A single-stranded RNA for detecting the activity of RNA adenosine deaminase ADAR1, its complementary strand, kit and application

Abstract

The present invention relates to the technical field of bioluminescence detection, and particularly relates to a single-stranded RNA for detecting the activity of RNA adenosine deaminase ADAR1, its complementary strand, a kit and an application. The single-stranded RNA has an RNA adenosine deaminase ADAR1 recognition site, and the nucleic acid sequences of the single-stranded RNA and its complementary strand are shown in SEQ ID No. 3-6. The present invention also provides a fluorescence kit and method for detecting the activity of RNA adenosine deaminase ADAR1 or screening for RNA adenosine deaminase ADAR1 inhibitors. Using this kit and method, the activity of different concentrations of ADAR1 can be sensitively and stably detected in vitro, and it has the advantages of being convenient and fast, having a low price, and using a small amount of raw materials. It can be used for high-throughput screening of ADAR1 inhibitors and avoid detecting false positive inhibitors.

Description

Technical Field

[0001] The present invention relates to the technical field of bioluminescence detection, and particularly relates to a single-stranded RNA for detecting the activity of RNA adenosine deaminase ADAR1, its complementary strand, a kit and applications thereof. Background Art

[0002] RNA adenosine deaminase (ADAR1) is an adenosine deaminase acting on double-stranded RNA (dsRNA), which can convert adenosine (A) into inosine (I), and this process is called A-to-I editing. ADAR1 is highly expressed in various tissues and has two subtypes: ADAR1p110 and ADAR1p150. ADAR1p110 is constitutively expressed and is mainly located in the nucleus; ADAR1p150 is interferon-inducible, with an N-terminal extension, containing a nuclear export signal (NES), which can shuttle between the nucleus and the cytoplasm, and also contains a Za domain that can bind to Z-RNA. The A-to-I editing function of ADAR1 can change the sequence of RNA, affect the post-transcriptional mechanisms of coding and non-coding dsRNA, and thus regulate RNA processing, protein expression and function.

[0003] In the nervous system, abnormal ADAR1 function may affect the RNA editing of key genes in nerve cells, such as the imbalance of mRNA editing of genes related to neurotransmitter transmission and synaptic plasticity, leading to disorder of nerve cell function and gradually causing neurodegeneration, and patients showing symptoms such as cognitive impairment and behavioral abnormalities. In the motor neurons of ALS patients, the level and RNA editing pattern of ADAR1 change. Abnormal RNA editing of specific genes may interfere with the normal functions of motor neurons, including processes such as protein synthesis and axonal transport, resulting in progressive death of motor neurons, and patients showing muscle weakness and atrophy, and ultimately respiratory and swallowing dysfunction.

[0004] In terms of immune response, ADAR1 can inhibit the pattern recognition receptor mechanism, prevent endogenous dsRNA from being recognized by cellular dsRNA sensors, thereby preventing the induction of antiviral signals and uncontrolled IFN-I production, and ensuring the activation of the innate immune response against foreign rather than self nucleic acids. The deletion or dysfunction of ADAR1 will lead to severe inflammatory reactions, affecting the maturation of T cells and B cells, and the development of dendritic cell subsets.

[0005] In the field of oncology, ADAR1 is closely related to the occurrence and development of various tumors. ADAR1 can promote the growth, angiogenesis, and metastasis of tumor cells by regulating the RNA editing of genes related to tumor cell proliferation, invasion, and metastasis. The ADAR1 RNA editing of specific transcripts is related to certain types of cancers and their pathogenicity, editing tumor suppressor miRNAs or exerting tumor suppressor effects on the activity of other miRNAs, and can also limit alternative splicing in cancers. The expression of ADAR1 is upregulated in various solid tumors such as ovarian cancer, colorectal cancer, and lung cancer.

[0006] Given the important role of ADAR1 in neurological diseases, immune diseases, and tumorigenesis, it is of great significance to explore technical means capable of detecting ADAR1 activity and apply them to the screening of ADAR1 inhibitors, with the expectation of screening out compounds that can effectively inhibit ADAR1 activity; and applying them to the detection of the content of ADAR1 in tissues / cells to assist in medical diagnosis.

[0007] Currently, there are not many means for detecting ADAR1 activity, mainly including PCR, gene sequencing, Western Blot, IHC, ELISA, TR-FRET analysis methods, etc. These means have disadvantages such as cumbersome operation, time-consuming, high price, large consumption of raw materials, and high requirements for special professional equipment and technology. Currently, the ADAR1 fluorescence reporter gene cell line detection method is reported more frequently for the high-throughput detection of ADAR1. The latest research reports the ADAR1 luciferase reporter gene HEK293 cell line developed by BPS Bioscience. This cell line expresses ADAR1 under the control of the CMV promoter and expresses the ADAR editing RNA reporter construct under the control of a separate CMV promoter. In the absence of ADAR1 activity, translation terminates at the stop codon, and the Renilla luciferase gene is not expressed. The reporter gene activity readout is the Renilla luciferase / firefly luciferase ratio, so that when ADAR activity is inhibited, the UAG (stop) codon is read, resulting in a decrease in Renilla luciferase expression and a decrease in the Renilla luciferase / firefly luciferase ratio. However, this method is an intracellular detection, with disadvantages such as cumbersome operation, expensive detection instruments, poor repeatability of conventional analysis, and substrate oxidation failure. Therefore, there is an urgent need in this field to develop a technical method that can specifically detect the ADAR1 enzyme activity in vitro and is convenient and fast. Summary of the Invention

[0008] In order to overcome the deficiencies and disadvantages of the prior art, the primary object of the present invention is to provide a single-stranded RNA and its complementary strand for detecting the activity of RNA adenosine deaminase ADAR1, which can be used to detect the activity of RNA adenosine deaminase ADAR1 or to screen RNA adenosine deaminase ADAR1 inhibitors, etc.

[0009] Another object of the present invention is to provide a fluorescence kit for detecting the activity of RNA adenosine deaminase ADAR1 or screening for inhibitors of RNA adenosine deaminase ADAR1.

[0010] Another object of the present invention is to provide the use of the above-mentioned single-stranded RNA, its complementary strand and the kit.

[0011] The fourth object of the present invention is to provide a method for detecting the activity of RNA adenosine deaminase ADAR1.

[0012] The fifth object of the present invention is to provide a method for screening for inhibitors of RNA adenosine deaminase ADAR1.

[0013] The objects of the present invention are achieved by the following technical solutions:

[0014] A single-stranded RNA for detecting the activity of RNA adenosine deaminase ADAR1 and its complementary strand, the single-stranded RNA has an RNA adenosine deaminase ADAR1 recognition site, and the nucleic acid sequence of the single-stranded RNA is as shown in SEQ ID No. 6:

[0015] SEQ ID No. 6: 5’-AAAAAAAAAAAAAUAUGUAGCAAGGAUGCGAG-3’;

[0016] The complementary strand of the single-stranded RNA is at least one of SEQ No. 3-5, and its nucleic acid sequence is as follows:

[0017] SEQ ID No. 3: 5’-AAAAAAAACUCGCAUCCUACCCA-3’;

[0018] SEQ ID No. 4: 5’-AAAAAAAACUCGCAUCCGACCCA-3’;

[0019] SEQ ID No. 5: 5’-AAAAAAAACUCGCAUCAGACCCA-3’;

[0020] The 5’ end of the single-stranded RNA is preferably linked to a labeling group, which is used to detect whether the single-stranded RNA and its complementary strand form stable dsRNA under the action of a labeling reagent; the 5’ end of the complementary strand of the single-stranded RNA is preferably linked to a chemical modification group, which is used to immobilize the complementary strand of the single-stranded RNA;

[0021] The labeling group is preferably biotin or digoxin; the chemical modification group is preferably a carboxyl group.

[0022] A fluorescence kit for detecting the activity of RNA adenosine deaminase ADAR1 or screening for inhibitors of RNA adenosine deaminase ADAR1, comprising the above single-stranded RNA and its complementary strand;

[0023] The kit preferably further comprises at least one of the following components: a labeling reagent that specifically binds to the labeling group at the 5'-end of the single-stranded RNA, a chromogenic solution, a chromogenic termination solution, a buffer solution, and an elution solution;

[0024] The labeling reagent that specifically binds to the labeling group at the 5'-end of the single-stranded RNA is preferably SA-HRP (streptavidin peroxidase labeled with horseradish peroxidase) or a digoxin antibody;

[0025] The chromogenic solution is used to develop color when the labeling group and the labeling reagent combine to form a complex, thereby detecting the nucleic acid linked to the labeling group;

[0026] The chromogenic solution is preferably a TMB chromogenic solution;

[0027] The buffer solution is preferably a deamination buffer solution;

[0028] The elution solution is preferably a 0.2×PBST solution or a 0.5×PBST solution;

[0029] The kit preferably further comprises RNA adenosine deaminase ADAR1.

[0030] A method for detecting the activity of RNA adenosine deaminase ADAR1, comprising the following steps:

[0031] (1) After thoroughly mixing the complementary strand of the above single-stranded RNA with EDCI in a PBS solution, incubate;

[0032] (2) Add the mixed solution after incubation in step (1) to an enzyme-labeled plate coated with BSA for incubation. After incubation, wash the plate with a PBST solution, then add a blocking solution for blocking. After blocking, wash the plate with a PBST solution to obtain an enzyme-labeled plate conjugated with the complementary strand of the single-stranded RNA;

[0033] (3) Add a PBS solution containing the above single-stranded RNA to the enzyme-labeled plate conjugated with the complementary strand of the single-stranded RNA in step (2) for incubation. After incubation, wash the plate with a PBST solution;

[0034] (4) Mix the RNA adenosine deaminase ADAR1 to be tested with a deamination buffer solution and incubate to obtain an incubated mixed solution;

[0035] (5) Add the incubated mixed solution in step (4) to the enzyme-labeled plate in step (3) for incubation. After incubation, wash the plate with a PBST solution;

[0036] (6) Add SA-HRP (Streptavidin-Horseradish Peroxidase) or digoxin antibody to the enzyme-linked immunosorbent assay (ELISA) plate. After incubation, wash the plate with PBST solution.

[0037] (7) Add chromogenic solution to the ELISA plate. After incubation in the dark, add chromogenic stop solution and measure the absorbance at a wavelength of 450 nm.

[0038] The concentration of the single-stranded RNA complementary strand described in step (1) in PBS solution is preferably 2 - 50 nM.

[0039] The dosage of EDCI described in step (1) is preferably 2 - 50 times the equivalent amount of the single-stranded RNA complementary strand.

[0040] The incubation temperature described in step (1) is preferably 25 °C, and the incubation time is preferably 10 - 60 min.

[0041] The blocking solution described in step (2) is preferably a 1% skim milk powder solution or a 2% PEG-4000 solution by mass fraction.

[0042] The blocking solution described in step (2) is preferably prepared with PBS solution.

[0043] The ELISA plate coated with BSA described in step (2) is obtained through the following preparation method:

[0044] Prepare a 1% BSA solution with 50 mM carbonate buffer at pH 9.4. Add the 1% BSA solution to the ELISA plate. After static coating overnight at 4 °C, wash the plate with PBST solution 2 - 4 times. The addition amount of the 1% BSA solution is preferably 200 μL.

[0045] The addition amount of the incubated mixed solution described in step (2) is 100 μL, and the addition amount of the blocking solution is preferably 200 μL.

[0046] The incubation temperature described in step (2) is preferably 37 °C, and the incubation time is preferably 1 - 3 h.

[0047] The blocking temperature described in step (2) is preferably 37 °C, and the blocking time is preferably 1 - 3 h.

[0048] The concentration of single-stranded RNA in the PBS solution containing single-stranded RNA described in step (3) is preferably 2 - 50 nM.

[0049] The addition amount of the PBS solution containing single-stranded RNA described in step (3) is preferably 100 μL.

[0050] The incubation temperature described in step (3) is preferably 37 °C, and the incubation time is preferably 1 - 3 h.

[0051] The deamination buffer described in step (4) is composed of: 0.1 mM 2-(N-Morpholino)ethanesulfonic acid (MES) and 0.1 mM Triethylamine (TEA), pH = 8.5;

[0052] The incubation temperature in step (4) is preferably 37 °C, and the incubation time is preferably 10 - 60 min;

[0053] The addition amount of the incubated mixed solution in step (5) is preferably 300 μL;

[0054] The incubation temperature in step (5) is preferably 37 °C, and the incubation time is preferably 1 - 3 h;

[0055] The dilution factor of SA-HRP described in step (6) is preferably 12,000, and the addition amount is preferably 100 μL;

[0056] The incubation temperature in step (6) is 25 °C, and the incubation time is 10 - 60 min;

[0057] The incubation temperature in step (7) is preferably 25 °C, and the incubation time is preferably 10 - 60 min;

[0058] The color development termination solution in step (7) is preferably 2M H 2 SO 4 ;

[0059] The absorbance measurement in step (7) is preferably carried out within 15 min after adding the color development termination solution;

[0060] The PBST solution described in steps (2), (3), (5) and (6) is preferably 0.2× or 0.5× PBST solution, and the pH value is pH 8.5;

[0061] The number of times of washing the enzyme-linked immunosorbent assay (ELISA) plate with the PBST solution in steps (2), (3), (5) and (6) is preferably 1 - 6 times.

[0062] A method for screening RNA adenosine deaminase ADAR1 inhibitors, comprising the following steps:

[0063] S1) After fully mixing the single-stranded RNA complementary strand with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) in PBS solution, incubate;

[0064] S2) Add the mixed solution after incubation in step S1) to an ELISA plate coated with Bovine Serum Albumin (BSA) for incubation. After incubation, wash the plate with PBST solution, then add a blocking solution for blocking. After blocking, wash the plate with PBST solution to obtain an ELISA plate conjugated with the single-stranded RNA complementary strand;

[0065] S3) Add single-stranded RNA to the enzyme-linked immunosorbent assay (ELISA) plate described in step S2) and incubate. After incubation, wash the plate with PBST solution;

[0066] S4) Mix the test compound with RNA adenosine deaminase ADAR1 in deamination buffer and incubate;

[0067] S5) Add the mixed solution after incubation in step S4) to the ELISA plate in step S3) for incubation. After incubation, wash the plate with PBST solution;

[0068] S6) Add SA-HRP or digoxin antibody to the ELISA plate for incubation. After incubation, wash the plate with PBST solution;

[0069] S7) Add chromogenic solution to the ELISA plate, incubate in the dark, add chromogenic stop solution, and measure the absorbance at a wavelength of 450 nm;

[0070] The incubation temperature described in step S1) is preferably 25 °C, and the incubation time is preferably 10 - 60 min;

[0071] The incubation temperature described in step S2) is preferably 37 °C, and the incubation time is preferably 1 - 3 h;

[0072] The blocking temperature described in step S2) is preferably 37 °C, and the blocking time is preferably 1 - 3 h;

[0073] The incubation temperature described in step S3) is preferably 37 °C, and the incubation time is preferably 1 - 3 h;

[0074] The incubation temperature described in step S4) is preferably 37 °C, and the pre-incubation time is 10 - 60 min;

[0075] The incubation temperature described in step S5) is preferably 37 °C, and the incubation time is preferably 1 - 3 h;

[0076] The incubation temperature described in step S6) is preferably 25 °C, and the incubation time is preferably 10 - 60 min;

[0077] The incubation temperature described in step S7) is preferably 25 °C, and the incubation time is preferably 10 - 60 min;

[0078] The PBST solution described in steps S2), S3), S5) and S6) is preferably 0.2× or 0.5× PBST solution;

[0079] Use of the single-stranded RNA and its complementary strand, and the kit in the preparation of products for detecting the activity of RNA adenosine deaminase ADAR1 or screening for inhibitors of RNA adenosine deaminase ADAR1.

[0080] Technical principle of the present invention:

[0081] The present invention designs a single-stranded RNA containing an RNA adenosine deaminase ADAR1 recognition site, with biotin linked to its 5' end and its complementary strand's 5' end modified with carboxyl group; an ADAR1 recognition site - adenine A is designed in the single-stranded RNA sequence, and a mismatched base cytosine C is designed at the corresponding position on the RNA complementary strand. The complementary strand of the single-stranded RNA is added to an enzyme-linked immunosorbent assay (ELISA) plate for incubation, and then the ELISA plate is washed with an eluent to form an ELISA plate coupled with the complementary strand of the single-stranded RNA. On this basis, the single-stranded RNA is added to the above ELISA plate, and it forms a double-stranded RNA with its complementary strand through base complementary pairing, and a mismatch is formed at the ADAR1 recognition site of the double-stranded RNA. ADAR1 recognizes adenine A in the single-stranded RNA sequence and mutates it into inosine I. Since adenine A mutates into inosine I and can pair with cytosine C at the corresponding site in the complementary strand, the base mismatch between the single-stranded RNA and its complementary strand is repaired. Utilizing the difference in binding ability caused by the different numbers of mismatches between the single-stranded RNA and its complementary strand, the biotin-linked single-stranded RNA with fewer mismatches or no mismatches cannot be eluted. At this time, SA-HRP is added for incubation and TMB chromogenic solution is added for color development, and then a significant absorbance can be detected.

[0082] The present invention has the following advantages and effects compared with the prior art:

[0083] (1) The present invention provides a single-stranded RNA and its complementary strand for detecting the activity of RNA adenosine deaminase ADAR1. The single-stranded RNA and its complementary strand can sensitively and stably detect the activities of different concentrations of ADAR1 in vitro. More importantly, when applied to screening inhibitors, color development using TMB, etc., can effectively avoid the quenching interference of compounds on traditional bioluminescence and prevent the detection of false positive inhibitors.

[0084] (2) The present invention provides a fluorescence kit for detecting the activity of RNA adenosine deaminase ADAR1 or screening RNA adenosine deaminase ADAR1 inhibitors based on the above single-stranded RNA and its complementary strand. The kit has simple components and low cost, and can be used for detecting the activity of RNA adenosine deaminase ADAR1 or screening RNA adenosine deaminase ADAR1 inhibitors.

[0085] (3) The present invention provides a method for detecting the activity of RNA adenosine deaminase ADAR1 or screening RNA adenosine deaminase ADAR1 inhibitors. This method can detect the activity of RNA adenosine deaminase ADAR1 or screen RNA adenosine deaminase ADAR1 inhibitors in vitro, with simple and convenient operation, low price, and small consumption of raw materials, and can be applicable to high-throughput screening of ADAR1 inhibitors. Brief Description of the Drawings

[0086] Figure 1 It is a schematic diagram for detecting the activity of RNA adenosine deaminase ADAR1 with single-stranded RNA and its complementary strand (SEQ ID No.1).

[0087] Figure 2 It is a schematic diagram for detecting the activity of RNA adenosine deaminase ADAR1 with single-stranded RNA and its complementary strand (SEQ ID No.2).

[0088] Figure 3 It is a schematic diagram for detecting the activity of RNA adenosine deaminase ADAR1 with single-stranded RNA and its complementary strand (SEQ ID No.3).

[0089] Figure 4 It is a schematic diagram for detecting the activity of RNA adenosine deaminase ADAR1 with single-stranded RNA and its complementary strand (SEQ ID No.4).

[0090] Figure 5 It is a schematic diagram for detecting the activity of RNA adenosine deaminase ADAR1 with single-stranded RNA and its complementary strand (SEQ ID No.5).

[0091] Figure 6 It is the OD for detecting the activity of RNA adenosine deaminase ADAR1 with single-stranded RNA and its complementary strand (SEQ ID No.1). 450 Result analysis diagram.

[0092] Figure 7 It is the OD for detecting the activity of RNA adenosine deaminase ADAR1 with single-stranded RNA and its complementary strand (SEQ ID No.2). 450 Result analysis diagram.

[0093] Figure 8 It is the OD for detecting the activity of RNA adenosine deaminase ADAR1 with single-stranded RNA and its complementary strand (SEQ ID No.3). 450 Result analysis diagram.

[0094] Figure 9 It is the OD for detecting the activity of RNA adenosine deaminase ADAR1 with single-stranded RNA and its complementary strand (SEQ ID No.4). 450 Result analysis diagram.

[0095] Figure 10 It is the result analysis diagram of OD450 for detecting the activity of RNA adenosine deaminase ADAR1 with single-stranded RNA and its complementary strand (SEQ ID No.5).

[0096] Figure 11It is a linear fitting graph of the OD450 results for detecting the activity of RNA adenosine deaminase ADAR1 by the single-stranded RNA and its complementary strand (SEQ ID No.3) in Example 2.

[0097] Figure 12 It is a linear fitting graph of the OD450 results for detecting the activity of RNA adenosine deaminase ADAR1 by the single-stranded RNA and its complementary strand (SEQ ID No.4) in Example 2.

[0098] Figure 13 It is a linear fitting graph of the OD450 results for detecting the activity of RNA adenosine deaminase ADAR1 by the single-stranded RNA and its complementary strand (SEQ ID No.5) in Example 2.

[0099] Figure 14 It is a result analysis graph for screening the optimal concentration of PBST solution for plate washing in Example 3.

[0100] Figure 15 It is a chemical structural formula of an ADAR1 inhibitor and a result analysis graph for screening, where A: chemical structural formula, B: screening result. Detailed implementation mode

[0101] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the implementation modes of the present invention are not limited thereto.

[0102] Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0103] In the examples, EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) is a condensing agent, purchased from Macklin; BSA, 12000×SA-HRP, TMB chromogenic solution, etc. are purchased from Beyotime, and Fludarabine-Cl is purchased from MCE;

[0104] In the examples, ADAR1 is expressed and purified by the recombinant plasmid pGEX-ADAR1 according to the conventional method. The recombinant plasmid pGEX-ADAR1 is purchased from Sangon Biotech (Shanghai) Co., Ltd.; this enzyme can also be directly purchased commercially;

[0105] In the examples, the composition of the deamination buffer is: 0.1 mM 2-(N-morpholino)ethanesulfonic acid (MES) and 0.1 mM triethylamine (TEA), pH = 8.5, where both MES and TEA are purchased from Macklin;

[0106] In the examples, the pH values of 5×PBST, 2×PBST solution, 1×PBST solution, 0.5×PBST solution, and 0.1×PBST solution are all 8.5; the pH value of 50 mM carbonate buffer solution is 9.4; PBS solution is a common buffer solution in the art with a pH of 7.4.

[0107] Example 1 Design and Synthesis of Single-stranded RNA and Its Complementary Strand

[0108] In the present invention, a total of 1 single-stranded RNA and 5 complementary strands of single-stranded RNA with different sequences were designed and screened, and their nucleic acid sequences are shown in Table 1. The 5' end of the single-stranded RNA sequence was biotin-labeled, and the 5' end of the complementary strand of the single-stranded RNA was modified with a carboxylic acid group. The synthesis of the primer, the labeling of biotin, and the modification of the carboxylic acid group were all completed by Sangon Biotech (Shanghai) Co., Ltd.

[0109]

[0110] Example 2 Screening of the Optimal Single-stranded RNA Sequence

[0111] (1) Add EDCI to a PBS solution (pH = 7.4) containing a final concentration of 5 nM complementary strand of single-stranded RNA (any one of SEQ ID No.1 - 5 in Table 1), mix well, and incubate at 25 °C for 20 min at room temperature to obtain an incubated mixed solution; among them, the dosage of EDCI is 50-fold equivalent of the complementary strand of single-stranded RNA, and its function is to activate the carboxyl group at the 5' end of the complementary strand of single-stranded RNA, enabling it to react with the amino group in BSA protein, thereby stably immobilizing the complementary strand of single-stranded RNA on the 96-well microplate;

[0112] (2) Prepare a 1% BSA solution with 50 mM carbonate buffer solution as the solvent; add 200 μL of the 1% BSA solution to each of the 96 wells of the microplate, let it stand at 4 °C for overnight coating, and then wash the plate 3 times with 0.2×PBST solution to obtain a microplate coated with BSA for use; take the incubated mixed solution in step (1) and add 100 μL to each well of the microplate coated with BSA, incubate at 37 °C for 2 h, and after incubation, wash the plate once with 0.2×PBST solution (300 μL); add 200 μL of 1% skim milk powder (prepared with PBS solution) to each well, incubate at 37 °C for 1 h, and after the blocking is completed, wash the plate 3 times with 0.2×PBST solution, 300 μL each time;

[0113] (3) Prepare a PBS solution containing a final concentration of 2.5 nM single-stranded RNA (Table 1, SEQ ID No.6), take 100 μL each and add it to the blocked microplate in step (2), incubate at 37 °C for 2 h; after incubation, wash the plate 3 times with 0.2×PBST solution, 300 μL each time;

[0114] (4) Add deamination buffer and ADAR1 at different final concentrations to an EP tube. The total volume of the reaction system is 300 μL. After thorough mixing, incubate at 37 °C for 1 h. In this example, a total of 6 ADAR1 concentration groups were set, with final concentrations of 0 nM, 200 nM, 400 nM, 600 nM, 800 nM, and 1000 nM respectively. Each concentration group was set with 3 replicate experiments.

[0115] (5) Add the mixed solution incubated in step (4) to the ELISA plate conjugated with dsRNA in step (3), and incubate at 37 °C for 1 h. After incubation, wash the plate 6 times with 0.2×PBST solution, 300 μL each time.

[0116] (6) Add 100 μL of 12,000×SA-HRP to each well of the ELISA plate, and incubate at 25 °C for 20 min. After incubation, wash the plate 6 times with 0.2×PBST solution, 300 μL each time.

[0117] (7) Add 100 μL of TMB chromogenic solution to each well of the ELISA plate, and incubate at 25 °C in the dark for 60 min. Finally, add 100 μL of chromogenic termination solution (2 M H 2 SO 4 ) to each well, and measure the absorbance at 450 nm within 15 min.

[0118] Figure 1 It is a schematic diagram for detecting the activity of RNA adenosine deaminase ADAR1 with the single-stranded RNA of SEQ ID No.6 and the complementary strand of the single-stranded RNA of SEQ ID No.1. As shown in the figure, if the ADAR1 enzyme activity is functional, it can mutate the adenine (A) of the single-stranded RNA of SEQ ID No.6 to inosine (I). The mismatch generated by the 19th base of the single-stranded RNA of SEQ ID No.6 and the complementary strand of the single-stranded RNA of SEQ ID No.1 is repaired. According to the design expectation, this strand will not be eluted. At this time, the biotin at the 5' end of this single-stranded RNA can be detected by coloring with TMB chromogenic solution, and absorbance can be detected at 450 nm. If ADAR1 is inactivated, the mismatch originally generated by the 19th base of this strand and the complementary strand of the single-stranded RNA of SEQ ID No.1 cannot be repaired. According to the design expectation, this strand will be eluted. At this time, no absorbance can be detected at 450 nm when coloring with TMB chromogenic solution. The experimental results are as Figure 6 shown. There is no concentration gradient shown in the absorbance between different concentration groups, indicating that the activity of ADAR1 at different concentrations cannot show a stable and regular result in the mutation of this single-stranded RNA. Therefore, the complementary strand of the single-stranded RNA of SEQ ID No.1 is not suitable as the complementary strand of the single-stranded RNA for detecting ADAR1 activity.

[0119] Figure 2 It is a schematic diagram for detecting the activity of RNA adenosine deaminase ADAR1 using the single-stranded RNA of SEQ ID No.6 and the complementary strand of the single-stranded RNA of SEQ ID No.2. As shown in the figure, if the ADAR1 enzyme activity is functional, the adenine of the single-stranded RNA of SEQ ID No.6 is mutated to hypoxanthine, and the mismatch generated between the 19th base of the single-stranded RNA of SEQ ID No.6 and the complementary strand of the single-stranded RNA of SEQ ID No.2 is repaired. There will be 1 base among the 18-25 bases that generates a mismatch with the complementary strand of the single-stranded RNA of SEQ ID No.2. According to the design expectation, this strand will not be eluted. At this time, when developed with the TMB chromogenic solution, the biotin at the 5' end of this single-stranded RNA can be detected, and the absorbance can be detected at 450 nm; if ADAR1 is inactivated, there will be 2 bases among the 18-25 bases of this strand that generate a mismatch with the complementary strand of the single-stranded RNA. According to the design expectation, this strand will be eluted. At this time, when developed with the TMB chromogenic solution, the absorbance cannot be detected at 450 nm. The experimental results are as Figure 7 shown. There is no concentration gradient shown in the absorbance among different concentration groups, indicating that the influence of the activity of ADAR1 at different concentrations on the mutation of this single-stranded RNA cannot be presented with stable and regular results. Therefore, the complementary strand of the single-stranded RNA of SEQ ID No.2 is not suitable as the complementary strand of the single-stranded RNA for detecting the activity of ADAR1.

[0120] Figure 3 It is a schematic diagram for detecting the activity of RNA adenosine deaminase ADAR1 using the single-stranded RNA of SEQ ID No.6 and the complementary strand of the single-stranded RNA of SEQ ID No.3. As shown in the figure, if the ADAR1 enzyme activity is functional, the adenine of the single-stranded RNA of SEQ ID No.6 is mutated to hypoxanthine, and the mismatch generated between the 19th base of the single-stranded RNA of SEQ ID No.6 and the complementary strand of the single-stranded RNA of SEQ ID No.3 is repaired. There will be 2 bases among the 18-25 bases that generate a mismatch with the complementary strand of the single-stranded RNA of SEQ ID No.3. According to the design expectation, this strand will not be eluted. At this time, when developed with the TMB chromogenic solution, the biotin at the 5' end of this single-stranded RNA can be detected, and the absorbance can be detected at 450 nm; if ADAR1 is inactivated, there will be 3 bases among the 18-25 bases of this strand that generate a mismatch with the complementary strand of the single-stranded RNA. According to the design expectation, this strand will be eluted. At this time, when developed with the TMB chromogenic solution, the absorbance cannot be detected at 450 nm. The experimental results are as Figure 8As shown, the absorbance among different concentration groups shows an obvious concentration gradient, indicating that the influence of the activity of ADAR1 at different concentrations on the mutation of this single-stranded RNA can be presented as stable and regular results. Therefore, the complementary strand of the single-stranded RNA of SEQ ID No.3 is suitable as the complementary strand of the single-stranded RNA for this kit.

[0121] Figure 4 It is a schematic diagram for detecting the activity of RNA adenosine deaminase ADAR1 using the complementary strand of the single-stranded RNA of SEQ ID No.6 and the single-stranded RNA of SEQ ID No.4. As shown in the figure, if the ADAR1 enzyme activity takes effect, the adenine of the single-stranded RNA of SEQ ID No.6 is mutated into hypoxanthine, and the mismatch generated by the 19th base of the single-stranded RNA of SEQ ID No.6 and the complementary strand of the single-stranded RNA of SEQ ID No.4 is repaired. There will be 3 bases among the 18-25 bases that generate mismatches with the complementary strand of the single-stranded RNA of SEQ ID No.4. According to the design expectation, this strand will not be eluted. At this time, when color-developed with TMB color-developing solution, the biotin at the 5' end of this single-stranded RNA strand can be detected, and the absorbance can be detected at 450 nm; if ADAR1 is inactivated, there will be 4 bases among the 18-25 bases of this strand that generate mismatches with the complementary strand of the single-stranded RNA of SEQ ID No.4. According to the design expectation, this strand will be eluted. At this time, when color-developed with TMB color-developing solution, no absorbance can be detected at 450 nm. The experimental results are as Figure 9 shown, the absorbance among different concentration groups shows a certain concentration gradient, indicating that the influence of the activity of ADAR1 at different concentrations on the mutation of this single-stranded RNA can be presented as stable and regular results. Therefore, the complementary strand of the single-stranded RNA of SEQ ID No.4 is suitable as the complementary strand of the single-stranded RNA for this kit.

[0122] Figure 5Schematic diagram for detecting the activity of RNA adenosine deaminase ADAR1 with the single-stranded RNA of SEQ ID No.6 and the complementary strand of the single-stranded RNA of SEQ ID No.5. As shown in the figure, if the ADAR1 enzyme activity is functional, the adenine of the single-stranded RNA of SEQ ID No.6 is mutated to hypoxanthine, and the mismatch generated by the 19th base of the single-stranded RNA of SEQ ID No.6 and the complementary strand of the single-stranded RNA of SEQ ID No.5 is repaired. Among the bases at positions 18 - 25, 4 bases will generate mismatches with the complementary strand of the single-stranded RNA of SEQ ID No.5. According to the design expectation, this strand will not be eluted. At this time, when developed with TMB chromogenic solution, the biotin at the 5' end of this single-stranded RNA can be detected, and the absorbance can be detected at 450 nm; if ADAR1 is inactivated, among the bases at positions 18 - 25 of this strand, 5 bases will generate mismatches with the complementary strand of the single-stranded RNA of SEQ ID No.5. According to the design expectation, this strand will be eluted. At this time, when developed with TMB chromogenic solution, no absorbance can be detected at 450 nm. The experimental results are as Figure 10 shown. The absorbance among different concentration groups shows an obvious concentration gradient, indicating that the activity of ADAR1 at different concentrations can show the mutation effect on this single-stranded RNA with stable and regular results. Therefore, the complementary strand of the single-stranded RNA of SEQ ID No.5 is suitable as the complementary strand of the single-stranded RNA for this kit.

[0123] Further, linear fitting is performed on the results corresponding to SEQ ID No.3, SEQ ID No.4, and SEQ ID No.5 above, and the results are respectively as Figure 11 , Figure 12 , Figure 13 shown. Comparing the three, there is a good linear relationship between the OD values corresponding to SEQ ID No.3 and SEQ ID No.5 and the ADAR1 enzyme, indicating that the activity of ADAR1 at different concentrations has strong stability and regularity on the mutation effect of this single-stranded RNA. Therefore, SEQ ID No.3 and SEQ ID No.5 are more suitable as the complementary strand of the single-stranded RNA for this kit.

[0124] Example 3 Screening for the optimal PBST concentration for plate washing

[0125] I. Preparation of PBST solutions with different concentrations

[0126] Six PBST concentration groups are designed, and 5× concentrated PBST is diluted to 2×PBST solution, 1×PBST solution, 0.5×PBST solution, and 0.1×PBST solution respectively.

[0127] II. Screening for the optimal PBST concentration for plate washing

[0128] Using the single-stranded RNA shown in SEQ ID No.6 and the complementary strand of the single-stranded RNA shown in SEQ ID No.3 as the objects, refer to Example 2 to conduct an optimal PBST concentration screening test for plate washing. The specific method is as follows:

[0129] (1) Add EDCI to the PBS solution containing the single-stranded RNA complementary strand with a final concentration of 5 nM, mix well, and incubate at 25 °C for 20 min at room temperature to obtain the incubated mixed solution; among them, the dosage of EDCI is 50-fold equivalent of the single-stranded RNA;

[0130] (2) Prepare a 1% BSA solution with 50 mM carbonate buffer as the solvent; add 200 μL of the 1% BSA solution to each of the 96 wells of the enzyme-linked immunosorbent assay (ELISA) plate, and let it stand at 4 °C for overnight coating; then wash the plate 3 times with PBST solutions of different concentrations to obtain the ELISA plate coated with BSA for standby; take the incubated mixed solution in step (1) and add 100 μL to each well of the ELISA plate coated with BSA, incubate at 37 °C for 2 h, and after incubation, wash the plate 1 time with PBST solutions of different concentrations (300 μL); add 200 μL of 1% skim milk powder (prepared with PBS solution) to each well, incubate at 37 °C for 1 h, and after the blocking is completed, wash the plate 3 times with PBST of different concentrations, 300 μL each time;

[0131] (3) Prepare a PBS solution containing the single-stranded RNA with a final concentration of 2.5 nM, and take 100 μL respectively and add them to the ELISA plate in step (3), incubate at 37 °C for 2 h; after incubation, wash the plate 3 times with PBST solutions of different concentrations, 300 μL each time;

[0132] (4) Add deamination buffer and different final concentrations of ADAR1 to an EP tube, and the total volume of the reaction system is 300 μL. Mix well and incubate at 37 °C for 1 h; in this example, a total of 6 ADAR1 concentration groups are set, which are final concentrations of 0 nM, 200 nM, 400 nM, 600 nM, 800 nM, and 1000 nM respectively, and each concentration group is set with 3 repeated experiments;

[0133] (5) Add the mixed solution incubated in step (4) to the ELISA plate coupled with dsRNA in step (3), and incubate at 37 °C for 1 h; after incubation, wash the plate 6 times with PBST solutions of different concentrations, 300 μL each time;

[0134] (6) Add 100 μL of 12000×SA-HRP to each well of the ELISA plate, incubate at 25 °C for 20 min, and after incubation, wash the plate 6 times with PBST solutions of different concentrations, 300 μL each time;

[0135] (7) Add 100 μL of TMB chromogenic solution to each well of the enzyme-linked immunosorbent assay (ELISA) plate and incubate at 25 °C in the dark for 60 min; finally, add 100 μL of chromogenic stop solution (2 M H 2 SO 4 ) and measure the absorbance at 450 nm within 15 min.

[0136] Ideally, the best washing effect of PBST concentration is as follows: if the ADAR1 enzyme activity takes effect and adenine of single-stranded RNA is mutated into hypoxanthine, the mismatch generated by the 19th base of single-stranded RNA with the complementary strand is repaired, and the stability of double-stranded RNA is enhanced. According to the design expectation, as the concentration of ADAR1 enzyme increases, the probability of this strand being eluted gradually decreases. After coloring with TMB chromogenic solution, the absorbance detected at 450 nm shows a concentration-dependent increasing trend as the concentration of ADAR1 enzyme increases. The experimental results are as Figure 14 shown. The absorbance between different concentration groups of 0.2×PBST solution group and 0.5×PBST solution group shows an obvious concentration gradient, indicating that these two concentrations of PBST have relatively ideal washing effects; although the absorbance between different concentration groups of 1×PBST solution group and 2×PBST solution group shows a concentration-dependent increasing trend, it is not obvious, indicating that the 1×PBST solution group and 2×PBST solution group cannot achieve the ideal washing effect.

[0137] Example 4 Verification of the ability of the ADAR1 enzyme inhibitor Fludarabine-Cl to detect the ADAR1 enzyme activity of this kit

[0138] Fludarabine-Cl ( Figure 15 ) is a compound that has been proven to inhibit the activity of RNA adenosine deaminase ADAR1 enzyme, and its intracellular IC 50 is 0.87 μM. In this example, this compound is used as the object to verify the feasibility of the present invention and the feasibility of screening ADAR1 inhibitors. The specific method is as follows:

[0139] (1) The specific operation is the same as that in Example 3;

[0140] (2) The specific operation is the same as that in Example 3, where the PBST solution is 0.2×PBST solution;

[0141] (3) The specific operation is the same as that in Example 3, where the PBST solution is 0.2×PBST solution;

[0142] (4) Add deamination buffer, inhibitor Fludarabine-Cl at different concentrations, and 1000 nM of ADAR1 into an EP tube and mix well. The total volume of the reaction system is 300 μL. After mixing well, incubate at 37 °C for 1 h. In this example, 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, and 4 μM respectively. Each concentration group is set with 3 repeated experiments.

[0143] (5) The specific operation is the same as that in Example 3, where the PBST solution is 0.2×PBST solution.

[0144] (6) The specific operation is the same as that in Example 3, where the PBST solution is 0.2×PBST solution.

[0145] (7) The same as Example 3.

[0146] The experimental results are as Figure 15 shown. It can be seen from the figure that as the concentration of the inhibitor Fludarabine-Cl increases, the absorbance of the nucleic acid substrate at 450 nm also decreases, indicating that the enzyme activity of ADAR1 weakens with the increase of the Fludarabine-Cl concentration. After calculation, the IC 50 of the inhibitor Fludarabine-Cl for ADAR1 is 1.656 μM, which proves the feasibility of the present invention for screening ADAR1 enzyme inhibitors.

[0147] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A kit for detecting RNA adenosine deaminase ADAR1 activity or screening RNA adenosine deaminase ADAR1 inhibitors, characterized in that: The kit comprises a single-stranded RNA and its complementary strand for detecting the activity of RNA adenosine deaminase ADAR1; The single-stranded RNA has an RNA adenosine deaminase ADAR1 recognition site, and the nucleic acid sequence of the single-stranded RNA is shown in SEQ ID No. 6: SEQ ID No. 6: 5'-AAAAAAAAAAAAAUAUGUAGCAAGGAUGCGAG-3'; The single-stranded RNA complementary strand is at least one of SEQ ID No.3-5, and its nucleic acid sequence is as follows: SEQ ID No.3: 5'-AAAAAAAAACUCGCAUCCUACCCA-3'; SEQ ID No.4: 5'-AAAAAAAAACUCGCAUCCGACCCA-3'; SEQ ID No.5: 5'-AAAAAAAAACUCGCAUCAGACCCA-3'; The 5' end of the single-stranded RNA is connected to a labeling group, which is used to detect whether the single-stranded RNA and its complementary chain form a stable dsRNA under the action of a labeling agent; the 5' end of the complementary chain of the single-stranded RNA is connected to a chemical modification group, which is used to fix the complementary chain of the single-stranded RNA; The kit also comprises a labeling reagent that specifically binds to the labeling group at the 5' end of the single-stranded RNA and a color developing solution.

2. The kit according to claim 1, characterized in that: The labeling group is biotin or digoxigenin; the chemical modification group is carboxyl.

3. The kit according to claim 1, characterized in that The method further comprises at least one of the following components: a color stop solution, a buffer solution and an eluent.

4. The kit according to claim 1, characterized in that: The labeling reagent specifically combined with the labeling group at the 5' end of the single-stranded RNA is SA-HRP or digoxigenin antibody-HRP.

5. The kit according to claim 3, characterized in that: The color developing solution is TMB color developing solution; The buffer is a deamination buffer; The eluent is 0.2×PBST solution or 0.5×PBST solution.

6. A method for detecting RNA adenosine deaminase ADAR1 activity for non-diagnostic purposes, characterized in that The following steps are included: (1) mixing the single-stranded RNA complementary strand described in any one of claims 1 to 2 with EDCI in a PBS solution and then incubating; (2) adding the mixed solution after incubation in step (1) to an ELISA plate coated with BSA for incubation, washing the plate with a PBST solution after the incubation, and then adding a blocking solution for blocking. After the blocking, washing the plate with a PBST solution to obtain an ELISA plate coupled with a single-stranded RNA complementary chain; (3) adding a PBS solution containing the single-stranded RNA according to any one of claims 1 to 2 to the ELISA plate coupled with the single-stranded RNA complementary chain in step (2) and incubating the plate, and washing the plate with a PBST solution after the incubation; (4) mixing the RNA adenosine deaminase ADAR1 to be tested with a deamination buffer, and incubating the mixture to obtain a mixed solution after incubation; (5) adding the mixed solution after incubation in step (4) to the ELISA plate in step (3) for incubation, and washing the plate with PBST solution after the incubation; (6) Add SA-HRP or digoxigenin antibody-HRP to the ELISA plate for incubation. After the incubation, wash the plate with PBST solution. (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.

7. A method for screening RNA adenosine deaminase ADAR1 inhibitors, characterized in that The following steps are included: S1) mixing the single-stranded RNA complementary strand according to any one of claims 1 to 2 with EDCI in a PBS solution and then incubating; S2) adding the mixed solution after incubation in step S1) to the BSA-coated ELISA plate for incubation, washing the plate with PBST solution after the incubation, adding blocking solution for blocking, and washing the plate with PBST solution after the blocking to obtain an ELISA plate coupled with the single-stranded RNA complementary chain; S3) adding the single-stranded RNA described in any one of claims 1 to 2 to the ELISA plate described in step S2) for incubation, and washing the plate with PBST solution after the incubation; S4) mixing the test compound with RNA adenosine deaminase ADAR1 in a 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; S6) Add SA-HRP or digoxigenin antibody-HRP to the ELISA plate for incubation, and wash the plate with PBST solution after the incubation. S7) 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.

8. Use of the kit according to any one of claims 1 to 5 in preparing a product for detecting RNA adenosine deaminase ADAR1 activity or screening RNA adenosine deaminase ADAR1 inhibitors.

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