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

Abstract

The invention relates to the technical field of biological fluorescence detection, in particular to a single-stranded RNA for detecting the activity of RNA adenosine deaminase ADAR1 as well as a complementary strand, a kit and application of the single-stranded RNA. The single-stranded RNA has an RNA adenosine deaminase ADAR1 recognition site, and the nucleic acid sequences of the single-stranded RNA and the complementary strand of the single-stranded RNA are as shown in SEQ ID No. 3-6. The invention also provides a fluorescent kit and a method for detecting the activity of the RNA adenosine deaminase ADAR1 or screening the RNA adenosine deaminase ADAR1 inhibitor, the activity of ADAR1 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 ADAR1 inhibitor can be screened in a high-throughput manner, and the application prospect is wide. The detection of a false positive inhibitor is avoided.

Description

Technical Field

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

[0002] RNA adenosine deaminase (ADAR1) is an adenosine deaminase that acts on double-stranded RNA (dsRNA) and can convert adenosine (A) to inosine (I), a process known as A-to-I editing. ADAR1 is highly expressed in a variety of tissues and has two subtypes: ADAR1p110 and ADAR1p150. ADAR1p110 is constitutively expressed and mainly located in the nucleus; ADAR1p150 is interferon-inducible, has an N-terminal extension, contains a nuclear export signal (NES), 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 thereby regulate RNA processing, protein expression and function.

[0003] In the nervous system, ADAR1 dysfunction may affect the RNA editing of key genes in nerve cells, such as imbalance in mRNA editing of genes related to neurotransmitter transmission and synaptic plasticity, leading to dysfunction of nerve cells and gradually inducing neurodegeneration. Patients experience symptoms such as cognitive impairment and abnormal behavior. In the motor neurons of ALS patients, ADAR1 levels and RNA editing patterns change. Abnormal RNA editing of specific genes may interfere with the normal function of motor neurons, including processes such as protein synthesis and axonal transport, leading to progressive death of motor neurons. Patients show muscle weakness and atrophy, and ultimately respiratory and swallowing dysfunction.

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

[0005] In the field of tumors, 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 RNA editing of genes related to tumor cell proliferation, invasion and metastasis. ADAR1 RNA editing of specific transcripts is associated with certain types of cancer and their pathogenicity. It edits tumor suppressor miRNAs or exerts tumor suppressor effects on the activity of other miRNAs, and can also limit selective splicing in cancer. ADAR1 expression is upregulated in various solid tumors such as ovarian cancer, colorectal cancer, and lung cancer.

[0006] In view of the important role of ADAR1 in neurological diseases, immune diseases and tumorigenesis, it is of great significance to explore technical means to detect ADAR1 activity and apply them to the screening of ADAR1 inhibitors in order to screen out compounds that can effectively inhibit ADAR1 activity; as well as to apply them to the detection of ADAR1 content in tissues / cells to assist in medical diagnosis.

[0007] There are currently few methods for detecting ADAR1 activity, mainly including PCR, gene sequencing, Western Blot, IHC, ELISA, TR-FRET analysis, etc. These methods have the disadvantages of cumbersome operation, time-consuming, high price, large amount of raw materials, special professional equipment and high technical requirements. At present, the most commonly used method for high-throughput detection of ADAR1 is the ADAR1 fluorescent reporter gene cell line detection method. The latest study reported the ADAR1 luciferase reporter gene HEK293 cell line developed by BPS Bioscience, which expresses ADAR1 under the control of the CMV promoter and the ADAR edited 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 fluorescent gene is not expressed. The reporter gene activity is read as the Renilla luciferase / firefly luciferase ratio, so that the UAG (stop) codon is read when ADAR activity is inhibited, resulting in reduced expression of Renilla luciferase and reduced expression of Renilla luciferase / firefly luciferase ratio. However, this method is an intracellular detection method, which is cumbersome to operate and has the disadvantages of expensive detection instruments, poor reproducibility of conventional analysis, and failure of substrate oxidation. Therefore, there is an urgent need in the art to develop a technical method that can specifically detect ADAR1 enzyme activity in vitro and is convenient and fast. Summary of the invention

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

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

[0010] Another object of the present invention is to provide applications of the above-mentioned single-stranded RNA, its complementary strand and 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 RNA adenosine deaminase ADAR1 inhibitors.

[0013] The purpose of the present invention is achieved through the following technical solutions: A single-stranded RNA for detecting RNA adenosine deaminase ADAR1 activity and its complementary strand, wherein 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 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 preferably 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 single-stranded RNA complementary chain is preferably connected to a chemical modification group, which is used to fix the single-stranded RNA complementary chain; The labeling group is preferably biotin or digoxigenin; the chemical modification group is preferably a carboxyl group.

[0014] A fluorescent kit for detecting RNA adenosine deaminase ADAR1 activity or screening RNA adenosine deaminase ADAR1 inhibitors, comprising the above-mentioned single-stranded RNA and its complementary strand; 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 color developing solution, a color development stop solution, a buffer solution, and an eluent; The labeling agent that specifically binds to the labeling group at the 5' end of the single-stranded RNA is preferably SA-HRP (horseradish peroxidase labeled streptavidin) or digoxigenin antibody; The color developing solution is used to develop color when the labeling group and the labeling reagent combine to form a complex, thereby detecting the nucleic acid connected to the labeling group; The color developing solution is preferably TMB color developing solution; The buffer is preferably a deamination buffer; The eluent is preferably 0.2×PBST solution or 0.5×PBST solution; The kit preferably further comprises RNA adenosine deaminase ADAR1.

[0015] A method for detecting RNA adenosine deaminase ADAR1 activity comprises the following steps: (1) The single-stranded RNA complementary chain is fully mixed with EDCI in a PBS solution and then incubated; (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 to the ELISA plate coupled with the complementary strand of the single-stranded RNA in step (2) and incubating the plate. After the incubation, wash the plate with a PBST solution. (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 (horseradish peroxidase-labeled streptavidin) or digoxigenin antibody to the ELISA plate and wash the plate with PBST solution after incubation. (7) Add the color developing solution to the ELISA plate, incubate in the dark, then add the color stop solution, and measure the absorbance at a wavelength of 450 nm; The concentration of the single-stranded RNA complementary strand in step (1) in the PBS solution is preferably 2-50 nM; The amount of EDCI used in step (1) is preferably 2-50 times the equivalent of the complementary strand of the single-stranded RNA; The incubation temperature in step (1) is preferably 25°C, and the incubation time is preferably 10-60 min; The blocking solution in step (2) is preferably a 1% by mass skimmed milk powder solution or a 2% by mass PEG-4000 solution; The blocking solution in step (2) is preferably prepared with PBS solution; The BSA-coated ELISA plate described in step (2) is prepared by the following method: A 1% BSA solution was prepared using a 50 mM carbonate buffer solution at pH 9.4, and the 1% BSA solution was added to the ELISA plate. After the plate was coated overnight at 4°C, the plate was washed 2-4 times with a PBST solution. The amount of the 1% BSA solution added was preferably 200 μL. The amount of the mixed solution added after incubation in step (2) is 100 μL, and the amount of the blocking solution added is preferably 200 μL; The incubation temperature in step (2) is preferably 37° C., and the incubation time is preferably 1-3 h; The sealing temperature in step (2) is preferably 37°C, and the sealing time is preferably 1-3 hours; The concentration of the single-stranded RNA in the PBS solution containing the single-stranded RNA in step (3) is preferably 2-50 nM; The amount of the PBS solution containing single-stranded RNA added in step (3) is preferably 100 μL; The incubation temperature in step (3) is preferably 37° C., and the incubation time is preferably 1-3 h; The composition of the deamination buffer in step (4) is: 0.1 mM tris(acrylamido)methylpropanesulfonic acid (MES) and 0.1 mM trimethylaminoethane (TEA), pH = 8.5; The incubation temperature in step (4) is preferably 37° C., and the incubation time is preferably 10-60 min; The amount of the mixed solution added after incubation in step (5) is preferably 300 μL; The incubation temperature in step (5) is preferably 37° C., and the incubation time is preferably 1-3 h; The dilution factor of SA-HRP in step (6) is preferably 12000, and the added amount is preferably 100 μL; The incubation temperature in step (6) is 25°C and the incubation time is 10-60 min; The incubation temperature in step (7) is preferably 25°C, and the incubation time is preferably 10-60 min; The color stop solution in step (7) is preferably 2M H2SO4; The absorbance measurement in step (7) is preferably performed within 15 minutes after the addition of the color stop solution. The PBST solution in steps (2), (3), (5) and (6) is preferably a 0.2× or 0.5× PBST solution having a pH value of pH 8.5; The number of times the plate is washed with PBST solution in steps (2), (3), (5) and (6) is preferably 1-6 times.

[0016] A method for screening RNA adenosine deaminase ADAR1 inhibitors comprises the following steps: S1) The single-stranded RNA complementary strand is fully mixed with EDCI in a PBS solution and then incubated; 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 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 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 to the ELISA plate for incubation, and wash the plate with PBST solution after the incubation. S7) Add color development solution to the ELISA plate, incubate in the dark, add color stop solution, and measure the absorbance at a wavelength of 450 nm; The incubation temperature in step S1) is preferably 25°C, and the incubation time is preferably 10-60 min; The incubation temperature in step S2) is preferably 37°C, and the incubation time is preferably 1-3h; The sealing temperature in step S2) is preferably 37°C, and the sealing time is preferably 1-3 hours; The incubation temperature in step S3) is preferably 37°C, and the incubation time is preferably 1-3 hours; The incubation temperature in step S4) is preferably 37°C, and the pre-incubation time is 10-60 min; The incubation temperature in step S5) is preferably 37°C, and the incubation time is preferably 1-3h; The incubation temperature in step S6) is preferably 25°C, and the incubation time is preferably 10-60 min; The incubation temperature in step S7) is preferably 25°C, and the incubation time is preferably 10-60 min; The PBST solution in steps S2), S3), S5) and S6) is preferably 0.2× or 0.5× PBST solution; The single-stranded RNA and its complementary strand, and the kit are used in preparing products for detecting RNA adenosine deaminase ADAR1 activity or screening RNA adenosine deaminase ADAR1 inhibitors.

[0017] Technical principle of the present invention: The present invention designs a single-stranded RNA containing an RNA adenosine deaminase ADAR1 recognition site, and its 5' end is connected to biotin, and its complementary chain 5' end is modified by a carboxyl group; the ADAR1 recognition site-adenine A is designed in the single-stranded RNA sequence, and the mismatched base cytosine C is designed at the corresponding position on the RNA complementary chain. The single-stranded RNA complementary chain is added to an ELISA plate for incubation, and then the ELISA plate is washed with an eluent to form an ELISA plate coupled with the single-stranded RNA complementary chain. On this basis, the single-stranded RNA is added to the above-mentioned ELISA plate, and the double-stranded RNA is formed through base complementary pairing with the complementary chain, and the double-stranded RNA forms a mismatch at the ADAR1 recognition site. ADAR1 recognizes adenine A in the single-stranded RNA sequence and mutates it into hypoxanthine I. Since adenine A mutates into hypoxanthine I, it can be complementary paired with cytosine C at the corresponding site in the complementary chain, and the base mismatch between the single-stranded RNA and its complementary chain is repaired. By utilizing the difference in binding ability caused by the different number of mismatches between single-stranded RNA and complementary chains, single-stranded RNA connected to biotin with few or no mismatches cannot be eluted. At this time, SA-HRP is added for incubation and TMB color development solution is added, and obvious absorbance can be detected.

[0018] Compared with the prior art, the present invention has the following advantages and effects: (1) The present invention provides a single-stranded RNA and its complementary chain for detecting the activity of RNA adenosine deaminase ADAR1. The single-stranded RNA and its complementary chain can sensitively and stably detect the activity of different concentrations of ADAR1 in vitro. More importantly, they are used in screening inhibitors. Using TMB or the like for color development can effectively avoid the quenching interference of compounds on traditional bioluminescence and prevent the detection of false positive inhibitors.

[0019] (2) The present invention provides a fluorescent kit for detecting RNA adenosine deaminase ADAR1 activity or screening RNA adenosine deaminase ADAR1 inhibitors based on the above-mentioned single-stranded RNA and its complementary strand. The kit has simple components and low cost, and can be used to detect RNA adenosine deaminase ADAR1 activity or screen RNA adenosine deaminase ADAR1 inhibitors.

[0020] (3) The present invention provides a method for detecting RNA adenosine deaminase ADAR1 activity or screening RNA adenosine deaminase ADAR1 inhibitors. The method can detect RNA adenosine deaminase ADAR1 activity or screen RNA adenosine deaminase ADAR1 inhibitors in vitro. The method is simple and convenient to operate, has a low price, uses a small amount of raw materials, and can be suitable for high-throughput screening of ADAR1 inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of detecting RNA adenosine deaminase ADAR1 activity using single-stranded RNA and its complementary strand (SEQ ID No.1).

[0022] Figure 2 It is a schematic diagram of detecting RNA adenosine deaminase ADAR1 activity using single-stranded RNA and its complementary strand (SEQ ID No. 2).

[0023] Figure 3 It is a schematic diagram of detecting RNA adenosine deaminase ADAR1 activity using single-stranded RNA and its complementary strand (SEQ ID No.3).

[0024] Figure 4 It is a schematic diagram of detecting RNA adenosine deaminase ADAR1 activity using single-stranded RNA and its complementary strand (SEQ ID No. 4).

[0025] Figure 5 It is a schematic diagram of detecting RNA adenosine deaminase ADAR1 activity using single-stranded RNA and its complementary strand (SEQ ID No.5).

[0026] Figure 6 It is the OD of single-stranded RNA and its complementary strand (SEQ ID No.1) to detect the activity of RNA adenosine deaminase ADAR1 450 Result analysis chart.

[0027] Figure 7 It is the OD of single-stranded RNA and its complementary strand (SEQ ID No.2) to detect the activity of RNA adenosine deaminase ADAR1 450 Result analysis chart.

[0028] Figure 8It is the OD of single-stranded RNA and its complementary strand (SEQ ID No.3) to detect the activity of RNA adenosine deaminase ADAR1 450 Result analysis chart.

[0029] Fig. 9 It is the OD of single-stranded RNA and its complementary strand (SEQ ID No.4) to detect the activity of RNA adenosine deaminase ADAR1 450 Result analysis chart.

[0030] Fig.10 This is an analysis chart of the OD450 results of detecting RNA adenosine deaminase ADAR1 activity using single-stranded RNA and its complementary strand (SEQ ID No.5).

[0031] Fig.11 It is a linear fitting graph of the OD450 results of detecting RNA adenosine deaminase ADAR1 activity using single-stranded RNA and its complementary strand (SEQ ID No. 3) in Example 2.

[0032] Fig.12 It is a linear fitting graph of the OD450 results of detecting RNA adenosine deaminase ADAR1 activity using single-stranded RNA and its complementary strand (SEQ ID No. 4) in Example 2.

[0033] Fig.13 It is a linear fitting graph of the OD450 results of detecting RNA adenosine deaminase ADAR1 activity using single-stranded RNA and its complementary strand (SEQ ID No. 5) in Example 2.

[0034] Fig.14 This is an analysis chart of the results of screening for the optimal plate washing PBST solution concentration in Example 3.

[0035] Fig.15 It is the chemical structure formula of ADAR1 inhibitor and the analysis diagram of screening results, wherein A: chemical structure formula, B: screening results. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0037] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0038] In the examples, EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) was used as a condensing agent and was purchased from Maclean; BSA, 12000×SA-HRP, TMB colorimetric solution, etc. were purchased from Beyotime, and Fludarabine-Cl was purchased from MCE; In the embodiment, ADAR1 is expressed and purified using the recombinant plasmid pGEX-ADAR1 according to conventional methods, and the recombinant plasmid pGEX-ADAR1 is purchased from Sangon Biotech (Shanghai) Co., Ltd.); the enzyme can also be directly purchased from the market; In the embodiment, the composition of the deamination buffer is: 0.1 mM tris(acrylamido)methylpropanesulfonic acid (MES) and 0.1 mM trimethylaminoethane (TEA), pH=8.5, wherein MES and TEA are purchased from Maclean; In the examples, the pH of 5×PBST, 2×PBST solution, 1×PBST solution, 0.5×PBST solution, and 0.1×PBST solution are all 8.5; the pH of 50 mM carbonate buffer is 9.4; and PBS solution is a common buffer in the art, with a pH of 7.4.

[0039] Example 1 Design and synthesis of single-stranded RNA and its complementary strand The present invention designed and screened a single-stranded RNA and five single-stranded RNA complementary chains with different sequences, 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 single-stranded RNA complementary chain was modified with a carboxylic acid group. The synthesis of primers, biotin labeling, and carboxylic acid group modification were all completed by Sangon Biotech (Shanghai) Co., Ltd.

[0040]

[0041] Example 2 Screening of the best single-stranded RNA sequence (1) EDCI was added to a PBS solution (pH = 7.4) containing a single-stranded RNA complementary chain (any one of SEQ ID No. 1-5, Table 1) with a final concentration of 5 nM, and the mixture was incubated at room temperature 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 RNA complementary chain, and its function was to activate the carboxyl group at the 5' end of the single-stranded RNA complementary chain, so that it reacted with the amino group in the BSA protein, thereby stably fixing the single-stranded RNA complementary chain on the 96-well ELISA plate; (2) Prepare a 1% BSA solution using 50 mM carbonate buffer as solvent; add 200 μL of the 1% BSA solution to each of the 96 wells of the ELISA plate, incubate at 4°C overnight, and then wash the plate three times with 0.2×PBST solution to obtain a BSA-coated ELISA plate for standby use; take the mixed solution after incubation in step (1) and add 100 μL to each well of the BSA-coated ELISA plate, incubate at 37°C for 2 h, and 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, block at 37°C for 1 h, and wash the plate three times with 0.2×PBST solution (300 μL each time) after blocking; (3) Prepare a PBS solution containing a single-stranded RNA (Table 1, SEQ ID No. 6) with a final concentration of 2.5 nM, take 100 μL of each and add it to the ELISA plate blocked in step (2), and incubate at 37°C for 2 h; after the incubation, wash the plate three times with 0.2× PBST solution, 300 μL each time; (4) Add deamination buffer and different final concentrations of ADAR1 to the EP tube. The total volume of the reaction system is 300 μL. After thorough mixing, incubate at 37°C for 1 h. In this embodiment, a total of 6 ADAR1 concentration groups were set up, with final concentrations of 0 nM, 200 nM, 400 nM, 600 nM, 800 nM and 1000 nM, and 3 repeated experiments were set up for each concentration group. (5) Add the mixed solution after incubation in step (4) to the ELISA plate coupled with dsRNA in step (3) and incubate at 37°C for 1 h. After the incubation, wash the plate 6 times with 0.2×PBST solution, 300 μL each time. (6) Add 100 μL of 12000× SA-HRP to each well of the ELISA plate and incubate at 25°C for 20 min. After incubation, wash the plate six times with 0.2× PBST solution, 300 μL each time. (7) Add 100 μL of TMB colorimetric solution to each well of the ELISA plate and incubate at 25°C in the dark for 60 min. Finally, add 100 μL of color stop solution (2 M H2SO4) to each well and measure the absorbance at 450 nm within 15 min.

[0042] Figure 1The figure is a schematic diagram of detecting the activity of RNA adenosine deaminase ADAR1 by using the single-stranded RNA of SEQ ID No.6 and the complementary chain of the single-stranded RNA of SEQ ID No.1. As shown in the figure, if the ADAR1 enzyme activity is functional, the adenine (A) of the single-stranded RNA of SEQ ID No.6 can be mutated into hypoxanthine (I), and the mismatch between the 19th base of the single-stranded RNA of SEQ ID No.6 and the complementary chain of the single-stranded RNA of SEQ ID No.1 is repaired. As expected by the design, the chain will not be eluted. At this time, the biotin at the 5' end of the single-stranded RNA can be detected by color development with TMB colorimetric solution, and the absorbance can be detected at 450nm; if ADAR1 is inactivated, the mismatch between the 19th base of the chain and the complementary chain of the single-stranded RNA of SEQ ID No.1 cannot be repaired. As expected by the design, the chain will be eluted. At this time, the absorbance cannot be detected by color development with TMB colorimetric solution at 450nm. The experimental results are as follows. Figure 6 As shown, the absorbance between different concentration groups did not show a concentration gradient, indicating that the effect of the activity of different concentrations of ADAR1 on the mutation of the single-stranded RNA could not be displayed in a stable and regular result. Therefore, the single-stranded RNA complementary chain of SEQ ID No.1 is not suitable as a single-stranded RNA complementary chain for detecting ADAR1 activity.

[0043] Figure 2 It is a schematic diagram of detecting the activity of RNA adenosine deaminase ADAR1 using the single-stranded RNA of SEQ ID No.6 and the single-stranded RNA complementary strand of SEQ ID No.2. As shown in the figure, if the ADAR1 enzyme activity is functional, the adenine in the single-stranded RNA of SEQ ID No.6 will be mutated into hypoxanthine, and the mismatch between the 19th base of the single-stranded RNA of SEQ ID No.6 and the complementary chain of the single-stranded RNA of SEQ ID No.2 will be repaired. One base among the bases at positions 18-25 will have a mismatch with the complementary chain of the single-stranded RNA of SEQ ID No.2. As expected by the design, this chain will not be eluted. At this time, the biotin at the 5' end of the single-stranded RNA chain can be detected by color development with TMB colorimetric solution, and the absorbance can be detected at 450nm; if ADAR1 is inactivated, two bases among the bases at positions 18-25 of the chain will have a mismatch with the complementary chain of the single-stranded RNA. As expected by the design, this chain will be eluted. At this time, the absorbance cannot be detected at 450nm by color development with TMB colorimetric solution. The experimental results are as follows. Figure 7 As shown, the absorbance between different concentration groups did not show a concentration gradient, indicating that the effect of the activity of different concentrations of ADAR1 on the mutation of the single-stranded RNA could not be displayed in a stable and regular result. Therefore, the single-stranded RNA complementary chain of SEQ ID No. 2 is not suitable as a single-stranded RNA complementary chain for detecting ADAR1 activity.

[0044] Figure 3 It is a schematic diagram of detecting the activity of RNA adenosine deaminase ADAR1 using the single-stranded RNA of SEQ ID No.6 and the single-stranded RNA complementary strand 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 will be mutated into hypoxanthine, and the mismatch between the 19th base of the single-stranded RNA of SEQ ID No.6 and the complementary chain of the single-stranded RNA of SEQ ID No.3 will be repaired. Two bases at positions 18-25 will mismatch with the complementary chain of the single-stranded RNA of SEQ ID No.3. As expected by the design, this chain will not be eluted. At this time, the biotin at the 5' end of the single-stranded RNA chain can be detected by color development with TMB colorimetric solution, and the absorbance can be detected at 450nm; if ADAR1 is inactivated, three bases at positions 18-25 of the chain will mismatch with the complementary chain of the single-stranded RNA. As expected by the design, this chain will be eluted. At this time, the absorbance cannot be detected at 450nm by color development with TMB colorimetric solution. The experimental results are as follows. Figure 8 As shown, the absorbance between different concentration groups shows an obvious concentration gradient, indicating that the effect of the activity of different concentrations of ADAR1 on the mutation of the single-stranded RNA can be displayed in a stable and regular result. Therefore, the single-stranded RNA complementary chain of SEQ ID No.3 is suitable as the single-stranded RNA complementary chain of the kit.

[0045] Figure 4 It is a schematic diagram of detecting the activity of RNA adenosine deaminase ADAR1 using the single-stranded RNA of SEQ ID No.6 and the single-stranded RNA complementary strand of SEQ ID No.4. As shown in the figure, if the ADAR1 enzyme activity is functional, the adenine in the single-stranded RNA of SEQ ID No.6 will be mutated into hypoxanthine, and the mismatch between the 19th base of the single-stranded RNA of SEQ ID No.6 and the complementary chain of the single-stranded RNA of SEQ ID No.4 will be repaired. Three bases from the 18th to the 25th bases will have mismatches with the complementary chain of the single-stranded RNA of SEQ ID No.4. As expected by the design, this chain will not be eluted. At this time, the biotin at the 5' end of the single-stranded RNA chain can be detected by using TMB colorimetric solution, and the absorbance can be detected at 450nm. If ADAR1 is inactivated, four bases from the 18th to the 25th bases of this chain will have mismatches with the complementary chain of the single-stranded RNA of SEQ ID No.4. As expected by the design, this chain will be eluted. At this time, the absorbance cannot be detected at 450nm by using TMB colorimetric solution. The experimental results are as follows. Fig. 9As shown, the absorbance between different concentration groups shows a certain concentration gradient, indicating that the effect of the activity of different concentrations of ADAR1 on the mutation of the single-stranded RNA can be displayed in a stable and regular result. Therefore, the single-stranded RNA complementary chain of SEQ ID No. 4 is suitable as the single-stranded RNA complementary chain of the kit.

[0046] Figure 5 It is a schematic diagram of detecting the activity of RNA adenosine deaminase ADAR1 using the single-stranded RNA of SEQ ID No.6 and the single-stranded RNA complementary strand of SEQ ID No.5. As shown in the figure, if the ADAR1 enzyme activity is functional, the adenine in the single-stranded RNA of SEQ ID No.6 will be mutated into hypoxanthine, and the mismatch between the 19th base of the single-stranded RNA of SEQ ID No.6 and the complementary chain of the single-stranded RNA of SEQ ID No.5 will be repaired. Four bases from bases 18 to 25 will have mismatches with the complementary chain of the single-stranded RNA of SEQ ID No.5. As expected by the design, this chain will not be eluted. At this time, the biotin at the 5' end of the single-stranded RNA chain can be detected by using TMB colorimetric solution, and the absorbance can be detected at 450nm. If ADAR1 is inactivated, five bases from bases 18 to 25 of the chain will have mismatches with the complementary chain of the single-stranded RNA of SEQ ID No.5. As expected by the design, this chain will be eluted. At this time, the absorbance cannot be detected at 450nm by using TMB colorimetric solution. The experimental results are as follows. Fig.10 As shown, the absorbance between different concentration groups shows an obvious concentration gradient, indicating that the effect of the activity of different concentrations of ADAR1 on the mutation of the single-stranded RNA can be displayed in a stable and regular result. Therefore, the single-stranded RNA complementary chain of SEQ ID No.5 is suitable as the single-stranded RNA complementary chain of the kit.

[0047] The results corresponding to the above SEQ ID No.3, SEQ ID No.4, and SEQ ID No.5 were further linearly fitted, and the results were as follows: Fig.11 , Fig.12 , Fig.13 As shown. Compared with the three, the OD values ​​corresponding to SEQ ID No.3 and SEQ ID No.5 have a good linear relationship with the ADAR1 enzyme, indicating that the activity of different concentrations of ADAR1 has a strong stability and regularity in affecting the mutation of the single-stranded RNA. Therefore, SEQ ID No.3 and SEQ ID No.5 are more suitable as the single-stranded RNA complementary chains of the kit.

[0048] Example 3 Screening of the Optimal PBST Concentration for Washing Plates 1. Preparation of PBST solutions of different concentrations 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.

[0049] 2. Screening of the optimal PBST concentration for washing The single-stranded RNA shown in SEQ ID No.6 and the single-stranded RNA complementary strand shown in SEQ ID No.3 were used as the objects, and the optimal plate washing PBST concentration screening test was performed with reference to Example 2. The specific method is as follows: (1) Add EDCI to a PBS solution containing a single-stranded RNA complementary chain at a final concentration of 5 nM, mix thoroughly, and incubate at room temperature 25°C for 20 min to obtain a mixed solution after incubation; wherein the amount of EDCI used is 50 times the equivalent of the single-stranded RNA; (2) Prepare a 1% BSA solution using 50 mM carbonate buffer as solvent; add 200 μL of the 1% BSA solution to each of the 96 wells of the ELISA plate and incubate at 4°C overnight for coating; then wash the plate three times with PBST solutions of different concentrations to obtain a BSA-coated ELISA plate for standby 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 solutions of different concentrations, 300 μL each time after blocking; (3) Prepare a PBS solution containing single-stranded RNA 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 the incubation, wash the plate three times with PBST of different concentrations, 300 μL each time; (4) Add deamination buffer and different final concentrations of ADAR1 to the EP tube. The total volume of the reaction system is 300 μL. Mix thoroughly and incubate at 37°C for 1 h. In this example, a total of 6 ADAR1 concentration groups were set up, with final concentrations of 0 nM, 200 nM, 400 nM, 600 nM, 800 nM and 1000 nM, and 3 repeated experiments were set up for each concentration group. (5) Add the mixed solution after incubation in step (4) to the ELISA plate coupled with dsRNA in step (3), and incubate at 37°C for 1 h. After the incubation, wash the plate 6 times with PBST solution of different concentrations, 300 μL each time. (6) Add 100 μL of 12000× SA-HRP to each well of the ELISA plate and incubate at 25°C for 20 min. After incubation, wash the plate six times with PBST solution of different concentrations, 300 μL each time. (7) Add 100 μL of TMB colorimetric solution to each well of the ELISA plate and incubate at 25°C in the dark for 60 min. Finally, add 100 μL of color stop solution (2 M H2SO4) to each well and measure the absorbance at 450 nm within 15 min.

[0050] The ideal washing effect of the optimal PBST concentration is: if the ADAR1 enzyme activity works, the adenine of the single-stranded RNA will be mutated into hypoxanthine, the mismatch between the 19th base of the single-stranded RNA and the complementary chain will be repaired, and the stability of the double-stranded RNA will be enhanced. According to the design expectations, as the concentration of the ADAR1 enzyme increases, the probability of the chain being eluted gradually decreases. The absorbance detected at 450nm, which was developed with TMB colorimetric solution, showed a concentration-dependent upward trend as the concentration of the ADAR1 enzyme increased. The experimental results are as follows Fig.14 As shown, the absorbance between different concentration groups of 0.2×PBST solution group and 0.5×PBST solution group showed an obvious concentration gradient, indicating that these two concentrations of PBST have a relatively ideal plate washing effect; although the absorbance between different concentration groups of 1×PBST solution group and 2×PBST solution group showed a concentration-dependent increasing trend, it was not obvious, indicating that 1×PBST solution group and 2×PBST solution group could not achieve an ideal plate washing effect.

[0051] Example 4 ADAR1 enzyme inhibitor Fludarabine-Cl verifies the ability of this kit to detect ADAR1 enzyme activity Fludarabine-Cl( Fig.15 ) is a compound that has been shown to inhibit the activity of RNA adenosine deaminase ADAR1, with an IC of 50 The concentration of 1 μM is 0.87 μM. This example uses this compound as the object to verify the feasibility of the present invention and the feasibility of using it for screening ADAR1 inhibitors. The specific method is as follows: (1) The specific operation is the same as that of Example 3; (2) The specific operation is the same as that in Example 3, wherein the PBST solution is 0.2×PBST solution; (3) The specific operation is the same as that in Example 3, wherein the PBST solution is 0.2×PBST solution; (4) Add deamination buffer, different concentrations of inhibitor Fludarabine-Cl and 1000 nM ADAR1 to the EP tube and mix thoroughly. The total volume of the reaction system is 300 μL. After thorough mixing, incubate at 37°C for 1 h. In this example, the inhibitor is set to 8 concentration groups, namely 0 μM, 0.25 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, and 4 μM. Each concentration group is set to perform 3 repeated experiments.

[0052] (5) The specific operation is the same as that in Example 3, wherein the PBST solution is 0.2×PBST solution; (6) The specific operation is the same as that in Example 3, wherein the PBST solution is 0.2×PBST solution; (7) Same as Example 3.

[0053] The experimental results are as follows Fig.15 As shown in the figure, it can be seen 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 decreases with the increase of Fludarabine-Cl concentration. After calculation, the IC value of the inhibitor Fludarabine-Cl on ADAR1 is 50 The result showed that the concentration of ADAR1 in the mixture was 1.656 μM, which proved that the present invention was feasible for screening ADAR1 enzyme inhibitors.

[0054] The above embodiments are preferred implementation modes of the present invention, but the implementation modes 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 modes and shall be included in the protection scope of the present invention.

Claims

1. A single-stranded RNA and its complementary strand for detecting RNA adenosine deaminase ADAR1 activity, characterized in that 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 6: SEQ 6:5'-AAAAAAAAAAAAAUAUGUAGCAAGGAUGCGAG-3'; The single-stranded RNA complementary strand is at least one of SEQ 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'.

2. The single-stranded RNA and its complementary strand according to claim 1, characterized in that: 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 stable dsRNA under the action of a labeling agent; the 5' end of the single-stranded RNA complementary chain is connected to a chemical modification group, which is used to fix the single-stranded RNA complementary chain.

3. The single-stranded RNA and its complementary strand according to claim 2, characterized in that: The labeling group is biotin or digoxigenin; the chemical modification group is carboxyl.

4. A fluorescent kit for detecting RNA adenosine deaminase ADAR1 activity or screening RNA adenosine deaminase ADAR1 inhibitors, comprising the single-stranded RNA according to any one of claims 1 to 3 and its complementary strand.

5. The kit according to claim 4, characterized in that The method further comprises at least one of the following components: a labeling agent specifically binding to the labeling group at the 5' end of the single-stranded RNA, a color developing solution, a color stopping solution, a buffer solution and an eluent.

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

7. The kit according to claim 5, 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.

8. A method for detecting RNA adenosine deaminase ADAR1 activity, characterized in that The following steps are included: (1) mixing the single-stranded RNA complementary strand described in any one of claims 1 to 3 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 3 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 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 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 3 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 3 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 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.

10. Use of the single-stranded RNA 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 preparing a product for detecting RNA adenosine deaminase ADAR1 activity or screening RNA adenosine deaminase ADAR1 inhibitors.

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