μL each time.

[0103] (3) Prepare a PBS solution containing a final concentration of 2.5nM single-stranded DNA (Table 1, SEQ ID NO: 6), and add 100μL to the blocked enzyme-labeled plate in step (2), and then incubate at 37℃ for 2h; then wash the plate 3 times with 0.5×PBST solution, 300μL each time after incubation.

[0104] (4) Add deamination buffer and different final concentrations of AID into the EP tube, and mix well. Incubate at 37°C for 1 h. In this embodiment, 6 AID concentration groups are set, i.e., 0 nM, 200 nM, 400 nM, 600 nM, 800 nM and 1000 nM, and each concentration group has 3 repeated experiments.

[0105] (5) Add the mixed solution after incubation in step (4) into the enzyme-labeled plate coupled with dsDNA in step (3), and incubate at 37°C for 1 h. After incubation, wash the plate 6 times with 0.5×PBST solution, 300 μL each time.

[0106] (6) Add 100 μL of SA-HRP 12000-fold dilution solution to each well of the enzyme-labeled plate, and incubate at 25°C for 20 min. After incubation, wash the plate 6 times with 0.5×PBST solution, 300 μL each time.

[0107] (7) Add 100 μL of TMB color developing solution to each well of the enzyme-labeled plate, and incubate at 25°C in the dark for 60 min. Finally, add 100 μL of color developing termination solution (2M H2SO4) to each well, and measure the absorbance at 450 nm within 15 min.

[0108] Figure 1 is a schematic diagram for detecting DNA cytosine deaminase AID activity by using the single-stranded DNA of SEQ ID NO: 6 and the complementary strand of the single-stranded DNA of SEQ ID NO: 1. As shown in the figure, if AID enzyme activity works, the adenine (A) of the single-stranded DNA of SEQ ID NO: 6 can be mutated to hypoxanthine (I), and the mismatch between the 21st base of the single-stranded DNA of SEQ ID NO: 6 and the complementary strand of the single-stranded DNA of SEQ ID NO: 1 is repaired. According to the design expectation, the strand will not be eluted, and the biotin at the 5' end of the single-stranded DNA can be detected by color development with TMB color developing solution, and the absorbance at 450 nm can be detected. If AID is inactivated, the mismatch between the 21st base of the strand and the complementary strand of the single-stranded DNA of SEQ ID NO: 1 cannot be repaired, and according to the design expectation, the strand will be eluted, and the absorbance at 450 nm cannot be detected by color development with TMB color developing solution. The experimental results are shown in Figure 6 As shown in the figure, the absorbance between different concentration groups does not show a concentration gradient, indicating that the activity of different concentrations of AID cannot affect the mutation of the single-stranded DNA in a stable and regular manner, and therefore the complementary strand of the single-stranded DNA of SEQ ID NO: 1 is not suitable as a complementary strand of the single-stranded DNA for detecting AID activity.

[0109] Figure 2is a schematic diagram of detecting DNA cytosine deaminase AID activity by single-stranded DNA of SEQ ID NO: 6 and single-stranded DNA complementary strand of SEQ ID NO: 2. As shown in the figure, if AID enzyme activity works, the adenine of single-stranded DNA of SEQ ID NO: 6 is mutated into hypoxanthine, the mismatch between the 21st base of single-stranded DNA of SEQ ID NO: 6 and the single-stranded DNA complementary strand of SEQ ID NO: 2 is repaired, and 1 base in the 20-28th base will mismatch with the single-stranded DNA complementary strand of SEQ ID NO: 2, according to the design expectation, the chain will not be eluted, and at this time the biotin at the 5' end of the single-stranded DNA chain can be detected by coloring with TMB color developing solution, and the absorbance at 450 nm can be detected; if AID is inactivated, 2 bases in the 20-28th base of the chain will mismatch with the single-stranded DNA complementary strand, according to the design expectation, the chain will be eluted, and at this time the absorbance at 450 nm cannot be detected by coloring with TMB color developing solution. The experimental results are shown in Figure 7 As shown in the figure, the absorbance between different concentration groups does not show a concentration gradient, indicating that the activity of AID of different concentrations cannot affect the mutation of the single-stranded DNA in a stable and regular result, and therefore the single-stranded DNA complementary strand of SEQ ID NO: 2 is not suitable as a single-stranded DNA complementary strand for detecting AID activity.

[0110] Figure 3 is a schematic diagram of detecting DNA cytosine deaminase AID activity by single-stranded DNA of SEQ ID NO: 6 and single-stranded DNA complementary strand of SEQ ID NO: 3. As shown in the figure, if AID enzyme activity works, the adenine of single-stranded DNA of SEQ ID NO: 6 is mutated into hypoxanthine, the mismatch between the 21st base of single-stranded DNA of SEQ ID NO: 6 and the single-stranded DNA complementary strand of SEQ ID NO: 3 is repaired, and 2 bases in the 20-28th base will mismatch with the single-stranded DNA complementary strand of SEQ ID NO: 3, according to the design expectation, the chain will not be eluted, and at this time the biotin at the 5' end of the single-stranded DNA chain can be detected by coloring with TMB color developing solution, and the absorbance at 450 nm can be detected; if AID is inactivated, 3 bases in the 20-28th base of the chain will mismatch with the single-stranded DNA complementary strand, according to the design expectation, the chain will be eluted, and at this time the absorbance at 450 nm cannot be detected by coloring with TMB color developing solution. The experimental results are shown in Figure 8 As shown in the figure, the absorbance between different concentration groups shows a clear concentration gradient, indicating that the activity of AID of different concentrations can affect the mutation of the single-stranded DNA in a stable and regular result, and therefore the single-stranded DNA complementary strand of SEQ ID NO: 3 is suitable as a single-stranded DNA complementary strand for the kit.

[0111] Figure 4 This is a schematic diagram of detecting DNA cytidine deaminase AID activity using the single-stranded DNA of SEQ ID NO: 6 and the complementary strand of the single-stranded DNA of SEQ ID NO: 4. As shown in the figure, if the AID enzyme activity is active, the adenine in the single-stranded DNA of SEQ ID NO: 6 is mutated into hypoxanthine, and the mismatch between the 21st base of the single-stranded DNA of SEQ ID NO: 6 and the single-stranded DNA complementary chain of SEQ ID NO: 4 is repaired. Three bases in the 20-25 bases will have mismatches with the single-stranded DNA complementary chain of SEQ ID NO: 4. As expected, this chain will not be eluted. At this time, the biotin at the 5' end of the single-stranded DNA chain can be detected by color development with TMB solution, and absorbance can be detected at 450nm. If AID is inactivated, four bases in the 20-28 bases of this chain will have mismatches with the single-stranded DNA complementary chain of SEQ ID NO: 4. As expected, this chain will be eluted. At this time, absorbance cannot be detected at 450nm by color development with TMB solution. The experimental results are as follows. Figure 9 As shown, the absorbance between different concentration groups showed a certain concentration gradient, indicating that the effect of the activity of different concentrations of AID on the mutation of the single-stranded DNA could not be displayed in a stable and regular result. Therefore, the single-stranded DNA complementary chain of SEQ ID NO: 4 is not suitable as the single-stranded DNA complementary chain of the kit.

[0112] Figure 5 This is a schematic diagram of detecting DNA cytidine deaminase AID activity using the single-stranded DNA of SEQ ID NO: 6 and the complementary strand of the single-stranded DNA of SEQ ID NO: 5. As shown in the figure, if the AID enzyme activity is active, the adenine in the single-stranded DNA of SEQ ID NO: 6 is mutated into hypoxanthine, and the mismatch between the 21st base of the single-stranded DNA of SEQ ID NO: 6 and the single-stranded DNA complementary chain of SEQ ID NO: 5 is repaired. Four bases from bases 20 to 28 will have mismatches with the single-stranded DNA complementary chain of SEQ ID NO: 5. As expected, this chain will not be eluted. At this time, the biotin at the 5' end of the single-stranded DNA chain can be detected by color development with TMB solution, and absorbance can be detected at 450nm. If AID is inactivated, five bases from bases 20 to 28 of this chain will have mismatches with the single-stranded DNA complementary chain of SEQ ID NO: 5. As expected, this chain will be eluted. At this time, no absorbance can be detected by color development with TMB solution at 450nm. The experimental results are shown in Figure 2. Figure 10As shown, the absorbance between different concentration groups showed an obvious concentration gradient, indicating that the effect of the activity of different concentrations of AID on the mutation of the single-stranded DNA could not be displayed in a stable and regular result. Therefore, the single-stranded DNA complementary chain of SEQ ID NO: 5 is not suitable as the single-stranded DNA complementary chain of the kit.

[0113] Example 3 Screening of the Optimal PBST Concentration for Washing Plates

[0114] 1. Preparation of PBST solutions of different concentrations

[0115] Six PBST concentration groups were designed, and 5× concentration PBST was diluted to 2× PBST solution, 1× PBST solution, 0.5× PBST solution, and 0.1× PBST solution, respectively.

[0116] 2. Screening of the optimal PBST concentration for plate washing

[0117] Using the single-stranded DNA represented by SEQ ID NO: 6 and the complementary strand of the single-stranded DNA represented by SEQ ID NO: 3 as the test objects, a screening test for the optimal plate washing PBST concentration was performed with reference to Example 2. The specific method is as follows:

[0118] (1) EDCI was added to a PBS solution containing a single-stranded DNA complementary chain at a final concentration of 5 nM, mixed thoroughly, and incubated at room temperature at 25°C for 20 min to obtain a mixed solution after incubation; wherein, the amount of EDCI used was 50 times the equivalent of the single-stranded DNA.

[0119] (2) Prepare a 1% BSA solution using 50 mM carbonate buffer as solvent; add 200 μL of 1% BSA solution to each of the 96 wells of the ELISA plate and let it sit at 4°C for overnight coating; then wash the plate three times with PBST solutions of different concentrations to obtain a BSA-coated ELISA plate for use; take the mixed solution after incubation in step (1) and add it to each well of the BSA-coated ELISA plate, 100 μL per well, incubate at 37°C for 2 h, and wash the plate once with PBST solutions of different concentrations (300 μL) after incubation; then add 200 μL of 1% skim milk powder (prepared with PBS solution) to each well, block at 37°C for 1 h, and wash the plate three times with PBST of different concentrations, 300 μL each time.

[0120] (3) Prepare a PBS solution containing single-stranded DNA at a final concentration of 2.5 nM, take 100 μL of each solution and add it to the ELISA plate in step (3), and incubate at 37°C for 2 h. After incubation, wash the plate three times with different concentrations of PBST, 300 μL each time.

[0121] (4) Add deamination buffer and different final concentrations of AID into the EP tube, and mix well. Incubate at 37°C for 1 h. In this example, 6 groups of AID concentrations are set, i.e. 0 nM, 200 nM, 400 nM, 600 nM, 800 nM and 1000 nM, and each group is set in triplicate.

[0122] (5) Add the mixed solution after incubation in step (4) into the enzyme-labeled plate coupled with dsDNA in step (3), and incubate at 37°C for 1 h. After incubation, wash the plate 6 times with different concentrations of PBST solution, 300 μL each time.

[0123] (6) Add 100 μL of 12000×SA-HRP to each well of the enzyme-labeled plate, and incubate at 25°C for 20 min. After incubation, wash the plate 6 times with different concentrations of PBST solution, 300 μL each time.

[0124] (7) Add 100 μL of TMB color developing solution to each well of the enzyme-labeled plate, and incubate at 25°C in the dark for 60 min. Finally, add 100 μL of color developing termination solution (2M H2SO4) to each well, and measure the absorbance at 450 nm within 15 min.

[0125] The ideal PBST concentration for plate washing is as follows: if AID enzyme activity works, adenine of single-stranded DNA is mutated into hypoxanthine, and the mismatch between the 21st base of single-stranded DNA and the complementary strand is repaired, thereby enhancing the stability of double-stranded DNA. According to the design expectation, as the AID enzyme concentration increases, the probability of the strand being eluted gradually decreases. Color development with TMB color developing solution shows that the absorbance detected at 450 nm presents a concentration-dependent upward trend as the AID enzyme concentration increases. The experimental results are shown in Figure 11 The absorbance between different concentration groups of 0.2×PBST solution group and 0.5×PBST solution group shows a clear concentration gradient, indicating that the two concentrations of PBST have a relatively ideal plate washing effect. Linearization of the experimental results of 0.2× and 0.5× PBST plate washing is shown in Figure 12 , 13 The OD value of 0.5×PBST has a good linear relationship with the AID enzyme concentration, indicating that the results of 0.5×PBST plate washing have stronger stability and regularity. Although the absorbance between different concentration groups of 0.1×PBST solution group, 1×PBST solution group and 2×PBST solution group presents a concentration-dependent increasing trend, it is not obvious, indicating that 1×PBST solution group and 2×PBST solution group cannot achieve the ideal plate washing effect.

[0126] Example 4 Verification of AID enzyme inhibitor aurantricarboxylic acid on the detection of AID enzyme activity by the kit

[0127] aurin tricarboxylic acid (Aurin) Figure 14 Aurin is a compound proved to have the activity of inhibiting DNA cytidine deaminase AID enzyme, IC 50 3.0, the embodiment takes the compound as the object to verify the feasibility of the application and the feasibility for screening AID inhibitors, and the specific method is as follows:

[0128] (1) The specific operation is the same as that in Example 3.

[0129] (2) The specific operation is the same as that in Example 3, wherein the PBST solution is 0.5×PBST solution.

[0130] (3) The specific operation is the same as that in Example 3, wherein the PBST solution is 0.5×PBST solution.

[0131] (4) The deamination buffer, different concentrations of the inhibitor aurin tricarboxylic acid and 1000 nM of AID are added in an EP tube and fully mixed, the total volume of the reaction system is 300 μL, and after fully mixed, the mixture is incubated at 37℃ for 1 h; in the embodiment, 8 concentration groups of the inhibitor are set, which are 0 μM, 0.25 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 4 μM, and 3 repeated tests are set for each concentration group.

[0132] (5) The specific operation is the same as that in Example 3, wherein the PBST solution is 0.5×PBST solution.

[0133] (6) The specific operation is the same as that in Example 3, wherein the PBST solution is 0.5×PBST solution.

[0134] (7) The same as Example 3.

[0135] The experimental results are shown in Table A, B and C, and the results are shown in the following table A, B and C: Figure 14 From the figure, it can be seen that with the increase of the concentration of the inhibitor aurin tricarboxylic acid, the absorbance of the nucleic acid substrate at 450 nm also decreases, indicating that the enzyme activity of AID decreases with the increase of the concentration of aurin tricarboxylic acid, and the calculated IC Figure 14 of the inhibitor aurin tricarboxylic acid to AID is shown in Table C. 50 2.6, which proves that the application has the feasibility for screening AID enzyme inhibitors.

[0136] The embodiments of the application are described in detail above in combination with the drawings, but the application is not limited to the above embodiments, and various changes can be made within the knowledge range of ordinary skilled in the art without departing from the purpose of the application. In addition, the embodiments of the application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A single-stranded DNA and its complementary strand for detecting DNA cytidine deaminase AID activity, characterized in that: The single-stranded DNA has a DNA cytidine deaminase AID recognition site; The nucleic acid sequence of the single-stranded DNA is shown in SEQ ID: 6: The single-stranded DNA complementary chain is shown in SEQ ID NO:

3.

2. The single-stranded DNA and its complementary strand according to claim 1, characterized in that: The 5' end of the single-stranded DNA is connected to a labeling group; The 5' end of the single-stranded DNA complementary chain is connected to a chemically modified group.

3. The single-stranded DNA and its complementary strand according to claim 2, characterized in that: The labeling group includes one of biotin and digoxin; The chemical modification groups include carboxyl groups.

4. A fluorescent kit for detecting DNA cytidine deaminase AID activity or screening DNA cytidine deaminase AID inhibitors, comprising the single-stranded DNA according to any one of claims 1 to 3 and its complementary strand.

5. The kit according to claim 4, wherein: The method comprises at least one of the following components: a labeling reagent that specifically binds to the 5'-end labeling group of the single-stranded DNA, a color development solution, a color development stop solution, a buffer solution, and an eluent.

6. The kit according to claim 5, wherein: The labeling test agent that specifically binds to the 5' end labeling group of the single-stranded DNA is SA-HRP or digoxigenin antibody.

7. The kit according to claim 5, wherein: The chromogenic solution is TMB chromogenic solution; The buffer is a deamination buffer; The eluent is PBST solution; The PBST solution is (0.2-0.5)×PBST.

8. A method for detecting DNA cytidine deaminase AID activity, characterized in that The following steps are included: (1) The single-stranded DNA complementary strand according to any one of claims 1 to 3 is thoroughly mixed with EDCI in a PBS solution and then incubated; (2) adding the mixed solution after incubation in step (1) to the enzyme-labeled plate coated with BSA for incubation, washing the plate with PBST solution after the incubation, and then adding blocking solution for blocking. After the blocking, washing the plate with PBST solution to obtain an enzyme-labeled plate coupled with the single-stranded DNA complementary chain; (3) adding a PBS solution containing the single-stranded DNA described in any one of claims 1 to 3 to the enzyme labeling plate coupled with the complementary strand of the single-stranded DNA in step (2) and incubating the plate, and washing the plate with a PBST solution after the incubation is completed; (4) mixing the DNA cytidine deaminase AID to be tested with the deamination buffer and incubating to obtain a mixed solution after incubation; (5) Add the mixed solution after incubation in step (4) to the ELISA plate in step (3) for incubation, and wash the plate with PBST solution after the incubation is completed; (6) Add SA-HRP or digoxigenin antibody to the ELISA plate and wash the plate with PBST solution after incubation. (7) Add TMB colorimetric solution to the ELISA plate, incubate in the dark, then add colorimetric stop solution and measure the absorbance at a wavelength of 450 nm.

9. A method for screening DNA cytidine deaminase AID inhibitors, characterized in that The following steps are included: S1) thoroughly mixing the complementary strand of the single-stranded DNA according to any one of claims 1 to 3 with EDCI in a PBS solution and incubating the mixture; S2) adding the mixed solution after incubation in step S1) to a BSA-coated ELISA plate for incubation, washing the plate with PBST solution after the incubation, adding a blocking solution for blocking, and washing the plate with PBST solution after the blocking to obtain an ELISA plate coupled with the single-stranded DNA complementary chain; S3) adding the single-stranded DNA according to any one of claims 1 to 3 to the ELISA plate described in step S2) and incubating the plate, and washing the plate with PBST solution after the incubation; S4) mixing the test compound with DNA cytidine deaminase AID in deamination buffer and incubating; S5) adding the mixed solution after incubation in step S4) to the ELISA plate in step S3) for incubation, and washing the plate with PBST solution after the incubation is completed; S6) Add SA-HRP to the ELISA plate and incubate. After incubation, wash the plate with PBST solution. S7) Add TMB colorimetric solution to the ELISA plate, incubate in the dark, then add colorimetric stop solution and measure the absorbance at 450 nm.

10. Use of the single-stranded DNA and its complementary strand according to any one of claims 1 to 3, and the kit according to any one of claims 4 to 7 in the preparation of a product for detecting DNA cytidine deaminase AID activity or screening DNA cytidine deaminase AID inhibitors.

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