A method for detecting the enzyme activity of a DNA 5mC or RNA m6A methylase / demethylase

Abstract

The present application relates to a DNA 5mC or RNA m6A methylation enzyme / demethylase enzyme activity detection method. The method comprises: in the presence of the DNA 5mC methylation enzyme / demethylase or RNA m6A methylation enzyme / demethylase, the change of the binding state of the DNA methylation binding protein or the RNA methylation binding protein with the fluorescently labeled substrate with time is monitored by using the signal of fluorescence polarization, time-resolved fluorescence energy resonance transfer (TR-FRET) or homogeneous photoexcitation chemiluminescence, and then the enzyme activity of the DNA 5mC methylation enzyme / demethylase or the RNA m6A methylation enzyme / demethylase is detected. The method has the characteristics of high efficiency and rapidness, high sensitivity, less required sample and low cost.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a method for detecting the enzyme activity of a DNA 5mC methylase / demethylase or a RNA m6A methylase / demethylase. BACKGROUND

[0002] DNA methylation is an important epigenetic modification, which usually occurs on the 5th carbon atom of cytosine, and is referred to as 5mC modification. 5mC is an important epigenetic marker, which is involved in gene transcription regulation, genomic imprinting, X chromosome inactivation and many other biological processes. In mammalian cells, 5mC is mainly catalyzed by DNMT family proteins with S-adenosyl methionine (SAM) as a methyl donor, in which DNMT1 is a maintenance methylase, which catalyzes the formation of double-stranded fully methylated DNA from hemimethylated DNA, and DNMT3A / 3B is a de novo methylase, which is involved in the formation of the initial methylation pattern with unmethylated DNA as a substrate. 5mC is a stable chemical marker, which can be reversed to unmodified cytosine in cells through a series of enzyme reactions. This process is mainly catalyzed by TET family proteins. TET family proteins are 2-OG and Fe 2+ dependent dioxygenases, which can continuously oxidize 5-methylcytosine on DNA into 5hmC, 5fC and 5caC. The oxidation products of 5hmC, 5fC and 5caC will then form unmodified cytosine through base excision repair process.

[0003] There are various methylation modifications on RNA, among which m 6 A is the first discovered reversible methylation modification occurring on the N6 position of adenosine, which is involved in many RNA metabolic processes, such as post-transcriptional processing of RNA, degradation of RNA, and transcriptional regulation. m 6 A is mainly catalyzed by protein complex METTL3-METTL14 with SAM as a methyl donor. m 6 A can be reversed to an unmodified state, and the enzyme catalyzing this process is FTO and ALKBH5, which are also 2-OG and Fe 2+ dependent dioxygenases.

[0004] Methylation modification on macromolecular nucleic acids is an important epigenetic marker, and abnormal methylation is closely related to various diseases. Therefore, targeting DNA / RNA methylation modification is currently a research hotspot internationally. Currently reported DNA methyltransferase inhibitors are mainly nucleoside analogs. These compounds act by being incorporated into the synthesis of genomic DNA, thus exhibiting strong toxic side effects. Non-nucleoside compounds often suffer from poor activity, poor selectivity, and unclear mechanisms of action. Inhibitors of DNA demethylases, RNA methyltransferases, and RNA demethylases are rarely reported. Therefore, research on small-molecule regulators of DNA / RNA-related methylation modification enzymes is of great significance to life sciences and clinical medicine.

[0005] Currently, the methods for screening enzymes that target nucleic acid methylation modification are mainly heterogeneous and low-throughput methods: (1) Radioactive isotope method: through 3 Using H-labeled SAM as a substrate, the radioactivity of the nucleic acid substrate immobilized on a solid support after the reaction is tested to determine the enzyme's reactivity. This method is the gold standard for methyltransferase testing, with high sensitivity, accuracy, and low false positives. However, this method cannot be performed in ordinary laboratories and will cause radioactive contamination. In addition, the high cost of reagents and instruments, as well as the complex experimental operation, limit its application in high-throughput screening. (2) ELISA: ELISA is also a classic method for testing nucleic acid methylation. The nucleic acid to be detected is immobilized on a plate using a specific antibody, and then the amount of modified substrate captured by the antibody is used to determine the enzyme's reactivity. (3) High-performance liquid chromatography (HPLC): Currently, this is the classic method used to test nucleic acid demethylases. The reaction product is digested to obtain individual nucleotides, and then the content of methylated and unmethylated bases is detected by HPLC. This method has low false positives, but due to the many sample processing steps, high concentrations of substrate and enzyme are often required for the reaction, resulting in low sensitivity and high cost. At the same time, the limitations of the instruments make this method very time-consuming.

[0006] DNA methylation binding proteins (MBPs) can be divided into three families: the MBD protein family, the methylated CpG binding zinc finger protein family, and the SRA protein family. The MBD family protein members mainly include MeCP2, MBD1, MBD2, MBD3, MBD4, MBD5, MBD6, etc., which all contain a homologous MBD domain. The MBD domain tends to recognize DNA that is methylated on both strands. The methylated CpG binding zinc finger protein family contains 8 members, and its recognition of DNA depends on the zinc finger domain at the C-terminus, which can bind to methylated or non-methylated DNA. The SRA family of methylation binding proteins includes two members: UHRF1 and UHRF2. They all contain at least five different functional domains, of which the SRA domain is responsible for binding to methylated DNA. Unlike the MBD domain, the SRA domain prefers to bind to hemimethylated DNA, and has weaker binding ability to non-methylated and fully methylated DNA.

[0007] m 6 A modification is accomplished by binding to specific m 6 A recognition proteins (also known as RNA methylation binding proteins) to decode the signal and then exert and execute its biological effects. m6A recognition proteins mainly include YTH domains, and there are five YTH domain-containing proteins in mammals, which are YTHDC1, YTHDC2, YTHDF1, YTHDF2, and YTHDF3. The most common YTH recognition motif is GGACU, and it is worth noting that this sequence is compatible with the catalytic motif of METTL3-METTL14. Therefore, the m6A substrates catalyzed by METTL3-METTL14 can be selectively recognized by YTH family proteins.

[0008] Fluorescence polarization assay is a commonly used method for analyzing protein-protein and protein-nucleic acid interactions in the field of biochemistry. The principle of this method is that when a fluorescent molecule is excited by polarized light, if the molecule remains stationary during excitation, the fluorescent molecule will maintain the original polarization of the excitation light. If the molecule is in a rotating state, the polarization plane of the emitted light will be different from that of the initial excitation light, i.e., the fluorescence depolarization phenomenon. In solution, the polarization of the molecule is proportional to the molecular rotation relaxation time, and the molecular rotation relaxation time is related to the solution viscosity, absolute temperature, molecular volume, and gas constant. Therefore, when intermolecular interactions occur, the molecular volume will increase, thereby causing a change in the molecular polarization.

[0009] In fluorescence resonance energy transfer (TR-FRET), the lifetime of the fluorescence emitted by the acceptor is equivalent to the lifetime of the fluorescence emitted by the donor. Because the fluorescence decay period of Eu is long, the Eu-containing donor induces the XL665 acceptor to emit fluorescence for a long time, and the fluorescence generated after excitation of the acceptor can last for a long time, so that the short-lived self-scattered fluorescence can be distinguished by time resolution, and the FRET signal can be easily distinguished from the short-lived fluorescence background. When the two fluorescent groups are close due to biomolecular interaction, part of the energy captured by the cryptate at the time of excitation is released, with an emission wavelength of 620 nm; another part of the energy is transferred to the acceptor, with an emission wavelength of 665 nm. The 665 nm emission light is only generated by FRET caused by the donor. Therefore, when biomolecular interaction occurs, there are two excitation lights of 620 nm and 665 nm; when there is no interaction, there is only one excitation light of 620 nm. Therefore, the TR-FRET detection technology can very sensitively detect the light signal generated by molecular interaction.

[0010] The light initiated chemiluminesence assay technology uses a homogeneous system chemiluminescence detection technology, a photosensitive bead is coated on one of the antibodies participating in the immune reaction; a luminescent bead is coated on the other antibody, and contains a dimethyl thiophene derivative and a rare earth element chelate. In the presence of the target antigen, a sandwich immune complex can be formed, and the target antigen can tightly connect the photosensitive bead and the luminescent bead labeled on the two antibodies together, and under the excitation of 680 nm light, the chemiluminescence process can be completed. SUMMARY

[0011] The technical purpose of the present application is to provide a DNA 5mC methylase / demethylase and RNA m6A methylase / demethylase enzyme activity detection method, which has the characteristics of high efficiency and rapidity, high sensitivity, less required sample, and low cost.

[0012] In one aspect, the present application provides a DNA 5mC methylase / demethylase or RNA m6A methylase / demethylase enzyme activity detection method,

[0013] The method comprises: in the presence of the DNA 5mC methylase / demethylase or the RNA m6A methylase / demethylase, monitoring the binding state of the DNA methylation binding protein or the RNA methylation binding protein with the fluorescently labeled substrate over time by using the signal of fluorescence polarization, time-resolved fluorescence energy resonance transfer (TR-FRET) or homogeneous light excitation chemiluminescence, and then detecting the enzyme activity of the DNA 5mC methylase / demethylase or the RNA m6A methylase / demethylase,

[0014] wherein the substrate is a fluorescently labeled methylated modified nucleic acid substrate or a fluorescently labeled unmethylated modified nucleic acid substrate, and for the DNA 5mC methylase / demethylase, the substrate is a DNA substrate, and for the RNA m6A methylase / demethylase, the substrate is an RNA substrate,

[0015] wherein the DNA methylation binding protein is a member of the MBD family protein, for example, MBD1, preferably GST-[MBD1(1-105)]n, wherein n is an integer from 1 to 5, more preferably GST-MBD1(1-105)-MBD1(1-105)-MBD1(1-105) recombinant protein (SEQ ID No.: 9), and

[0016] the RNA methylation binding protein is a YTH domain-containing protein, preferably selected from YTHDC1, YTHDC2, YTHDF1, YTHDF2 and YTHDF3, more preferably GST-YTHDF1(361-559) protein (SEQ ID No.: 10).

[0017] In a specific embodiment, in the method for detecting the enzyme activity of the DNA 5mC methylase / demethylase, the length of the DNA substrate is 11-12 bp, and the length of the DNA sequence will affect the signal value, a longer sequence will reduce the signal value, and a shorter sequence will affect the formation of double-stranded DNA.

[0018] In a specific embodiment, in the method for detecting the enzyme activity of the RNA m6A methylase / demethylase, the sequence of the RNA substrate is a GGACU catalytic motif.

[0019] In a specific embodiment, in the method for detecting the enzyme activity of the DNA 5mC methylase / demethylase, the sequence of the DNA substrate can be a fully methylated sequence, a semi-methylated sequence, a hydroxymethylated sequence or an unmethylated sequence, depending on the function of the enzyme. In some embodiments, the sequence of the DNA substrate is as shown in the following table:

[0020]

[0021]

[0022] In the specific embodiments, in the method for detecting the enzyme activity of RNA m6A methylase / demethylase, the sequence of the RNA substrate comprises a non-modified RNA sequence and an m6A modified RNA sequence. In some embodiments, the sequence of the RNA substrate is shown in the following table:

[0023]

[0024] In the specific embodiments, the fluorescent label can include, but is not limited to, FAM, FITC, CY3, CY5, BODIPY.

[0025] In the specific embodiments, the method comprises the following steps:

[0026] 1. Determine the enzyme activity system;

[0027] 2. Dilute the DNA or RNA substrate with fluorescent label, the DNA 5mC methylase / demethylase to be detected or the RNA m6A methylase / demethylase to be detected, and the cofactor corresponding to the DNA 5mC methylase / demethylase to be detected or the RNA m6A methylase / demethylase to be detected with the reaction solution, and dilute the DNA or RNA methylation binding protein with the termination solution; the cofactor includes SAM, 2-OG.

[0028] 3. Add the DNA 5mC methylase / demethylase to be detected or the RNA m6A methylase / demethylase to be detected to the multi-well reactor, then add the mixed solution of the fluorescently labeled DNA or RNA substrate and the cofactor to start the reaction at different reaction starting times, and incubate at a certain temperature for a certain period of time;

[0029] 4. After incubation, add the diluted DNA or RNA binding protein to the multi-well reactor, finally mix on the shaker, and read the data on the multifunctional enzyme marker,

[0030] 5. Result analysis:

[0031] The FP signal value obtained by the multifunctional enzyme marker represents the amount of substrate reduction or product generation, thereby determining the enzyme activity of the DNA 5mC methylase / demethylase to be detected or the RNA m6A methylase / demethylase to be detected.

[0032] In a specific embodiment, the DNA 5mC methyltransferase is DNMT1 (SEQ ID No.:4); the DNA 5mC demethyltransferase is TET2 (SEQ ID No.:5); the RNA m6A methyltransferase is the METTL3 (SEQ ID No.:6)-METTL14 (SEQ ID No.:7) complex; and the RNA m6A demethyltransferase is ALKBH5 (SEQ ID No.:8).

[0033] In a specific embodiment, the porous reactor is a 384-well plate.

[0034] In a specific embodiment, in step 2, the reaction solution and the termination solution comprise one or more of the following: 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), citric acid / sodium citrate, 2-morpholine ethanesulfonic acid buffer (MES), and 3-morpholine propanesulfonic acid buffer (MOPS), preferably 4-hydroxyethylpiperazine ethanesulfonic acid buffer. The reaction solution and the termination solution may also comprise sodium chloride, EDTA, RNase inhibitors, DTT, ATP, vitamin C, etc.

[0035] In a specific embodiment, in step 3, the concentration of the FAM-labeled substrate is between 1 / 10 and 10 km, preferably between 1 / 2 and 2 km, the concentration of each enzyme is between 10 and 200 nM, preferably between 20 and 80 nM, and the reaction time is between 20 and 90 min, preferably 60 min.

[0036] In a specific implementation, in step 3,

[0037] When the enzyme being tested is a DNA 5mC methyltransferase, for example, when the DNA 5mC methyltransferase is DNMT1, the pH of the reaction system is between 6.5 and 10.5, preferably pH 7.4; the reaction temperature is between 20 and 50°C, preferably 37°C; and the salt concentration of the stop solution is between 300 mM and 1000 mM NaCl, preferably 800 mM NaCl.

[0038] When the enzyme being tested is a DNA 5mC demethylase, for example, when the DNA 5mC demethylase is TET2, the pH of the reaction system is between 6.5 and 10.5, preferably pH 8.0; the reaction temperature is between 20 and 50°C, preferably 37°C; the salt concentration of the reaction solution is between 0 and 300 mM NaCl, preferably 100 mM NaCl; and the salt concentration of the termination solution is between 300 and 1000 mM NaCl, preferably 500 mM NaCl.

[0039] When the enzyme being tested is an RNA m6A methyltransferase, for example, when the RNA m6A methyltransferase is METTL3 / 14, the pH of the reaction system is between 6.5 and 10.5, preferably pH 7.4; the reaction temperature is between 20 and 50°C, preferably 25°C; and the salt concentration of the stop solution is between 300 and 1000 mM NaCl, preferably 500 mM NaCl.

[0040] When the enzyme being tested is an RNA m6A demethylase, for example, ALKBH5, the pH of the reaction system is between 6.5 and 10.5, preferably pH 7.4; the reaction temperature is between 20 and 50°C, preferably 37°C; the salt concentration of the reaction solution is between 100 and 1000 mM NaCl, preferably 250 mM NaCl. The salt concentration of the stop solution is between 100 and 1000 mM NaCl, preferably 250 mM NaCl.

[0041] In a specific implementation,

[0042] When the enzyme being tested is a DNA 5mC methyltransferase, for example, when the DNA 5mC methyltransferase is DNMT1, the reaction solution is 1.20mM HEPES, pH 7.4, 2.1mM EDTA, and 3.1mM DTT; the stop solution is 20mM HEPES, pH 7.4, and 800mM NaCl.

[0043] When the enzyme being tested is a DNA 5mC demethylase, for example, when the DNA 5mC demethylase is TET2, the reaction solution is 20mM HEPES, pH 8.0, 100mM NaCl, 1mM DTT, 1mM ATP, 2mM vitamin C, and 100mM Fe. 2+ (NH4)2(SO4)2; the stop solution was 20 mM HEPES, pH 7.4, 500 mM NaCl;

[0044] When the enzyme being tested is an RNA m6A methyltransferase, for example, when the RNA m6A methyltransferase is METTL3 / 14, the reaction solution is 20 mM HEPES, pH 7.4, 1 mM DTT, and 0.4 U / μL RNase inhibitor; the stop solution is 20 mM HEPES, pH 7.4, and 500 mM NaCl.

[0045] When the enzyme being tested is an RNA m6A demethylase, for example, when the RNA m6A demethylase is ALKBH5, the reaction solution is 20 mM HEPES, pH 7.4, 250 mM NaCl, 300 μM vitamin C, and 50 μM Fe. 2+(NH4)2(SO4)2; the stop solution was 20 mM HEPES, pH 7.4, 250 mM NaCl.

[0046] On the other hand, the present invention provides a method for screening inhibitors or agonists targeting DNA 5mC methyltransferase / demethylase or RNA m6A methyltransferase / demethylase, the method comprising: screening the inhibitors or agonists by comparing the differences in enzyme activity of DNA 5mC methyltransferase / demethylase or RNA m6A methyltransferase / demethylase in the presence and absence of the inhibitor or agonist, wherein the enzyme activity of DNA 5mC methyltransferase / demethylase or RNA m6A methyltransferase / demethylase is detected according to the method described above.

[0047] In another aspect, the present invention provides a kit for detecting the activity of DNA 5mC methyltransferase / demethyltransferase or RNA m6A methyltransferase / demethyltransferase, which, depending on the enzyme to be tested, contains the reagents described above for preparing each reaction solution and stop solution, a DNA or RNA substrate, a methylation-binding protein, and a cofactor corresponding to the enzyme to be tested.

[0048] Beneficial effects

[0049] This invention establishes a method for detecting the activity of DNA 5mC methyltransferase / demethylase and RNA m6A methyltransferase / demethylase by means of fluorescence polarization analysis, fluorescence resonance energy transfer, or photo-induced chemiluminescence. This method is characterized by high efficiency, speed, high sensitivity, small sample requirement, and low cost. Attached Figure Description

[0050] Figure 1 Schematic diagram of the enzyme activity detection principle for DNA 5mC methyltransferase / demethyltransferase or RNA m6A methyltransferase / demethyltransferase.

[0051] Figure 2 The binding curves of DNA methylation binding proteins to fully methylated DNA substrates and hemimethylated DNA substrates (A) and the curves of FP signals generated by different ratios of hemimethylated substrate DNA and fully methylated product DNA (B).

[0052] Figure 3 The binding curves of DNA methylation binding proteins to fully methylated DNA substrates and hydroxymethylated DNA substrates (A) and the curves of FP signals generated by mixing fully methylated substrate DNA and hydroxymethylated product DNA in different proportions (B).

[0053] Figure 4The binding curves of RNA methylation-binding proteins to unmodified RNA substrates and m6A-modified RNA substrates (A) and the curves of FP signals generated by different ratios of unmethylated RNA and methylated RNA (B).

[0054] Figure 5 Enzyme activity reaction time-signal value curves of DNMT1(A) and TET2(B).

[0055] Figure 6 Enzyme activity reaction time-signal value curves of METTL3-METTL14(A) and ALKBH5(B).

[0056] Figure 7 S-adenosine-L-homocysteine ​​(SAH) competitive inhibition curve of DNMT1 (A) and IC50 of SAH 50 Linear relationship between vs. SAM concentrations (B).

[0057] Figure 8 : TET2's IOX1 (B) competition suppression curve (A) and IOX1's IC 50 Linear relationship between vs. 2-OG concentration (B).

[0058] Figure 9 SAH competition inhibition curves (A) of METTL3-METTL14 and IC50 of SAH 50 Linear relationship between vs. SAM concentrations (B).

[0059] Figure 10 : IOX1 competition suppression curve (A) of ALKBH5 and IC of IOX1 50 Linear relationship between vs. 2-OG concentration (B).

[0060] Figure 11 Z-factor experiments of DNMT1(A) and TET2(B).

[0061] Figure 12 Z-factor experiments of METTL3-METTL14(A) and ALKBH5(B). Detailed Implementation

[0062] The following embodiments will further illustrate the present invention, but are not intended to limit the invention.

[0063] Test Example 1: Binding curves of DNA methylation-binding proteins to hemimethylated substrate DNA and fully methylated product DNA

[0064] Intracellularly, the functional execution of methylation signaling primarily relies on the specific recognition of methylation by methylation-binding proteins (MBPs). These natural proteins, closely associated with DNMT1, can help specifically distinguish between substrates and products of DNA methylation reactions in vitro. In this test, once a methylation-binding protein specifically recognizes the modification state of the substrate or product, this change in binding state is converted into a signal output using fluorescence polarization. (In the following text, [M / M], [M / C], and [H / H] represent the modification states of the two strands of DNA, where C represents unmodified cytosine, M represents 5mC modification, and H represents 5hmC modification). Binding curves were plotted and dissociation constants were determined by titrating FAM-labeled substrate nucleic acids with different concentrations of protein. The affinity of methylation-binding proteins for different substrate and product modification states varied, thus confirming the feasibility of the experimental detection.

[0065]

[0066] (1) Material preparation:

[0067] Black 384-well plate (Corning #3575), FAM-labeled fully methylated DNA [M / M] (synthesized by Nanjing Genscript Biotech Co., Ltd.), FAM-labeled hemimethylated substrate DNA [M / C] (synthesized by Nanjing Genscript Biotech Co., Ltd.), reaction solution (20 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM DTT), DNA methylation binding protein GST-MBD1(1-105)-MBD1(1-105)-MBD1(1-105) recombinant protein (hereinafter referred to as tri-MBD1), stop solution (20 mM HEPES, pH 7.4, 800 mM NaCl), shaker (MixMate, eppendorf), multi-functional microplate reader (EnVision, PerkinElmer).

[0068] (2) Operation steps:

[0069] ① Establishment of the reaction system: The volume of the reaction system was 40 μL, and it was carried out in a black 384-well plate (Corning #3575).

[0070] ② Dilute the FAM-labeled [M / M] and [M / C] to 20 nM with the reaction solution, and dilute tri-MBD1 to 4.0 μM, 2.0 μM, 1.0 μM, 0.5 μM, 250.0 nM, 125.0 nM, 62.5 nM, and 31.3 nM with the stop solution (20 mM HEPES, pH 7.4, 800 mM NaCl).

[0071] ③ First, add 20 μL of diluted [M / M] or [M / C] solution to each well of a black 384-well plate (Corning #3575). Then, add 20 μL of Tri-MBD1 at different concentrations to each well. Mix thoroughly for 2 minutes at 1000 rpm using a shaker (MixMate, eppendorf) to ensure the reaction solution and stop solution are fully mixed. After mixing, the 384-well plate can be read using a multi-functional microplate reader (EnVision, PerkinElmer), and multiple readings can be performed.

[0072] (3) Analysis of experimental results

[0073] like Figure 2 As shown in Figure A, under a salt concentration of 400 mM, tri-MBD1 exhibits a good preference for both [M / M] and [M / C]. This is beneficial for detecting the substrate production in subsequent experiments, demonstrating the reliability of this method.

[0074] Test Example 2: Curves of FP signals generated by different ratios of hemimethylated substrate DNA and fully methylated product DNA mixed at a specific tri-MBD1 concentration.

[0075] In fluorescence polarization experiments, the signal originates from fluorescent labels added to small molecular weight probes. When these labels bind to large molecular weight proteins to form complexes, the fluorescence polarization value increases significantly, creating a signal window. Therefore, at specific concentrations of methylation-binding proteins, the reaction product [M / M] of the methylation-binding protein and the DNA methyltransferase DNMT1 forms a fluorescent probe-protein complex, generating a corresponding fluorescence polarization signal value. This binding process is a thermodynamic steady-state equilibrium; given a fixed set of parameters within the system, the final binding portion and the resulting signal value can be accurately determined. Conversely, by detecting the fluorescence polarization signal value of the system, the relative amounts of substrate and product in the system can be calculated using other known parameters, thus determining enzyme activity.

[0076] FAM-labeled [M / M] and [M / C] were mixed in ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10, respectively, while keeping the total amount of fluorescent probe constant. A standard curve of the FP signal generated by MBD1 was plotted. This curve was used to simulate the signal values ​​at different stages of the DNA methylation reaction catalyzed by DNMT1.

[0077] (1) Material preparation:

[0078] Black 384-well plate (Corning #3575), FAM-labeled semi-methylated substrate DNA (synthesized by Nanjing Genscript Biotech Co., Ltd.), FAM-labeled fully methylated product DNA (synthesized by Nanjing Genscript Biotech Co., Ltd.), cofactor S-adenosylmethionine (Sigma-Aldrich Trading Co., Ltd., catalog number: A7007), reaction solution (20mM HEPES, pH 7.4, 1mM EDTA, 1mM DTT), DNA methylation binding protein GST-MBD1(1-105)-MBD1(1-105)-MBD1(1-105) recombinant protein (hereinafter referred to as tri-MBD1), stop solution (20mM HEPES, pH 7.4, 800mM NaCl), shaker (MixMate, eppendorf), multi-functional microplate reader (EnVision, PerkinElmer).

[0079] (2) Operation steps:

[0080] ① Establishment of the reaction system: The volume of the reaction system was 40 μL, and it was carried out in a black 384-well plate (Corning #3575).

[0081] ② Dilute the FAM-labeled fully methylated product DNA and the FAM-labeled hemimethylated substrate DNA with the reaction solution, and mix them in ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 respectively (total probe concentration of 20 nM) to maintain a constant total amount of fluorescent probe. Dilute tri-MBD1 to 3 μM with stop solution (20 mM HEPES, pH 7.4, 800 mM NaCl).

[0082] ③ First, add 20 μL of the diluted [M / M] and [M / C] mixture to each well of a black 384-well plate (Corning #3575). Then, add 20 μL of Tri-MBD1 diluted with stop solution to each well. Mix thoroughly for 2 minutes at 1000 rpm using a MixMate (Eppendorf) plate reader to ensure the reaction solution and stop solution are fully mixed. After mixing, the 384-well plate can be read using a multi-functional microplate reader (EnVision, PerkinElmer), and multiple readings can be performed.

[0083] (3) Analysis of experimental results

[0084] like Figure 2 As shown in Figure B, the FP signal value and the increase of [M / M] are approximately linearly correlated, with a linear correlation coefficient R. 2 The value is 0.994. Due to the excellent linear relationship, its Hill coefficient can be considered to be 1.

[0085] Test Example 3: Binding curves of DNA methylation-binding proteins to fully methylated substrate DNA and hydroxymethylated DNA products.

[0086] (1) Material preparation:

[0087] Black 384-well plate (Corning #3575), FAM-labeled fully methylated substrate DNA [M / M] (synthesized by Nanjing Genscript Biotech Co., Ltd.), FAM-labeled hydroxymethylated product DNA [H / H] (synthesized by Nanjing Genscript Biotech Co., Ltd.), reaction solution (20 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM DTT), DNA methylation binding protein GST-MBD1(1-105)-MBD1(1-105)-MBD1(1-105) recombinant protein (hereinafter referred to as tri-MBD1), stop solution (20 mM HEPES, pH 7.4, 600 mM NaCl), shaker (MixMate, eppendorf), multi-functional microplate reader (EnVision, PerkinElmer).

[0088] (2) Operation steps:

[0089] ① Establishment of the reaction system: The volume of the reaction system was 40 μL, and it was carried out in a black 384-well plate (Corning #3575).

[0090] ② Dilute the FAM-labeled [M / M] and [H / H] to 20 nM with the reaction solution, and dilute tri-MBD1 with the stop solution (20 mM HEPES, pH 7.4, 600 mM NaCl) to 500.0 nM, 250.0 nM, 125.0 nM, 62.5 nM, 31.3 nM, 15.6 nM, 7.8 nM, and 3.9 nM.

[0091] ③ First, add 20 μL of diluted [M / M] or [H / H] solution to each well of a black 384-well plate (Corning #3575). Then, add 20 μL of Tri-MBD1 at different concentrations to each well. Mix thoroughly for 2 minutes at 1000 rpm using a shaker (MixMate, eppendorf) to ensure the reaction solution and stop solution are fully mixed. After mixing, the 384-well plate can be read using a multi-functional microplate reader (EnVision, PerkinElmer), and multiple readings can be performed.

[0092] (3) Analysis of experimental results

[0093] like Figure 3As shown in Figure A, under a salt concentration of 300 mM, tri-MBD1 exhibits a good preference for both [M / M] and [H / H]. This is beneficial for detecting the substrate production in subsequent experiments, demonstrating the reliability of this method.

[0094] Test Example 4: Curves of FP signals generated by fully methylated substrate DNA and hydroxymethylated product DNA mixed in different proportions at a specific tri-MBD1 concentration.

[0095] (1) Material preparation:

[0096] Black 384-well plate (Corning #3575), FAM-labeled [M / M] (synthesized by Nanjing Genscript Biotech Co., Ltd.), FAM-labeled [H / H] (synthesized by Nanjing Genscript Biotech Co., Ltd.), cofactor S-adenosylmethionine (Sigma-Aldrich Trading Co., Ltd., catalog number: A7007), reaction solution (20mM HEPES, pH 7.4, 1mM EDTA, 1mM DTT), DNA methylation binding protein GST-MBD1(1-105)-MBD1(1-105)-MBD1(1-105) recombinant protein (hereinafter referred to as tri-MBD1), stop solution (20mM HEPES, pH 7.4, 600mM NaCl), shaker (MixMate, eppendorf), multi-functional microplate reader (EnVision, PerkinElmer).

[0097] (2) Operation steps:

[0098] ① Establishment of the reaction system: The volume of the reaction system was 40 μL, and it was carried out in a black 384-well plate (Corning #3575).

[0099] ② Dilute the FAM-labeled [M / M] and [H / H] with the reaction solution, and mix them in ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 respectively (total probe concentration is 20 nM) to maintain a constant total amount of fluorescent probe. Dilute tri-MBD1 to 1.2 μM with stop solution (20 mM HEPES, pH 7.4, 600 mM NaCl).

[0100] ③ First, add 20 μL of the diluted [M / M] and [H / H] mixture to each well of a black 384-well plate (Corning #3575). Then, add 20 μL of Tri-MBD1 diluted with stop solution to each well. Mix thoroughly for 2 minutes at 1000 rpm using a MixMate (Eppendorf) plate reader to ensure the reaction solution and stop solution are fully mixed. After mixing, the 384-well plate can be read using a multi-functional microplate reader (EnVision, PerkinElmer), and multiple readings can be performed.

[0101] (3) Analysis of experimental results

[0102] like Figure 3 As shown in Figure B, the FP signal value and the increase of [M / M] are approximately linear, with a linear correlation coefficient R² of 0.994. Due to the excellent linear relationship, its Hill coefficient can be considered to be 1.

[0103] Test Example 5: Binding curves of RNA methylation-binding proteins with unmodified RNA and m6A-modified RNA.

[0104] (1) Material preparation:

[0105] Black 384-well plate (Corning #3575), FAM-labeled RNA containing GGACU catalytic motif (methylated) (synthesized by Nanjing GenScript Biotech Co., Ltd.), FAM-labeled RNA containing GGACU catalytic motif (unmethylated) (synthesized by Nanjing GenScript Biotech Co., Ltd.), reaction solution (20mM HEPES, pH 7.4, 1mM DTT, 0.4U / μL RNase Inhibitor), RNA methylation binding protein GST-YTHDF1 (361-559), stop solution (20mM HEPES, pH 7.4, 500mM NaCl), shaker (MixMate, eppendorf), multi-functional microplate reader (EnVision, PerkinElmer).

[0106] (2) Operation steps:

[0107] ① Establishment of the reaction system: The volume of the reaction system was 40 μL, and it was carried out in a black 384-well plate (Corning #3575).

[0108] ② Dilute FAM-labeled methylated RNA and unmethylated RNA to 50 nM with reaction solution. Dilute RNA methylation binding protein GST-YTHDF1 (361-559) to 4.0 μM, 2.0 μM, 1.0 μM, 0.5 μM, 250.0 nM, 125.0 nM, 62.5 nM, 31.3 nM, and 15.6 nM with stop solution (20 mM HEPES, pH 7.4, 500 mM NaCl).

[0109] ③ First, add 20 μL of diluted FAM-labeled methylated RNA or unmethylated RNA to each well of a black 384-well plate (Corning #3575). Then, add 20 μL of GST-YTHDF1 at different concentrations to each well. Mix thoroughly for 2 minutes at 1000 rpm using a MixMate (Eppendorf) microplate reader to ensure the reaction solution and stop solution are fully mixed. After mixing, the 384-well plate can be read using a multi-functional microplate reader (EnVision, PerkinElmer), and multiple reads can be performed.

[0110] (3) Analysis of experimental results

[0111] like Figure 4 As shown in Figure A, GST-YTHDF1 exhibits a good preference for both methylated and unmethylated RNA at a salt concentration of 250 mM. This is beneficial for detecting the substrate production in subsequent experiments, demonstrating the reliability of this method.

[0112] Test Example 6: Curves of FP signals generated by different ratios of unmethylated and methylated RNA mixed at a specific GST-YTHDF1 concentration.

[0113] (1) Material preparation:

[0114] Black 384-well plate (Corning #3575), FAM-labeled RNA containing GGACU catalytic motif (methylated) (synthesized by Nanjing GenScript Biotech Co., Ltd.), FAM-labeled RNA containing GGACU catalytic motif (unmethylated) (synthesized by Nanjing GenScript Biotech Co., Ltd.), reaction solution (20mM HEPES, pH 7.4, 1mM DTT, 0.4U / μL RNase Inhibitor), RNA methylation binding protein GST-YTHDF1 (361-559), stop solution (20mM HEPES, pH 7.4, 500mM NaCl), shaker (MixMate, eppendorf), multi-functional microplate reader (EnVision, PerkinElmer).

[0115] (2) Operation steps:

[0116] ① Establishment of the reaction system: The volume of the reaction system was 40 μL, and it was carried out in a black 384-well plate (Corning #3575).

[0117] ② Dilute FAM-labeled methylated RNA and unmethylated RNA with reaction solution, and mix them in ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 (total probe concentration of 25 nM) to maintain a constant total amount of fluorescent probe. Dilute GST-YTHDF1 to 1.5 μM with stop solution (20 mM HEPES, pH 7.4, 500 mM NaCl).

[0118] ③ First, add 20 μL of the diluted mixture of FAM-labeled methylated RNA and unmethylated RNA to each well of a black 384-well plate (Corning #3575). Then, add 20 μL of GST-YTHDF1 diluted with stop solution to each well. Mix thoroughly for 2 minutes at 1000 rpm using a MixMate (Eppendorf) plate reader to ensure the reaction solution and stop solution are fully mixed. After mixing, the 384-well plate can be read using a multi-functional microplate reader (EnVision, PerkinElmer), and multiple reads can be performed.

[0119] (3) Analysis of experimental results

[0120] like Figure 4 As shown in Figure B, the FP signal value and the increase of methylated RNA are approximately linearly correlated, with a linear correlation coefficient R. 2 The value is 0.994. Due to the excellent linearity, its Hill coefficient can be considered to be 1. The increase in methylated RNA indicates the amount of product generated by the RNA methyltransferase enzyme activity reaction, or the amount of substrate reduced by the RNA demethyltransferase enzyme activity reaction.

[0121] Verification Example 1: Enzyme reaction time-signal value curve of DNA methyltransferase DNMT1

[0122] The method was validated by detecting the enzyme reaction time-signal value curve of DNA methyltransferase DNMT1, demonstrating that it can accurately detect the state of the enzyme reaction process.

[0123] (1) Material preparation:

[0124] DNA methyltransferase DNMT1 (351-1600), black 384-well plate (Corning #3575), FAM-labeled hemimethylated substrate DNA (5'-FAM-TACGACCAGGAT-3', 3'-ATGCTGGTCCTA-5') (synthesized by Nanjing Genscript Biotech Co., Ltd.), cofactor S-adenosylmethionine (Sigma-Aldrich Trading Co., Ltd., catalog number: A7007), reaction solution (20 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM DTT), DNA methylation binding protein GST-MBD1(1-105)-MBD1(1-105)-MBD1(1-105) recombinant protein (hereinafter referred to as tri-MBD1), stop solution (20 mM HEPES, pH 7.4, 800 mM NaCl), shaker (MixMate, eppendorf), multi-functional microplate reader (EnVision, PerkinElmer).

[0125] (2) Operation steps:

[0126] ① Establishment of the enzyme activity reaction system: The volume of the enzyme activity reaction system was 20 μL, and the enzyme activity detection system was 40 μL. Both were carried out in black 384-well plates (Corning #3575).

[0127] ② Dilute DNMT1 (351-1600), FAM-labeled hemimethylated substrate DNA, and cofactor SAM with reaction buffer to achieve final concentrations of 160 nM, 80 nM, 40 nM, and 20 nM for DNMT1 (351-1600). The final concentration of FAM-labeled hemimethylated substrate DNA was 10 nM. The final concentration of cofactor SAM was 1 μM. Dilute tri-MBD1 to a final concentration of 1.5 μM using stop solution (20 mM HEPES, pH 7.4, 800 mM NaCl).

[0128] ③ First, add diluted DNMT1 (351-1600) to a black 384-well plate (Corning #3575). Then, at different reaction start times—0 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min—add a mixture of FAM-labeled hemimethylated substrate DNA and cofactor SAM to initiate the reaction. Incubate the 384-well plate at 37°C for 60 min.

[0129] ④ After incubation, add 20 μL of Tri-MBD1 diluted with stop solution to each well to terminate the reaction. Therefore, the corresponding reaction times are 60 min, 50 min, 40 min, 30 min, 20 min, 10 min, and 0 min. For samples at 0 min, adding the stop solution first, followed by the starting reaction components, avoids human error caused by delays in operation time. Finally, mix thoroughly for 2 min at 1000 rpm using a MixMate (Eppendorf) microplate reader to ensure complete mixing of the reaction solution and stop solution. After mixing, the 384-well plate can be read using a multi-functional microplate reader (EnVision, PerkinElmer), allowing for multiple reads.

[0130] (3) Analysis of experimental results

[0131] Simultaneously, reaction time-signal curves were tested at enzyme concentrations of 160 nM, 80 nM, 40 nM, and 20 nM. The FP signal value can linearly represent the decrease in substrate or the amount of product generated. For example... Figure 5 As shown in Figure A, for the time-signal curve of DNMT1, as time progresses, the absolute value of the signal difference (ΔFP, i.e., the absolute value of the signal value of the reaction group minus the signal value at time zero) continuously increases. The slope of the curve, i.e., the reaction rate, is the largest at the initial moment, and the curve becomes flatter as the reaction time increases. Since the reaction rate is closely related to the enzyme concentration, the higher the enzyme concentration, the faster the curve rises.

[0132] During the plotting of the reaction time-signal value curves, all samples had identical compositions, with the only difference being the reaction initiation time, which fully demonstrates the reliability of this method.

[0133] Verification Example 2: Enzyme reaction time-signal value curve of DNA demethylase TET2

[0134] The method was validated by detecting the enzyme reaction time-signal value curve of DNA demethylase TET2, which demonstrates that this method can accurately detect the state of the enzyme reaction process.

[0135] (1) Material preparation:

[0136] DNA demethylase TET2 catalyzes domain proteins (1129-1936Δ1481-1843), black 384-well plate (Corning #3575), FAM-labeled fully methylated substrate DNA (5'-FAM-TACGACCAGGAT-3', 3'-ATGCTGGTCCTA-5') (synthesized by Nanjing Genscript Biotech), cofactor 2-OG (Sigma-Aldrich, catalog number: 870451O), reaction solution (20mM HEPES, pH 8.0, 100mM NaCl, 1mM DTT, 1mM ATP, 2mM Vitamin C, 100mM MFe). 2+ (NH4)2(SO4)2). DNA methylation binding protein GST-MBD1(1-105)-MBD1(1-105)-MBD1(1-105) recombinant protein (hereinafter referred to as tri-MBD1), stop solution (20mM HEPES, pH 7.4, 500mM NaCl), shaker (MixMate, eppendorf), multi-functional microplate reader (EnVision, PerkinElmer).

[0137] (2) Operation steps:

[0138] ① Establishment of the enzyme activity reaction system: The volume of the enzyme activity reaction system was 20 μL, and the enzyme activity detection system was 40 μL. Both were carried out in black 384-well plates (Corning #3575).

[0139] ② Dilute the TET2 catalytic domain protein (1129-1936Δ1481-1843), FAM-labeled fully methylated substrate DNA, and cofactor 2-OG with reaction buffer to achieve final concentrations of 160 nM, 80 nM, 40 nM, and 20 nM for TET2, 10 nM for FAM-labeled fully methylated substrate DNA, and 1 mM for cofactor 2-OG. Dilute tri-MBD1 with stop solution to a final concentration of 500 nM.

[0140] ③ First, diluted TET2 catalytic domain protein (1129-1936Δ1481-1843) was added to a black 384-well plate (Corning #3575). Then, a mixture of FAM-labeled fully methylated substrate DNA and 2-OG was added at different reaction start times—0 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min—to initiate the reaction. The reaction was carried out at 37°C for 60 min.

[0141] ④ Subsequently, 20 μL of tri-MBD1 protein at a final concentration of 500 nM was added to each well to terminate the reaction. Therefore, the corresponding reaction times were 60 min, 50 min, 40 min, 30 min, 20 min, 10 min, and 0 min. For samples at 0 min, adding the termination solution first, followed by the starting reaction components, avoids human error caused by delays in operation time. Finally, the 384-well plate was mixed at 1000 rpm for 2 min using an Eppendorf plate reader. Readings were performed using a multi-functional microplate reader (EnVision, PerkinElmer), allowing for multiple readings.

[0142] (3) Analysis of experimental results

[0143] Simultaneously, reaction time-signal curves were tested at enzyme concentrations of 160 nM, 80 nM, 40 nM, and 20 nM. The FP signal value can linearly represent the decrease in substrate or the amount of product generated. For example... Figure 5 As shown in Figure B, for the time-signal curve of TET2, as time progresses, the absolute value of the signal difference (ΔFP, i.e., the absolute value of the signal value of the reaction group minus the signal value at time zero) continuously increases. The slope of the curve, i.e., the reaction rate, is the largest at the initial moment, and the curve becomes flatter as the reaction time increases. Since the reaction rate is closely related to the enzyme concentration, the higher the enzyme concentration, the faster the curve rises.

[0144] During the plotting of the reaction time-signal value curves, all samples had identical compositions, with the only difference being the reaction initiation time, which fully demonstrates the reliability of this method.

[0145] Verification Example 3: Enzyme reaction time-signal value curve of RNA methyltransferase METTL3-METTL14

[0146] The method was validated by detecting the reaction time-signal value curve of RNA methyltransferase METTL3-METTL14 to verify that it can accurately detect the state of the enzyme reaction process.

[0147] (1) Material preparation:

[0148] RNA methyltransferase METTL3(1-580)-METTL14(1-456) complex, black 384-well plate (Corning #3575), FAM-labeled substrate RNA containing GGACU catalytic motif (5'-FAM-GAACCGGACUGUCUUA-3') (synthesized by Nanjing Genscript Biotech Co., Ltd.), cofactor: S-adenosylmethionine (Sigma-Aldrich Trading Co., Ltd., catalog number: A7007), reaction solution (20mM HEPES, pH 7.4, 1mM DTT, 0.4U / μL RNase Inhibitor), RNA methylation recognition protein GST-YTHDF1 (361-559), stop solution (20mM HEPES, pH 7.4, 500mM NaCl), shaker (MixMate, eppendorf), multi-plate reader (EnVision, PerkinElmer).

[0149] (2) Operation steps:

[0150] ① Establishment of the enzyme activity reaction system: The volume of the enzyme activity reaction system was 20 μL, and the enzyme activity detection system was 40 μL. Both were carried out in black 384-well plates (Corning #3575).

[0151] ② Dilute the METTL3(1-580)-METTL14(1-456) protein complex, FAM-labeled substrate RNA containing the GGACU catalytic motif, and cofactor SAM with reaction buffer to achieve final concentrations of 80 nM, 40 nM, 20 nM, and 10 nM for METTL3(1-580)-METTL14(1-456) protein, 25 nM for the FAM-labeled substrate RNA containing the GGACU catalytic motif, and 1 μM for cofactor SAM. Dilute GST-YTHDF1(361-559) with stop solution to a final concentration of 750 nM.

[0152] ③ First, add diluted METTL3(1-580)-METTL14(1-456) protein complex to a black 384-well plate (Corning #3575). Then, at different reaction start times—0 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min—add a mixture of FAM-labeled substrate RNA containing the GGACU catalytic motif and cofactor SAM to initiate the reaction. Incubate the 384-well plate at room temperature for 60 min.

[0153] ④ Subsequently, 20 μL of GST-YTHDF1 protein at a final concentration of 750 nM was added to each well to terminate the reaction. Therefore, the corresponding reaction times were 60 min, 50 min, 40 min, 30 min, 20 min, 10 min, and 0 min. For samples at 0 min, adding the termination solution first, followed by the starting reaction components, avoids human error caused by delays in operation time. Finally, the 384-well plate was mixed at 1000 rpm for 2 min using an Eppendorf plate reader. After mixing, the plate was read using a multi-functional microplate reader (EnVision, PerkinElmer), allowing for multiple reads.

[0154] (3) Analysis of experimental results

[0155] Simultaneously, reaction time-signal curves were tested at enzyme concentrations of 80 nM, 40 nM, 20 nM, and 10 nM. The FP signal value can linearly represent the decrease in substrate or the amount of product generated. For example... Figure 6 As shown in Figure A, for the time-signal curve of METTL3-METTL14, as time progresses, the absolute value of the signal value difference (ΔFP, i.e., the absolute value of the signal value of the reaction group minus the signal value at time zero) continuously increases. The slope of the curve, i.e., the reaction rate, is the largest at the initial time, and the curve becomes flatter as the reaction time increases. Since the reaction rate is closely related to the enzyme concentration, the higher the enzyme concentration, the faster the curve rises.

[0156] During the plotting of the reaction time-signal value curves, all samples had identical compositions, differing only in the reaction initiation time. This demonstrates that the method can reliably detect m. 6 Changes of A during the enzyme reaction process.

[0157] Verification Example 4: Enzyme reaction time-signal value curve of RNA demethylase ALKBH5

[0158] The method was validated by detecting the enzyme reaction time-signal value curve of RNA demethylase ALKBH5, which demonstrates that this method can accurately detect the state of the enzyme reaction process.

[0159] (1) Material preparation:

[0160] RNA demethylase ALKBH5 (66-292), black 384-well plate (Corning #3575), FAM-labeled m6A-modified substrate RNA (5'-FAM-GAACCGGm6ACUGUCUUA-3') (synthesized by Nanjing Genscript Biotech), cofactor 2-OG (Sigma-Aldrich, catalog number: 870451O), reaction solution (20mM HEPES, pH 7.4, 250mM NaCl, 300μM Vitamin C, 50μM Fe). 2+ (NH4)2(SO4)2). RNA methylation binding protein GST-YTHDF1 (361-559), stop solution (20mM HEPES, pH 7.4, 250mM NaCl), shaker (MixMate, eppendorf), multi-functional microplate reader (EnVision, PerkinElmer).

[0161] (2) Operation steps:

[0162] ① Establishment of the enzyme activity reaction system: The volume of the enzyme activity reaction system was 20 μL, and the enzyme activity detection system was 40 μL. Both were carried out in black 384-well plates (Corning #3575).

[0163] ② Dilute RNA demethylase ALKBH5 (66-292), FAM-labeled m6A-modified substrate RNA, and cofactor 2-OG with reaction buffer to achieve final concentrations of 80 nM, 40 nM, 20 nM, and 10 nM for ALKBH5 (66-292), 25 nM for FAM-labeled m6A-modified substrate RNA, and 5 μM for cofactor 2-OG. Dilute GST-YTHDF1 (361-559) with stop buffer to a final concentration of 750 nM.

[0164] ③ First, add diluted ALKBH5 (66-292) to a black 384-well plate (Corning #3575). Then, at different reaction start times—0 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min—add a mixture of FAM-labeled m6A-modified substrate RNA and 2-OG to initiate the reaction. The reaction is carried out at 37°C for 60 min.

[0165] ④ Subsequently, 20 μL of GST-YTHDF1 protein at a final concentration of 750 nM was added to each well to terminate the reaction. Therefore, the corresponding reaction times were 60 min, 50 min, 40 min, 30 min, 20 min, 10 min, and 0 min. For samples at 0 min, adding the termination solution first, followed by the starting reaction components, avoids human error caused by delays in operation time. Finally, the 384-well plate was mixed at 1000 rpm for 2 min using an Eppendorf plate reader. Readings were performed using a multi-functional microplate reader (EnVision, PerkinElmer), allowing for multiple readings.

[0166] (3) Analysis of experimental results

[0167] Simultaneously, reaction time-signal curves were tested at enzyme concentrations of 80 nM, 40 nM, 20 nM, and 10 nM. The FP signal value can linearly represent the decrease in substrate or the amount of product generated. For example... Figure 6 As shown in Figure B, for the time-signal curve of ALKBH5, as time progresses, the absolute value of the signal difference (ΔFP, i.e., the absolute value of the signal value of the reaction group minus the signal value at time zero) continuously increases. The slope of the curve, i.e., the reaction rate, is the largest at the initial moment, and the curve becomes flatter as the reaction time increases. Since the reaction rate is closely related to the enzyme concentration, the higher the enzyme concentration, the faster the curve rises.

[0168] During the plotting of the reaction time-signal value curves, all samples had identical compositions, differing only in the reaction initiation time. This demonstrates that the method can reliably detect m. 6 Changes of A during the enzyme reaction process.

[0169] Screening and Validation Example 1: Positive Inhibitor Test for DNA Methyltransferase DNMT1

[0170] The reliability of this method was verified by testing the inhibitory activity of SAH, a natural inhibitor of DNA methyltransferase, against DNMT1.

[0171] (1) Material preparation:

[0172] DNA methyltransferase DNMT1 (351-1600), black 384-well plate (Corning #3575), FAM-labeled hemimethylated substrate DNA (synthesized by Nanjing Genscript Biotech Co., Ltd.), cofactors S-adenosylmethionine (SAM) (Sigma-Aldrich Trading Co., Ltd., catalog number: A7007), S-adenosyl-L-homocysteine ​​(SAH) (Sigma-Aldrich Trading Co., Ltd., catalog number: A9384), reaction solution (20mM HEPES, pH 7.4, 1mM EDTA, 1mM DTT), DNA methylation binding protein GST-MBD1(1-105)-MBD1(1-105)-MBD1(1-105) recombinant protein (hereinafter referred to as tri-MBD1), stop solution (20mM HEPES, pH 7.4, 800mM... NaCl), a shaking plate apparatus (MixMate, eppendorf), and a multi-functional microplate reader (EnVision, PerkinElmer).

[0173] (2) Operation steps:

[0174] ① Establishment of the enzyme activity reaction system: The volume of the enzyme activity reaction system was 20 μL, and the enzyme activity detection system was 40 μL. Both were carried out in black 384-well plates (Corning #3575).

[0175] ② Dilute DNMT1 (351-1600), FAM-labeled hemimethylated substrate DNA, and cofactor SAM with reaction solution, and dilute tri-MBD1 with stop solution (20mM HEPES, pH 7.4, 800mM NaCl).

[0176] ③ First, add 5 μL of SAH at different concentrations (final concentrations of 500.0 μM, 166.6 μM, 55.6 μM, 18.5 μM, 6.2 μM, 2.1 μM, 0.7 μM, 0.08 μM, 0.03 μM, 0.008 μM, 0.003 μM) to a black 384-well plate (Corning #3575). Then add 5 μL of DNMT1 (351-1600) (final concentration of 20 nM). After pre-incubating for 15 min, add 10 μL of a mixture of FAM-labeled hemimethylated substrate DNA (final concentration of 10 nM) and cofactor SAM (final concentrations of 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM) to start the reaction (final concentrations of enzyme and substrate DNA are 20 nM and 20 nM, respectively). The 384-well plate was then incubated at 37°C for 70 min.

[0177] ④ Add 20 μL of Tri-MBD1 diluted with stop solution to each well (final concentration 1.5 μM) to terminate the reaction. Then, mix thoroughly at 1000 rpm for 2 min using a MixMate (Eppendorf) plate shaker to ensure the reaction solution and stop solution are fully mixed. After mixing, the 384-well plate can be read using a multi-functional microplate reader (EnVision, PerkinElmer), and multiple readings can be performed.

[0178] (3) Analysis of experimental results

[0179] like Figure 7 As shown in Figure A, in the enzyme activity reaction of DNMT1, the IC50 of SAH was tested at SAM concentrations of 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, and 100 μM. 50 The concentrations were 1.100 μM, 1.560 μM, 15.19 μM, 30.16 μM, 148.8 μM, and 275.5 μM. For competitive inhibitors, according to the Cheng-Prusoff formula:

[0180]

[0181] IC 50 The half-inhibition concentration was measured experimentally.

[0182] K i The dissociation constant of the competitive inhibitor;

[0183] [S] represents the concentration of the competing substrate;

[0184] K m The Mie constant is the value of the substrate being competed for.

[0185] In the enzyme activity assay of DNMT1, the IC50 of SAH was... 50 The IC50 of SAH is linearly correlated with SAM concentration. 50 The concentration of SAM increased linearly with increasing concentration, with a linear correlation coefficient of 0.998 (see [link to relevant documentation]). Figure 7 B). Furthermore, the inhibition constant K of SAH can be calculated using the formula. i The Mie constant K of SAM is 1.549 μM. m The value was 0.5570. Furthermore, this method remains sensitive even at very low SAM concentrations (less than 1 μM), meaning that SAM-competing compounds with weak initial scaffold activity can also respond to the system. This is of great significance for screening competitive SAM inhibitors.

[0186] Screening and validation example 2: Positive inhibitor test for DNA demethylase TET2

[0187] The reliability of this method was verified by testing the activity of IOX1 (5-carboxy-8-hydroxyquinoline), a broad-spectrum inhibitor of 2-OG oxygenase, in inhibiting TET2.

[0188] (1) Material preparation:

[0189] DNA demethylase TET2 catalyzes domain proteins (1129-1936Δ1481-1843), black 384-well plate (Corning #3575), FAM-labeled fully methylated substrate DNA (synthesized by Nanjing Genscript Biotech Co., Ltd.), IOX1 (Shanghai Taosu Biotechnology Co., Ltd., catalog number: T6545), reaction solution (20mM HEPES, pH 8.0, 100mM NaCl, 1mM DTT, 1mM ATP, 2mM Vitamin C, 100mM Fe). 2+ (NH4)2(SO4)2). DNA methylation binding protein GST-MBD1(1-105)-MBD1(1-105)-MBD1(1-105) recombinant protein (hereinafter referred to as tri-MBD1), stop solution (20mM HEPES, pH 7.4, 500mM NaCl), shaker (MixMate, eppendorf), multi-functional microplate reader (EnVision, PerkinElmer).

[0190] (2) Operation steps:

[0191] ① Establishment of the enzyme activity reaction system: The volume of the enzyme activity reaction system was 20 μL, and the enzyme activity detection system was 40 μL. Both were carried out in black 384-well plates (Corning #3575).

[0192] ② Dilute the TET2 catalytic domain protein (1129-1936Δ1481-1843), FAM-labeled fully methylated substrate DNA, and cofactor 2-OG with the reaction solution, and dilute tri-MBD1 with the stop solution.

[0193] ③ First, add diluted IOX1 at different concentrations (final concentrations of 25.0 μM, 15.0 μM, 12.5 μM, 6.3 μM, 3.1 μM, 1.6 μM, 1.8 μM, 0.4 μM, 0.2 μM, 0.1 μM, and 0.05 μM) to a black 384-well plate (Corning #3575). Then add TET2 catalytic domain protein (1129-1936Δ1481-1843) (final concentration of 20 nM) and incubate at room temperature for 10–15 min. Next, add FAM-labeled fully methylated substrate DNA at a final concentration of 20 nM. Different concentrations of 2-OG (final concentrations of 20 μM, 100 μM, 200 μM, 1000 μM, and 2000 μM) are added last to initiate the reaction. The reaction is carried out at 37 °C for 60 min.

[0194] ④ Then, 20 μL of tri-MBD1 protein at a final concentration of 500 nM was added to each well to terminate the reaction. The 384-well plate was then mixed at 1000 rpm for 2 min on an Eppendorf plate shaker. The 384-well plate can then be read on a multi-functional microplate reader (EnVision, PerkinElmer), and multiple reads can be performed.

[0195] (3) Analysis of experimental results

[0196] like Figure 8 As shown in Figure A, in the enzyme activity reaction of TET2, the IC50 of 10X1 was tested at 2-OG concentrations of 20 μM, 100 μM, 200 μM, 1000 μM, and 2000 μM. 50 The concentrations were 2.805 μM, 4.373 μM, 4.916 μM, 4.513 μM, and 4.481 μM, respectively. It is noteworthy that the IC50 values ​​of IOX1 at different concentrations of 2-OG were... 50 It did not show a linear increase like SAH (see Figure 8 B). This may be because although IOX1 is a substrate-competitive inhibitor, it forms stable coordination bonds with metal ions in the protein. Therefore, this binding has properties similar to covalent bonds, making it difficult to generate substrate competition. In this case, the Cheng-Prusoff formula can no longer accurately describe it.

[0197] Screening and validation example 3: Positive inhibitor test for RNA methyltransferase METTL3-METTL14

[0198] The reliability of this method was verified by testing the inhibitory activity of SAH, a natural inhibitor of RNA methyltransferase, against METTL3-METTL14.

[0199] (1) Material preparation:

[0200] RNA methyltransferase METTL3(1-580)-METTL14(1-456) complex, black 384-well plate (Corning #3575), FAM-labeled substrate RNA containing GGACU catalytic motif (5'-FAM-GAACCGGACUGUCUUA-3') (synthesized by Nanjing Genscript Biotech Co., Ltd.), cofactors: S-adenosylmethionine (Sigma-Aldrich Trading Co., Ltd., catalog number: A7007), S-adenosyl-L-homocysteine ​​SAH (Sigma-Aldrich Trading Co., Ltd., catalog number: A9384), reaction solution (20mM HEPES, pH 7.4, 1mM DTT, 0.4U / μL RNase Inhibitor), RNA methylation binding protein GST-YTHDF1 (361-559), stop solution (20mM HEPES, pH 7.4, 500mM... NaCl), a shaking plate apparatus (MixMate, eppendorf), and a multi-functional microplate reader (EnVision, PerkinElmer).

[0201] (2) Operation steps:

[0202] ① Establishment of the enzyme activity reaction system: The volume of the enzyme activity reaction system was 20 μL, and the enzyme activity detection system was 40 μL. Both were carried out in black 384-well plates (Corning #3575).

[0203] ② Dilute the METTL3(1-580)-METTL14(1-456) protein complex, FAM-labeled substrate RNA containing GGACU catalytic motif, and cofactor SAM with reaction solution, and dilute GST-YTHDF1(361-559) with stop solution.

[0204] ③ First, add different concentrations of SAH (final concentrations of 500.0 μM, 166.6 μM, 55.6 μM, 18.5 μM, 6.2 μM, 2.1 μM, 0.7 μM, 0.08 μM, 0.03 μM, 0.008 μM, 0.003 μM) to a black 384-well plate (Corning #3575). Then add a diluted METTL3(1-580)-METTL14(1-456) protein complex and incubate at room temperature for 10-15 min. Then add a mixed solution of FAM-labeled substrate RNA containing GGACU catalytic motif and cofactor SAM to start the reaction (in the reaction system, the final concentrations of enzyme and substrate RNA are 20 nM and 40 nM, respectively, and the final concentrations of cofactor SAM are 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM). The 384-well plate was incubated at room temperature for 60 minutes.

[0205] ④ Subsequently, 20 μL of GST-YTHDF1 protein at a final concentration of 750 nM was added to each well to terminate the reaction. The 384-well plate was mixed at 1000 rpm for 2 min using an Eppendorf plate shaker. After mixing, the plate was read using a multi-functional microplate reader (EnVision, PerkinElmer), and multiple readings could be performed.

[0206] (3) Analysis of experimental results

[0207] like Figure 9 As shown in Figure A, the inhibitory activity of SAH was tested in the METTL3-METTL14 enzyme activity reaction at SAM concentrations of 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, and 100 μM. Its IC50... 50 The concentrations were 1.974 μM, 2.775 μM, 13.38 μM, 36.10 μM, 163.8 μM, and 304.9 μM, respectively. According to the Cheng-Prusoff formula...

[0208]

[0209] IC 50 The half-inhibition concentration was measured experimentally.

[0210] K i The dissociation constant of the competitive inhibitor;

[0211] [S] represents the concentration of the competing substrate;

[0212] K m The Mie constant is the value of the substrate being competed for.

[0213] SAH's IC 50 It has a good linear relationship with SAM concentration, and its linear correlation coefficient R 2 It is 0.998 (see Figure 9 B) Based on the formula, the inhibition constant Ki of SAH against METTL3-METTL14 can be further calculated to be 1.885 μM, while the Michaelis constant Km of SAM is 0.6134 μM. Furthermore, this method can be performed under low concentrations of cofactors, which is highly advantageous for screening competitive inhibitors of cofactors.

[0214] Screening and validation example 4: Positive inhibitor test for RNA demethylase ALKBH5

[0215] The reliability of this method was verified by testing the inhibitory activity of IOX1, a broad-spectrum inhibitor of 2-OG oxygenase, against ALKBH5.

[0216] (1) Material preparation:

[0217] RNA demethylase ALKBH5 (66-292), black 384-well plate (Corning #3575), FAM-labeled m6A-modified substrate RNA (5'-FAM-GAACCGGm6ACUGUCUUA-3') (synthesized by Nanjing Genscript Biotech Co., Ltd.), IOX1 (Shanghai Taosu Biotechnology Co., Ltd., catalog number: T6545), reaction solution (20mM HEPES, pH 7.4, 250mM NaCl, 300μM Vitamin C, 50μM Fe). 2+ (NH4)2(SO4)2). RNA methylation binding protein GST-YTHDF1 (361-559), stop solution (20mM HEPES, pH 7.4, 250mM NaCl), shaker (MixMate, eppendorf), multi-functional microplate reader (EnVision, PerkinElmer).

[0218] (2) Operation steps:

[0219] ① Establishment of the enzyme activity reaction system: The volume of the enzyme activity reaction system was 20 μL, and the enzyme activity detection system was 40 μL. Both were carried out in black 384-well plates (Corning #3575).

[0220] ② Dilute the RNA demethylase ALKBH5 (66-292) and FAM-labeled substrate RNA containing m6A modification with the reaction solution, and dilute GST-YTHDF1 (361-559) with the stop solution.

[0221] ③ First, add diluted IOX1 at different concentrations (final concentrations: 25.0 μM, 15.0 μM, 12.5 μM, 6.3 μM, 3.1 μM, 1.6 μM, 1.8 μM, 0.4 μM, 0.2 μM, 0.1 μM, 0.05 μM) to a black 384-well plate (Corning #3575). Then add ALKBH5 (66-292), followed by FAM-labeled m6A-modified substrate RNA. Finally, add different concentrations of cofactor 2-OG to initiate the reaction (in the reaction system, the final concentration of ALKBH5 is 20 nM, the final concentration of FAM-labeled m6A-modified substrate RNA is 40 nM, and the final concentrations of 2-OG are 2 μM, 5 μM, 20 μM, 50 μM, and 200 μM, respectively). The reaction is carried out at 37°C for 60 min.

[0222] ④ Subsequently, 20 μL of GST-YTHDF1 protein at a final concentration of 750 nM was added to each well to terminate the reaction. The 384-well plate was mixed at 1000 rpm for 2 min using an Eppendorf plate shaker. Readings were performed using a multi-functional microplate reader (EnVision, PerkinElmer), allowing for multiple readings.

[0223] (3) Analysis of experimental results

[0224] like Figure 10 As shown in Figure A, at 2-OG concentrations of 2 μM, 5 μM, 20 μM, 50 μM, and 200 μM, the IC50 of IOX1 on ALKB5 was... 50 The concentrations were 1.941 μM, 2.361 μM, 4.364 μM, 5.045 μM, and 5.564 μM, respectively. Similar to TET2, IOX1 showed different IC50 values ​​for ALKBH5 at different 2-OG concentrations. 50 It did not increase linearly, but rather increased slightly at lower concentrations, and remained essentially stable at higher concentrations (see [reference]). Figure 10 B). The possible reason is similar to that of TET2: IOX1 can competitively occupy the 2-OG binding site, while simultaneously binding with Fe in ALKBH5. 2+ It forms two mutually perpendicular coordinate bonds, thus exhibiting certain covalent bond properties and therefore does not show a competitive relationship similar to SAH.

[0225] General Screening Example 1: High-throughput screening targeting DNA methyltransferases / demethyltransferases or RNA methyltransferases / demethyltransferases

[0226] Z-factor is a key parameter for evaluating the reliability of a testing method, and it is usually determined statistically. The performance of this method in high-throughput screening of DNMT1, TET2, METTL3-METTL 14, and ALKBH5 enzymes was evaluated using Z-factor detection.

[0227] (1) Operation steps:

[0228] ① Establishment of enzyme activity reaction system: The volume of enzyme activity reaction system was 20 μL, and the volume of enzyme activity detection system was 40 μL. Both were carried out in black 384-well plates (Corning #3575).

[0229] ② Dilute enzymes DNMT1, TET2, METTL3-METTL 14, ALKBH5, FAM-labeled substrate, SAH, cofactor SAM, and 2-OG with reaction buffer. Dilute DNA methylation-binding protein tri-MBD1 and RNA methylation-binding protein GST-YTHDF1 (361-559) with stop buffer. Test the signal values ​​of DNMT1, TET2, METTL3-METTL 14, and ALKBH5 in parallel with 50 positive controls and 50 negative controls, respectively. The positive control system for the DNMT1 enzyme activity assay included 10 nM hemimethylated DNA + 1 μM SAM + 20 nM DNMT1 + 1.5 μM tri-MBD1, while the negative control system included 10 μM SAH + 20 nM DNMT1 + 1.5 μM tri-MBD1. The positive control system for the TET2 enzyme activity assay included 10 nM fully methylated DNA + 1 mM 2-OG + 500 nM tri-MBD1, while the negative control system included 20 nM TET2 + 10 nM fully methylated DNA + 1 mM 2-OG + 500 nM tri-MBD1. The positive control system for the METTL3-METTL14 enzyme activity assay included 25 nM unmethylated RNA + 1 μM SAM + 20 nM METTL3-METTL14 + 750 nM GST-YTHDF1, while the negative control system included 10 μM SAH + 25 nM unmethylated RNA + 20 nM tri-MBD1. The positive control for the ALKBH5 enzyme activity assay included 25 nM methylated RNA + 200 μM 2-OG + 750 nM GST-YTHDF1, and the negative control included 20 nM ALKBH5 + 25 nM methylated RNA + 200 μM 2-OG + 750 nM GST-YTHDF1.

[0230] First, add the diluted enzymes to each well in a black 384-well plate (Corning #3575), then add the corresponding FAM-labeled substrate and cofactor mixture to initiate the reaction. The reaction is carried out at 37°C for 60 min.

[0231] Subsequently, 20 μL of substrate-binding protein diluted with stop solution was added to each well to terminate the reaction. The 384-well plate was mixed at 1000 rpm for 2 min on an Eppendorf plate shaker. Readings were performed using a multi-functional microplate reader (EnVision, PerkinElmer), allowing for multiple readings.

[0232] (2) Analysis of experimental results

[0233] The formula for calculating Z-factor is:

[0234]

[0235] σ P The standard deviation of the positive control;

[0236] μ P This represents the average value of the positive control group;

[0237] σ N The standard deviation of the negative control;

[0238] μ N This represents the average value of the negative control group;

[0239] A larger Z-factor value indicates less variation among parallel test samples and a more stable method. Figures 11-12 As shown, in DNMT1 ( Figure 11 A) TET2 Figure 11 B), METTL3-METTL14 ( Figure 12 A), ALKBH5 Figure 12 In the enzyme activity assays (B), the Z-factors for DNMT1, TET2, METTL3-METTL14, and ALKBH5 were 0.81, all significantly higher than the lower limit of 0.5 and exceeding 0.8. This indicates that this method is highly robust in the enzyme activity assays of DNMT1, TET2, METTL3-METTL14, and ALKBH5, and is suitable for high-throughput screening experiments. sequence list <110> Shanghai Institute of Materia Medica, Chinese Academy of Sciences <120> A method for detecting the activity of DNA 5mC or RNA m6A methyltransferase / demethyltransferase <130> DI21-0551-XC03 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 12 <212> DNA <213> Artificial sequence <220> <223> DNA substrate <400> 1 tacgaccagg at 12 <210> 2 <211> 12 <212> DNA <213> Artificial sequence <220> <223> DNA substrate <400> 2 atgctggtcc ta 12 <210> 3 <211> 16 <212> RNA <213> Artificial sequence <220> <223> RNA substrate <400> 3 gaaccggacu gucuua 16 <210> 4 <211> 1250 <212> PRT <213> Artificial sequence <220> <223> DNMT1(351-1600) <400> 4 Pro Lys Cys Ile Gln Cys Gly Gln Tyr Leu Asp Asp Pro Asp Leu Lys 1 5 10 15 Tyr Gly Gln His Pro Pro Asp Ala Val Asp Glu Pro Gln Met Leu Thr 20 25 30 Asn Glu Lys Leu Ser Ile Phe Asp Ala Asn Glu Ser Gly Phe Glu Ser 35 40 45 Tyr Glu Ala Leu Pro Gln His Lys Leu Thr Cys Phe Ser Val Tyr Cys 50 55 60 Lys His Gly His Leu Cys Pro Ile Asp Thr Gly Leu Ile Glu Lys Asn 65 70 75 80 Ile Glu Leu Phe Phe Ser Gly Ser Ala Lys Pro Ile Tyr Asp Asp Asp 85 90 95 Pro Ser Leu Glu Gly Gly Val Asn Gly Lys Asn Leu Gly Pro Ile Asn 100 105 110 Glu Trp Trp Ile Thr Gly Phe Asp Gly Gly Glu Lys Ala Leu Ile Gly 115 120 125 Phe Ser Thr Ser Phe Ala Glu Tyr Ile Leu Met Asp Pro Ser Pro Glu 130 135 140 Tyr Ala Pro Ile Phe Gly Leu Met Gln Glu Lys Ile Tyr Ile Ser Lys 145 150 155 160 Ile Val Val Glu Phe Leu Gln Ser Asn Ser Asp Ser Thr Tyr Glu Asp 165 170 175 Leu Ile Asn Lys Ile Glu Thr Thr Val Pro Pro Ser Gly Leu Asn Leu 180 185 190 Asn Arg Phe Thr Glu Asp Ser Leu Leu Arg His Ala Gln Phe Val Val 195 200 205 Glu Gln Val Glu Ser Tyr Asp Glu Ala Gly Asp Ser Asp Glu Gln Pro 210 215 220 Ile Phe Leu Thr Pro Cys Met Arg Asp Leu Ile Lys Leu Ala Gly Val 225 230 235 240 Thr Leu Gly Gln Arg Arg Ala Gln Ala Arg Arg Gln Thr Ile Arg His 245 250 255 Ser Thr Arg Glu Lys Asp Arg Gly Pro Thr Lys Ala Thr Thr Thr Lys 260 265 270 Leu Val Tyr Gln Ile Phe Asp Thr Phe Phe Ala Glu Gln Ile Glu Lys 275 280 285 Asp Asp Arg Glu Asp Lys Glu Asn Ala Phe Lys Arg Arg Arg Cys Gly 290 295 300 Val Cys Glu Val Cys Gln Gln Pro Glu Cys Gly Lys Cys Lys Ala Cys 305 310 315 320 Lys Asp Met Val Lys Phe Gly Gly Ser Gly Arg Ser Lys Gln Ala Cys 325 330 335 Gln Glu Arg Arg Cys Pro Asn Met Ala Met Lys Glu Ala Asp Asp Asp 340 345 350 Glu Glu Val Asp Asp Asn Ile Pro Glu Met Pro Ser Pro Lys Lys Met 355 360 365 His Gln Gly Lys Lys Lys Lys Gln Asn Lys Asn Arg Ile Ser Trp Val 370 375 380 Gly Glu Ala Val Lys Thr Asp Gly Lys Lys Ser Tyr Tyr Lys Lys Val 385 390 395 400 Cys Ile Asp Ala Glu Thr Leu Glu Val Gly Asp Cys Val Ser Val Ile 405 410 415 Pro Asp Asp Ser Ser Lys Pro Leu Tyr Leu Ala Arg Val Thr Ala Leu 420 425 430 Trp Glu Asp Ser Ser Asn Gly Gln Met Phe His Ala His Trp Phe Cys 435 440 445 Ala Gly Thr Asp Thr Val Leu Gly Ala Thr Ser Asp Pro Leu Glu Leu 450 455 460 Phe Leu Val Asp Glu Cys Glu Asp Met Gln Leu Ser Tyr Ile His Ser 465 470 475 480 Lys Val Lys Val Ile Tyr Lys Ala Pro Ser Glu Asn Trp Ala Met Glu 485 490 495 Gly Gly Met Asp Pro Glu Ser Leu Leu Glu Gly Asp Asp Gly Lys Thr 500 505 510 Tyr Phe Tyr Gln Leu Trp Tyr Asp Gln Asp Tyr Ala Arg Phe Glu Ser 515 520 525 Pro Pro Lys Thr Gln Pro Thr Glu Asp Asn Lys Phe Lys Phe Cys Val 530 535 540 Ser Cys Ala Arg Leu Ala Glu Met Arg Gln Lys Glu Ile Pro Arg Val 545 550 555 560 Leu Glu Gln Leu Glu Asp Leu Asp Ser Arg Val Leu Tyr Tyr Ser Ala 565 570 575 Thr Lys Asn Gly Ile Leu Tyr Arg Val Gly Asp Gly Val Tyr Leu Pro 580 585 590 Pro Glu Ala Phe Thr Phe Asn Ile Lys Leu Ser Ser Pro Val Lys Arg 595 600 605 Pro Arg Lys Glu Pro Val Asp Glu Asp Leu Tyr Pro Glu His Tyr Arg 610 615 620 Lys Tyr Ser Asp Tyr Ile Lys Gly Ser Asn Leu Asp Ala Pro Glu Pro 625 630 635 640 Tyr Arg Ile Gly Arg Ile Lys Glu Ile Phe Cys Pro Lys Lys Ser Asn 645 650 655 Gly Arg Pro Asn Glu Thr Asp Ile Lys Ile Arg Val Asn Lys Phe Tyr 660 665 670 Arg Pro Glu Asn Thr His Lys Ser Thr Pro Ala Ser Tyr His Ala Asp 675 680 685 Ile Asn Leu Leu Tyr Trp Ser Asp Glu Glu Ala Val Val Asp Phe Lys 690 695 700 Ala Val Gln Gly Arg Cys Thr Val Glu Tyr Gly Glu Asp Leu Pro Glu 705 710 715 720 Cys Val Gln Val Tyr Ser Met Gly Gly Pro Asn Arg Phe Tyr Phe Leu 725 730 735 Glu Ala Tyr Asn Ala Lys Ser Lys Ser Phe Glu Asp Pro Pro Asn His 740 745 750 Ala Arg Ser Pro Gly Asn Lys Gly Lys Gly Lys Gly Lys Gly Lys Gly 755 760 765 Lys Pro Lys Ser Gln Ala Cys Glu Pro Ser Glu Pro Glu Ile Glu Ile 770 775 780 Lys Leu Pro Lys Leu Arg Thr Leu Asp Val Phe Ser Gly Cys Gly Gly 785 790 795 800 Leu Ser Glu Gly Phe His Gln Ala Gly Ile Ser Asp Thr Leu Trp Ala 805 810 815 Ile Glu Met Trp Asp Pro Ala Ala Gln Ala Phe Arg Leu Asn Asn Pro 820 825 830 Gly Ser Thr Val Phe Thr Glu Asp Cys Asn Ile Leu Leu Lys Leu Val 835 840 845 Met Ala Gly Glu Thr Thr Asn Ser Arg Gly Gln Arg Leu Pro Gln Lys 850 855 860 Gly Asp Val Glu Met Leu Cys Gly Gly Pro Pro Cys Gln Gly Phe Ser 865 870 875 880 Gly Met Asn Arg Phe Asn Ser Arg Thr Tyr Ser Lys Phe Lys Asn Ser 885,890,895 Leu Val Val Ser Phe Leu Ser Tyr Cys Asp Tyr Tyr Arg Pro Arg Phe 900 905 910 Phe Leu Leu Glu Asn Val Arg Asn Phe Val Ser Phe Lys Arg Ser Met 915,920,925 Val Leu Lys Leu Thr Leu Arg Cys Leu Val Leu Met Gly Tyr Gln Cys 930,935,940 Thr Phe Gly Val Leu Gln Ala Gly Gln Tyr Gly Val Ala Gln Thr Arg 945 950 955 960 Arg Arg Wing Leu Wing Wing Wing Pro Gly Glu Lys Leu Pro Leu 965,970,975 Phe Pro Glu Pro Leu His Val Phe Ala Pro Arg Ala Cys Gln Leu Ser 980,985,990 Val Val Val Asp Asp Lys Lys Phe Val Ser Asn Ile Thr Arg Leu Ser 995 1000 1005 Ser Gly Pro Phe Arg Thr Ile Thr Val Arg Asp Thr Met Ser Asp 1010 1015 1020 Leu Pro Glu Val Arg Asn Gly Ala Ser Ala Leu Glu Ile Ser Tyr 1025 1030 1035 Asn Gly Glu Pro Gln Ser Trp Phe Gln Arg Gln Leu Arg Gly Ala 1040 1045 1050 Gln Tyr Gln Pro Ile Leu Arg Asp His Ile Cys Lys Asp Met Ser 1055 1060 1065 Ala Leu Val Ala Ala Arg Met Arg His Ile Pro Leu Ala Pro Gly 1070 1075 1080 Ser Asp Trp Arg Asp Leu Pro Asn Ile Glu Val Arg Leu Ser Asp 1085 1090 1095 Gly Thr Met Ala Arg Lys Leu Arg Tyr Thr His His Asp Arg Lys 1100 1105 1110 Asn Gly Arg Ser Ser Ser Gly Ala Leu Arg Gly Val Cys Ser Cys 1115 1120 1125 Val Glu Ala Gly Lys Ala Cys Asp Pro Ala Ala Arg Gln Phe Asn 1130 1135 1140 Thr Leu Ile Pro Trp Cys Leu Pro His Thr Gly Asn Arg His Asn 1145 1150 1155 His Trp Ala Gly Leu Tyr Gly Arg Leu Glu Trp Asp Gly Phe Phe 1160 1165 1170 Ser Thr Thr Val Thr Asn Pro Glu Pro Met Gly Lys Gln Gly Arg 1175 1180 1185 Val Leu His Pro Glu Gln His Arg Val Val Ser Val Arg Glu Cys 1190 1195 1200 Ala Arg Ser Gln Gly Phe Pro Asp Thr Tyr Arg Leu Phe Gly Asn 1205 1210 1215 Ile Leu Asp Lys His Arg Gln Val Gly Asn Ala Val Pro Pro Pro 1220 1225 1230 Leu Ala Lys Ala Ile Gly Leu Glu Ile Lys Leu Cys Met Leu Ala 1235 1240 1245 Lys Ala 1250 <210> 5 <211> 509 <212> PRT <213> Artificial Sequence <220> <223> TET2 (1129-1936 Δ 1481-1843) <400> 5 Asp Phe Pro Ser Cys Arg Cys Val Glu Gln Ile Ile Glu Lys Asp Glu 1 5 10 15 Gly Pro Phe Tyr Thr His Leu Gly Ala Gly Pro Asn Val Ala Ala Ile 20 25 30 Arg Glu Ile Met Glu Glu Arg Phe Gly Gln Lys Gly Lys Ala Ile Arg 35 40 45 Ile Glu Arg Val Ile Tyr Thr Gly Lys Glu Gly Lys Ser Ser Gln Gly 50 55 60 Cys Pro Ile Ala Lys Trp Val Val Arg Arg Ser Ser Ser Glu Glu Lys 65 70 75 80 Leu Leu Cys Leu Val Arg Glu Arg Ala Gly His Thr Cys Glu Ala Ala 85 90 95 Val Ile Val Ile Leu Ile Leu Val Trp Glu Gly Ile Pro Leu Ser Leu 100 105 110 Ala Asp Lys Leu Tyr Ser Glu Leu Thr Glu Thr Leu Arg Lys Tyr Gly 115 120 125 Thr Leu Thr Asn Arg Arg Cys Ala Leu Asn Glu Glu Arg Thr Cys Ala 130 135 140 Cys Gln Gly Leu Asp Pro Glu Thr Cys Gly Ala Ser Phe Ser Phe Gly 145 150 155 160 Cys Ser Trp Ser Met Tyr Tyr Asn Gly Cys Lys Phe Ala Arg Ser Lys 165 170 175 Ile Pro Arg Lys Phe Lys Leu Leu Gly Asp Asp Pro Lys Glu Glu Glu 180 185 190 Lys Leu Glu Ser His Leu Gln Asn Leu Ser Thr Leu Met Ala Pro Thr 195 200 205 Tyr Lys Lys Leu Ala Pro Asp Ala Tyr Asn Asn Gln Ile Glu Tyr Glu 210 215 220 His Arg Ala Pro Glu Cys Arg Leu Gly Leu Lys Glu Gly Arg Pro Phe 225 230 235 240 Ser Gly Val Thr Ala Cys Leu Asp Phe Cys Ala His Ala His Arg Asp 245 250 255 Leu His Asn Met Gln Asn Gly Ser Thr Leu Val Cys Thr Leu Thr Arg 260 265 270 Glu Asp Asn Arg Glu Phe Gly Gly Lys Pro Glu Asp Glu Gln Leu His 275 280 285 Val Leu Pro Leu Tyr Lys Val Ser Asp Val Asp Glu Phe Gly Ser Val 290 295 300 Glu Ala Gln Glu Glu Lys Lys Arg Ser Gly Ala Ile Gln Val Leu Ser 305 310 315 320 Ser Phe Arg Arg Lys Val Arg Met Leu Ala Glu Pro Val Lys Thr Cys 325 330 335 Arg Gln Arg Lys Leu Glu Ala Lys Lys Ala Ala Ala Glu Lys Leu Ser 340 345 350 Asp Glu Val Trp Ser Asp Ser Glu Gln Ser Phe Leu Asp Pro Asp Ile 355 360 365 Gly Gly Val Ala Val Ala Pro Thr His Gly Ser Ile Leu Ile Glu Cys 370 375 380 Ala Lys Arg Glu Leu His Ala Thr Thr Pro Leu Lys Asn Pro Asn Arg 385 390 395 400 Asn His Pro Thr Arg Ile Ser Leu Val Phe Tyr Gln His Lys Ser Met 405 410 415 Asn Glu Pro Lys His Gly Leu Ala Leu Trp Glu Ala Lys Met Ala Glu 420 425 430 Lys Ala Arg Glu Lys Glu Glu Glu Cys Glu Lys Tyr Gly Pro Asp Tyr 435 440 445 Val Pro Gln Lys Ser His Gly Lys Lys Val Lys Arg Glu Pro Ala Glu[[ID=!24]] ! 450 455 460 Pro His Glu Thr Ser Glu Pro Thr Tyr Leu Arg Phe Ile Lys Ser Leu 465 470 475 480 Ala Glu Arg Thr Met Ser Val Thr Thr Asp Ser Thr Val Thr Thr Ser 485 490 495<00008!36>! [[ID=3!5]]Pro Tyr Ala Phe Thr Arg Val Thr Gly Pro Tyr Asn Arg ! 500 50! ! <210> 6 <211> 580 <212> PRT <!213> 人工序列! It seems there are some incorrect tags in the original text like <!213> which should be <213> and <00008!36> which should be etc. The above translation is based on the corrected understanding of the tags. If the tags are indeed meant to be incorrect as presented, please clarify. <220> <223> METTL3(1‑580) <400> 6 Met Ser Asp Thr Trp Ser Ser Ile Gln Ala His Lys Lys Gln Leu Asp 1 5 10 15 Ser Leu Arg Glu Arg Leu Gln Arg Arg Arg Lys Gln Asp Ser Gly His 20 25 30 Leu Asp Leu Arg Asn Pro Glu Ala Ala Leu Ser Pro Thr Phe Arg Ser 35 40 45 Asp Ser Pro Val Pro Thr Ala Pro Thr Ser Gly Gly Pro Lys Pro Ser 50 55 60 Thr Ala Ser Ala Val Pro Glu Leu Ala Thr Asp Pro Glu Leu Glu Lys 65 70 75 80 Lys Leu Leu His His Leu Ser Asp Leu Ala Leu Thr Leu Pro Thr Asp 85 90 95 Ala Val Ser Ile Cys Leu Ala Ile Ser Thr Pro Asp Ala Pro Ala Thr 100 105 110 Gln Asp Gly Val Glu Ser Leu Leu Gln Lys Phe Ala Ala Gln Glu Leu 115 120 125 Ile Glu Val Lys Arg Gly Leu Leu Gln Asp Asp Ala His Pro Thr Leu 130 135 140 Val Thr Tyr Ala Asp His Ser Lys Leu Ser Ala Met Met Gly Ala Val 145 150 155 160 Ala Glu Lys Lys Gly Pro Gly Glu Val Ala Gly Thr Val Thr Gly Gln 165 170 175 Lys Arg Arg Ala Glu Gln Asp Ser Thr Thr Val Ala Ala Phe Ala Ser 180 185 190 Ser Leu Val Ser Gly Leu Asn Ser Ser Ala Ser Glu Pro Ala Lys Glu 195 200 205 Pro Ala Lys Lys Ser Arg Lys His Ala Ala Ser Asp Val Asp Leu Glu 210 215 220 Ile Glu Ser Leu Leu Asn Gln Gln Ser Thr Lys Glu Gln Gln Ser Lys 225 230 235 240 Lys Val Ser Gln Glu Ile Leu Glu Leu Leu Asn Thr Thr Thr Ala Lys 245 250 255 Glu Gln Ser Ile Val Glu Lys Phe Arg Ser Arg Gly Arg Ala Gln Val 260 265 270 Gln Glu Phe Cys Asp Tyr Gly Thr Lys Glu Glu Cys Met Lys Ala Ser 275 280 285 Asp Ala Asp Arg Pro Cys Arg Lys Leu His Phe Arg Arg Ile Ile Asn 290 295 300 Lys His Thr Asp Glu Ser Leu Gly Asp Cys Ser Phe Leu Asn Thr Cys 305 310 315 320 Phe His Met Asp Thr Cys Lys Tyr Val His Tyr Glu Ile Asp Ala Cys 325 330 335 Met Asp Ser Glu Ala Pro Gly Ser Lys Asp His Thr Pro Ser Gln Glu 340 345 350 Leu Ala Leu Thr Gln Ser Val Gly Gly Asp Ser Ser Ala Asp Arg Leu 355 360 365 Phe Pro Pro Gln Trp Ile Cys Cys Asp Ile Arg Tyr Leu Asp Val Ser 370 375 380 Ile Leu Gly Lys Phe Ala Val Val Met Ala Asp Pro Pro Trp Asp Ile 385 390 395 400 His Met Glu Leu Pro Tyr Gly Thr Leu Thr Asp Asp Glu Met Arg Arg 405 410 415 Leu Asn Ile Pro Val Leu Gln Asp Asp Gly Phe Leu Phe Leu Trp Val 420 425 430 Thr Gly Arg Ala Met Glu Leu Gly Arg Glu Cys Leu Asn Leu Trp Gly 435 440 445 Tyr Glu Arg Val Asp Glu Ile Ile Trp Val Lys Thr Asn Gln Leu Gln 450 455 460 Arg Ile Ile Arg Thr Gly Arg Thr Gly His Trp Leu Asn His Gly Lys 465 470 475 480 Glu His Cys Leu Val Gly Val Lys Gly Asn Pro Gln Gly Phe Asn Gln 485 490 495 Gly Leu Asp Cys Asp Val Ile Val Ala Glu Val Arg Ser Thr Ser His 500 505 510 Lys Pro Asp Glu Ile Tyr Gly Met Ile Glu Arg Leu Ser Pro Gly Thr 515 520 525 Arg Lys Ile Glu Leu Phe Gly Arg Pro His Asn Val Gln Pro Asn Trp 530 535 540 Ile Thr Leu Gly Asn Gln Leu Asp Gly Ile His Leu Leu Asp Pro Asp 545 550 555 560 Val Val Ala Arg Phe Lys Gln Arg Tyr Pro Asp Gly Ile Ile Ser Lys 565 570 575 Pro Lys Asn Leu 580 <210> 7 <211> 456 <212> PRT <213> Artificial Sequence <220> <223> METTL14 (1-456) <400> 7 Met Asp Ser Arg Leu Gln Glu Ile Arg Glu Arg Gln Lys Leu Arg Arg 1 5 10 15 Gln Leu Leu Ala Gln Gln Leu Gly Ala Glu Ser Ala Asp Ser Ile Gly 20 25 30 Ala Val Leu Asn Ser Lys Asp Glu Gln Arg Glu Ile Ala Glu Thr Arg 35 40 45 Glu Thr Cys Arg Ala Ser Tyr Asp Thr Ser Ala Pro Asn Ala Lys Arg 50 55 60 Lys Tyr Leu Asp Glu Gly Glu Thr Asp Glu Asp Lys Met Glu Glu Tyr 65 70 75 80 Lys Asp Glu Leu Glu Met Gln Gln Asp Glu Glu Asn Leu Pro Tyr Glu 85 90 95 Glu Glu Ile Tyr Lys Asp Ser Ser Thr Phe Leu Lys Gly Thr Gln Ser 100 105 110 Leu Asn Pro His Asn Asp Tyr Cys Gln His Phe Val Asp Thr Gly His 115 120 125 Arg Pro Gln Asn Phe Ile Arg Asp Val Gly Leu Ala Asp Arg Phe Glu 130 135 140 Glu Tyr Pro Lys Leu Arg Glu Leu Ile Arg Leu Lys Asp Glu Leu Ile 145 150 155 160 Ala Lys Ser Asn Thr Pro Pro Met Tyr Leu Gln Ala Asp Ile Glu Ala 165 170 175 Phe Asp Ile Arg Glu Leu Thr Pro Lys Phe Asp Val Ile Leu Leu Glu 180 185 190 Pro Pro Leu Glu Glu Tyr Tyr Arg Glu Thr Gly Ile Thr Ala Asn Glu 195 200 205 Lys Cys Trp Thr Trp Asp Asp Ile Met Lys Leu Glu Ile Asp Glu Ile 210 215 220 Ala Ala Pro Arg Ser Phe Ile Phe Leu Trp Cys Gly Ser Gly Glu Gly 225 230 235 240 Leu Asp Leu Gly Arg Val Cys Leu Arg Lys Trp Gly Tyr Arg Arg Cys 245 250 255 Glu Asp Ile Cys Trp Ile Lys Thr Asn Lys Asn Asn Pro Gly Lys Thr 260 265 270 Lys Thr Leu Asp Pro Lys Ala Val Phe Gln Arg Thr Lys Glu His Cys 275 280 285 Leu Met Gly Ile Lys Gly Thr Val Lys Arg Ser Thr Asp Gly Asp Phe 290 295 300 Ile His Ala Asn Val Asp Ile Asp Leu Ile Ile Thr Glu Glu Pro Glu 305 310 315 320 Ile Gly Asn Ile Glu Lys Pro Val Glu Ile Phe His Ile Ile Glu His 325 330 335 Phe Cys Leu Gly Arg Arg Arg Leu His Leu Phe Gly Arg Asp Ser Thr 340 345 350 Ile Arg Pro Gly Trp Leu Thr Val Gly Pro Thr Leu Thr Asn Ser Asn 355 360 365 Tyr Asn Ala Glu Thr Tyr Ala Ser Tyr Phe Ser Ala Pro Asn Ser Tyr 370 375 380 Leu Thr Gly Cys Thr Glu Glu Ile Glu Arg Leu Arg Pro Lys Ser Pro 385 390 395 400 Pro Pro Lys Ser Lys Ser Asp Arg Gly Gly Gly Ala Pro Arg Gly Gly 405 410 415 Gly Arg Gly Gly Thr Ser Ala Gly Arg Gly Arg Glu Arg Asn Arg Ser 420 425 430 Asn Phe Arg Gly Glu Arg Gly Gly Phe Arg Gly Gly Arg Gly Gly Ala 435 440 445 His Arg Gly Gly Phe Pro Pro Arg 450 455 <210> 8 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> ALKBH5(66-​292) <400> 8 Pro Glu Arg Ser Asp Tyr Glu Glu Gln Gln Leu Gln Lys Glu Glu Glu 1 5 10 15 Ala Arg Lys Val Lys Ser Gly Ile Arg Gln Met Arg Leu Phe Ser Gln 20 25 30 Asp Glu Cys Ala Lys Ile Glu Ala Arg Ile Asp Glu Val Val Ser Arg 35 40 45 Ala Glu Lys Gly Leu Tyr Asn Glu His Thr Val Asp Arg Ala Pro Leu 50 55 60 Arg Asn Lys Tyr Phe Phe Gly Glu Gly Tyr Thr Tyr Gly Ala Gln Leu 65 70 75 80 Gln Lys Arg Gly Pro Gly Gln Glu Arg Leu Tyr Pro Pro Gly Asp Val 85 90 95 Asp Glu Ile Pro Glu Trp Val His Gln Leu Val Ile Gln Lys Leu Val 100 105 110 Glu His Arg Val Ile Pro Glu Gly Phe Val Asn Ser Ala Val Ile Asn 115 120 125 Asp Tyr Gln Pro Gly Gly Cys Ile Val Ser His Val Asp Pro Ile His 130 135 140 Ile Phe Glu Arg Pro Ile Val Ser Val Ser Phe Phe Ser Asp Ser Ala 145 150 155 160 Leu Cys Phe Gly Cys Lys Phe Gln Phe Lys Pro Ile Arg Val Ser Glu 165 170 175 Pro Val Leu Ser Leu Pro Val Arg Arg Gly Ser Val Thr Val Leu Ser 180 185 190 Gly Tyr Ala Ala Asp Glu Ile Thr His Cys Ile Arg Pro Gln Asp Ile 195 200 205 Lys Glu Arg Arg Ala Val Ile Ile Leu Arg Lys Thr Arg Leu Asp Ala 210 215 220 Pro Arg Leu 225 <210> 9 <211> 315 <212> PRT <213> Artificial Sequence <220> <223> MBD1(1-105)-MBD1(1-105)-MBD1(1-105) <400> 9 Met Ala Glu Asp Trp Leu Asp Cys Pro Ala Leu Gly Pro Gly Trp Lys 1 5 10 15 Arg Arg Glu Val Phe Arg Lys Ser Gly Ala Thr Cys Gly Arg Ser Asp 20 25 30 Thr Tyr Tyr Gln Ser Pro Thr Gly Asp Arg Ile Arg Ser Lys Val Glu 35 40 45 Leu Thr Arg Tyr Leu Gly Pro Ala Cys Asp Leu Thr Leu Phe Asp Phe 50 55 60 Lys Gln Gly Ile Leu Cys Tyr Pro Ala Pro Lys Ala His Pro Val Ala 65 70 75 80 Val Ala Ser Lys Lys Arg Lys Lys Pro Ser Arg Pro Ala Lys Thr Arg 85 90 95 Lys Arg Gln Val Gly Pro Gln Ser Gly Met Ala Glu Asp Trp Leu Asp 100 105 110 Cys Pro Ala Leu Gly Pro Gly Trp Lys Arg Arg Glu Val Phe Arg Lys 115 120 125 Ser Gly Ala Thr Cys Gly Arg Ser Asp Thr Tyr Tyr Gln Ser Pro Thr 130 135 140 Gly Asp Arg Ile Arg Ser Lys Val Glu Leu Thr Arg Tyr Leu Gly Pro 145 150 155 160 Ala Cys Asp Leu Thr Leu Phe Asp Phe Lys Gln Gly Ile Leu Cys Tyr 165 170 175 Pro Ala Pro Lys Ala His Pro Val Ala Val Ala Ser Lys Lys Arg Lys 180 185 190 Lys Pro Ser Arg Pro Ala Lys Thr Arg Lys Arg Gln Val Gly Pro Gln 195 200 205 Ser Gly Met Ala Glu Asp Trp Leu Asp Cys Pro Ala Leu Gly Pro Gly 210 215 220 Trp Lys Arg Arg Glu Val Phe Arg Lys Ser Gly Ala Thr Cys Gly Arg 225 230 235 240 Ser Asp Thr Tyr Tyr Gln Ser Pro Thr Gly Asp Arg Ile Arg Ser Lys 245 250 255 Val Glu Leu Thr Arg Tyr Leu Gly Pro Ala Cys Asp Leu Thr Leu Phe 260 265 270 Asp Phe Lys Gln Gly Ile Leu Cys Tyr Pro Ala Pro Lys Ala His Pro 275 280 285 Val Ala Val Ala Ser Lys Lys Arg Lys Lys Pro Ser Arg Pro Ala Lys 290 295 300 Thr Arg Lys Arg Gln Val Gly Pro Gln Ser Gly 305 310 315 <210> 10 <211> 199 <212> PRT <213> artificial sequence <220> <223> YTHDF1(361‑559) <400> 10 Ser Val Glu Ser His Pro Val Leu Glu Lys Leu Lys Ala Ala His Ser 1 5 10 15 Tyr Asn Pro Lys Glu Phe Glu Trp Asn Leu Lys Ser Gly Arg Val Phe 20 25 30 Ile Ile Lys Ser Tyr Ser Glu Asp Asp Ile His Arg Ser Ile Lys Tyr 35 40 45 Ser Ile Trp Cys Ser Thr Glu His Gly Asn Lys Arg Leu Asp Ser Ala 50 55 60 Phe Arg Cys Met Ser Ser Lys Gly Pro Val Tyr Leu Leu Phe Ser Val 65 70 75 80 Asn Gly Ser Gly His Phe Cys Gly Val Ala Glu Met Lys Ser Pro Val 85 90 95 Asp Tyr Gly Thr Ser Ala Gly Val Trp Ser Gln Asp Lys Trp Lys Gly 100 105 110 Lys Phe Asp Val Gln Trp Ile Phe Val Lys Asp Val Pro Asn Asn Gln 115 120 125 Leu Arg His Ile Arg Leu Glu Asn Asn Asp Asn Lys Pro Val Thr Asn 130 135 140 Ser Arg Asp Thr Gln Glu Val Pro Leu Glu Lys Ala Lys Gln Val Leu 145 150 155 160 Lys Ile Ile Ser Ser Tyr Lys His Thr Thr Ser Ile Phe Asp Asp Phe 165 170 175 Ala His Tyr Glu Lys Arg Gln Glu Glu Glu Glu Val Val Arg Lys Glu 180 185 190 Arg Gln Ser Arg Asn Lys Gln 195

Claims

1. A method for detecting the enzyme activity of DNA 5mC methyltransferase / demethyltransferase or RNA m6A methyltransferase / demethyltransferase. The method includes: In the presence of the DNA 5mC methyltransferase / demethylase or RNA m6A methyltransferase / demethylase, the binding state of DNA methylation-binding proteins or RNA methylation-binding proteins to fluorescently labeled substrates over time is monitored using fluorescence polarization, time-resolved fluorescence energy resonance transfer (TR-FRET), or homogeneous photoexcitation chemiluminescence signals. This allows for the detection of the enzyme activity of the DNA 5mC methyltransferase / demethylase or RNA m6A methyltransferase / demethylase. The substrate is either a fluorescently labeled methylated nucleic acid substrate or a fluorescently labeled unmethylated nucleic acid substrate. For DNA 5mC methyltransferases / demethylases, the substrate is a DNA substrate, and the sequence of the DNA substrate is a fully methylated, hemimethylated, hydroxymethylated, or unmethylated sequence. For RNA m6A methyltransferases / demethylases, the substrate is an RNA substrate, and the RNA substrate sequence contains a GGACU catalytic motif. The RNA substrate sequence includes both unmodified RNA sequences and m6A-modified RNA sequences. The DNA methylation-binding protein is a recombinant protein of GST-MBD1(1-105)-MBD1(1-105)-MBD1(1-105) (SEQ ID No.: 9), and The RNA methylation-binding protein is GST-YTHDF1(361-559) protein (SEQ ID No.: 10), and The DNA 5mC methyltransferase is DNMT1 (SEQ ID No.: 4); the DNA 5mC demethyltransferase is TET2 (SEQ ID No.: 5); the RNA m6A methyltransferase is the METTL3 (SEQ ID No.: 6)-METTL14 (SEQ ID No.: 7) complex; and the RNA m6A demethyltransferase is ALKBH5 (SEQ ID No.: 8).

2. The method according to claim 1, wherein, In the enzyme activity assay for DNA 5mC methyltransferase / demethyltransferase, the DNA substrate length is 11-12 bp.

3. The method according to claim 2, wherein, In the enzyme activity assay for DNA 5mC methyltransferase / demethyltransferase, the DNA substrate sequence is shown in the table below: In the enzyme activity assay for RNA m6A methyltransferase / demethyltransferase, the RNA substrate sequence is shown in the table below: 。 4. The method according to claim 1, wherein, The fluorescent labels include FAM, FITC, CY3, CY5, and BODIPY.

5. The method according to claim 1, wherein, The method includes the following steps: Step 1. Determine the enzyme activity system; Step 2. Dilute the fluorescently labeled DNA or RNA substrate, the DNA 5mC methyltransferase / demethylase or RNA m6A methyltransferase / demethylase to be detected, and the corresponding cofactors of the DNA 5mC methyltransferase / demethylase or RNA m6A methyltransferase / demethylase to be detected with the reaction solution, and dilute the DNA or RNA methylation binding protein with the stop solution; the cofactors include SAM and 2-OG; Step 3. Add the DNA 5mC methyltransferase / demethyltransferase or RNA m6A methyltransferase / demethyltransferase to be detected to the porous reactor, and then add a mixed solution of fluorescently labeled DNA or RNA substrate and cofactor at different reaction start times to start the reaction, and incubate at a certain temperature for a certain period of time. Step 4. After incubation, add diluted DNA or RNA methylation binding protein to the porous reactor, mix well on a shaker, and read the data on a multi-functional microplate reader. Step 5. Result Analysis: The FP signal value obtained by a multi-functional microplate reader is used to indicate the amount of substrate reduction or product formation, thereby determining the enzyme activity of the DNA 5mC methyltransferase / demethyltransferase or RNA m6A methyltransferase / demethyltransferase to be tested.

6. The method according to claim 5, wherein, In step 2, the reaction solution and the termination solution contain one or more of the following: 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), citric acid / sodium citrate, 2-morpholine ethanesulfonic acid buffer (MES), and 3-morpholine propanesulfonic acid buffer (MOPS). The reaction solution and the termination solution also contain sodium chloride, EDTA, RNase inhibitor, DTT, ATP, and vitamin C.

7. The method according to claim 5, wherein, In step 2, the reaction solution and the termination solution contain 4-hydroxyethylpiperazine ethanesulfonic acid buffer.

8. The method according to claim 5, wherein, In step 3, the concentration of the fluorescently labeled substrate is between 1 / 10 and 10 km, the concentration of each enzyme is between 10 and 200 nM, and the reaction time is between 20 and 90 min.

9. The method according to claim 5, wherein, In step 3, the concentration of the fluorescently labeled substrate is between 1 / 2 and 2 Km, and the concentration of each enzyme is 20 to 80 nM; the reaction time is 60 min.

10. The method according to claim 5, wherein, In step 3, When the enzyme being tested is DNMT1, the pH of the reaction system is between 6.5 and 10.5; the reaction temperature is between 20 and 50°C; and the salt concentration of the termination solution is between 300 mM and 1000 mM NaCl. When the enzyme being tested is TET2, the pH of the reaction system is between 6.5 and 10.5; the reaction temperature is between 20 and 50°C; the salt concentration of the reaction solution is between 0 and 300 mM NaCl; and the salt concentration of the termination solution is between 300 and 1000 mM NaCl. When the enzyme being tested is METTL3 / 14, the pH of the reaction system is between 6.5 and 10.5; the reaction temperature is between 20 and 50°C; and the salt concentration of the stop solution is between 300 and 1000 mM NaCl. When the enzyme being tested is ALKBH5, the pH of the reaction system is between 6.5 and 10.5; the reaction temperature is between 20 and 50°C; the salt concentration of the reaction solution is between 100 and 1000 mM NaCl; and the salt concentration of the termination solution is between 100 and 1000 mM NaCl.

11. The method according to claim 5, wherein, In step 3, When the enzyme being tested was DNMT1, the pH of the reaction system was 7.4; the reaction temperature was 37℃; and the salt concentration of the stop solution was 800 mM NaCl. When the enzyme being tested is TET2, the pH of the reaction system is 8.0; the reaction temperature is 37℃; the salt concentration of the reaction solution is 100 mM NaCl; and the salt concentration of the termination solution is 500 mM NaCl. When the enzyme being tested was METTL3 / 14, the pH of the reaction system was 7.4; the reaction temperature was 25℃; and the salt concentration of the stop solution was 500 mM NaCl. When the enzyme being tested is ALKBH5, the pH of the reaction system is 7.4; the reaction temperature is 37℃; the salt concentration of the reaction solution is 250 mM NaCl; and the salt concentration of the stop solution is 250 mM NaCl.

12. The method according to claim 5, wherein, In step 3, When the enzyme being tested is DNMT1, the reaction solution is 1.20 mM HEPES, pH 7.4, 2.1 mM EDTA, and 3.1 mM DTT; the stop solution is 20 mM HEPES, pH 7.4, and 800 mM NaCl. When the enzyme being tested is TET2, the reaction solution is 20 mM HEPES, pH 8.0, 100 mM NaCl, 1 mM DTT, 1 mM ATP, 2 mM Vitamin C, and 100 mM Fe. 2+ (NH4)2(SO4)2; the stop solution was 20 mM HEPES, pH 7.4, 500 mM NaCl; When the enzyme being tested is METTL3 / 14, the reaction solution is 20 mM HEPES, pH 7.4, 1 mM DTT, and 0.4 U / μL RNase inhibitor; the stop solution is 20 mM HEPES, pH 7.4, and 500 mM NaCl. When the enzyme being tested is ALKBH5, the reaction solution is 20 mM HEPES, pH 7.4, 250 mM NaCl, 300 μM Vitamin C, and 50 μM Fe. 2+ (NH4)2(SO4)2; the stop solution was 20 mM HEPES, pH 7.4, 250 mM NaCl.

13. A method for screening inhibitors or agonists targeting DNA 5mC methyltransferase / demethyltransferase or RNA m6A methyltransferase / demethyltransferase, the method comprising: The inhibitors or agonists are screened by comparing the differences in enzyme activities of DNA 5mC methyltransferase / demethyltransferase or RNA m6A methyltransferase / demethyltransferase in the presence and absence of the inhibitors or agonists, wherein the enzyme activities of DNA 5mC methyltransferase / demethyltransferase or RNA m6A methyltransferase / demethyltransferase are detected according to the method described in claim 1.

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