A method for detecting the content and / or activity of fto and its subfamily

Abstract

The application discloses a molecular structure, a long G-quadruplex DNAzyme chain and a supramolecular NMM-G-quadruplex structure. The application further discloses a kit containing DNA1, DNA2 and a circular template. The application further discloses application of the molecular structure, the long G-quadruplex DNAzyme chain, the supramolecular NMM-G-quadruplex structure or the kit in detection of FTO and / or activity of a subfamily thereof. The application further discloses a method for detecting content / activity of FTO and / or a subfamily thereof. The application can accurately recognize and cut demethylated dsDNA, effectively eliminate background signals and improve detection specificity; can realize label-free and sensitive detection of FTO, and the detection limit is as low as 3.10*10 ‑16 M; and has wide application prospects in RNA demethylase related biomedical research and clinical diagnosis.

Description

TECHNICAL FIELD

[0001] The present application relates to a molecular structure, a long G-quadruplex DNAzyme chain, a supramolecular NMM-G-quadruplex structure, a kit thereof and application in detecting FTO and / or its subfamily content and / or activity, and belongs to the field of biological analysis. BACKGROUND

[0002] N 6 -methyladenine(m 6 A) modification is one of the most common post-transcriptional modifications in RNA molecules, and plays an important role in cell processes such as translation initiation, alternative splicing, cell fate conversion, tumor metastasis, etc. Recent studies have shown that m 6 A modification is correctable and dynamically regulated. The m 6 A methyltransferase tri-complex (METTL3 / METTL14 / WTAP) and METTL16 can act as m 6 A modification writer to catalyze m 6 A modification, while two human demethylases including ALKBH5 and FTO (fat mass and obesity-associated enzyme) can act as m 6 A modification eraser to oxidatively remove m 6 A label in mRNA. FTO and ALKBH5 are both AlkB dioxygenase family, and their activities depend on Fe 2+ and 2-oxoglutarate (2-OG). FTO is the first discovered m 6 A demethylase, which can catalyze m 6 A demethylation in mRNA / single-stranded DNA (ssDNA). In addition, FTO is not only involved in adipocyte differentiation and energy homeostasis, but also closely related to various human diseases such as cardiovascular disease, Alzheimer's disease, acute myeloid leukemia (AML), cancer, etc., indicating that FTO can be not only a diagnostic biomarker, but also a potential therapeutic target. Therefore, developing sensitive and effective methods for FTO activity detection and FTO inhibitor screening is of great help to clinical diagnosis, drug discovery and prognosis treatment.

[0003] Currently, FTO detection methods include radioassay, HPLC-MS and gel electrophoresis. Radioassay is a gold standard method, but it requires specific radioisotope labeling and has the risk of dangerous radiation. Although HPLC-MS is the most commonly used method, its cumbersome and time-consuming operation limits its practical application. Gel electrophoresis based on DpnII digestion has been applied to the determination of RNA demethylase activity, but its sensitivity is poor and sample consumption is large. In addition, several new methods for detecting RNA demethylase activity have been reported. For example, methylation switchable probes and FTO-responsive DNAzymes can detect FTO activity based on induced conformational changes, but their sensitivity is poor, the probe design is complex, and incomplete fluorescence quenching leads to false positives. Recently, E. coli toxin MazF was introduced to detect RNA demethylase activity. E. coli toxin MazF is a m 6 A sensitive ACA-specific endoribonuclease that selectively cleaves unmethylated 5'-ACA-3' RNA sequences. Although the hybridization chain reaction based on MazF can selectively detect FTO activity, the expensive fluorescently labeled probe and capture / separation steps hinder its widespread application. Therefore, there is an urgent need for a new strategy to specifically and sensitively detect FTO activity. SUMMARY

[0004] The technical problem solved by the present application is to provide a new molecular structure, a long G-quadruplex DNAzyme chain and a supramolecular NMM-G-quadruplex structure.

[0005] The present application also provides a new kit comprising DNA1, said DNA2 and said circular template, wherein the sequence of the DNA1 is 5'-GTG TAA AGC GGA GTG ATG TTA GGA TCA GTG TCT CGA A-3', the bold A is N 6 -methyladenosine(m 6 A).

[0006] The present application also provides the use of the molecular structure, the long G-quadruplex DNAzyme chain, the supramolecular NMM-G-quadruplex structure or the kit in detecting the content and / or activity of FTO and / or its subfamily.

[0007] The present application also provides a method for detecting the content and / or activity of FTO and / or its subfamily.

[0008] Technical solution: To solve the above technical problems, the application provides a molecular structure, which comprises a circular template and a DNA cutting product which is partially complementary to the circular template, wherein the sequence of the circular template is 5'-CCA ACC CACCCT ACC CAC TTT TTT CTA ACA TCA CTC CGC TTT ACA CTT TTT TCC AAC CCA CCC TACCCA CTT TTT TTT T-3'; and the sequence of the DNA cutting product is 5'-GTGT AAA GCG GAGT GATG TTAG-3'.

[0009] The DNA cutting product is obtained by cutting dsDNA by DpnII.

[0010] The dsDNA is obtained by hybridizing demethylated DNA1 and its complementary DNA2, wherein the sequence of the demethylated DNA1 is 5'-GTG TAA AGC GGA GTG ATG TTA GGA TCA GTG TCT CGA A-3', and the sequence of the DNA2 is 5'-ACG ATA CAT CCC ACT GAT CCT AAC ATC CTC-3'.

[0011] The application further provides a long G-quadruplex DNAzyme chain, which is obtained by combining the molecular structure with FTO.

[0012] The application further provides a supramolecular NMM-G-quadruplex structure, which is obtained by combining N-methyl mesoporphyrin IX and the long G-quadruplex DNAzyme chain.

[0013] The application further provides a kit, which comprises DNA1, the DNA2 and the circular template, wherein the sequence of the DNA1 is 5'-GTG TAA AGC GGA GTG ATG TTA GGA TCA GTG TCT CGA A-3', the bold A is N 6 -methyladenosine (m 6 A).

[0014] The kit further comprises L-ascorbic acid, (NH4)2Fe(SO4)2, 2-ketoglutaric acid, DpnII, dNTP, phi29 DNA polymerase, 1×phi29 buffer, 1×DpnII buffer, N-methyl mesoporphyrin IX.

[0015] The application also provides application of the molecular structure, the long G-quadruplex DNAzyme chain, the supramolecular NMM-G-quadruplex structure or the kit in detecting FTO and / or content and / or activity of a subfamily thereof.

[0016] The application also provides a method for detecting content / activity of FTO and / or a subfamily thereof, comprising the following steps:

[0017] (1) reacting DNA1 with FTO and / or a subfamily thereof in a buffer solution to obtain demethylated DNA, wherein the buffer solution comprises L-ascorbic acid, (NH4)2Fe(SO4)2 and α-ketoglutaric acid;

[0018] (2) mixing the demethylated DNA obtained in step (1) with its complementary strand, incubating, cooling to generate a dsDNA product;

[0019] (3) adding the dsDNA product obtained in step (2) to a reaction system containing DpnII and 1×DpnII buffer solution, inactivating to obtain a DNA cleavage product;

[0020] (4) adding the DNA cleavage product obtained in step (3) to a reaction system containing a circular template, dNTP, phi29 DNA polymerase and 1×phi29 reaction buffer solution to perform rolling circle amplification, incubating to generate a long G-quadruplex DNAzyme chain;

[0021] (5) adding N-methyl mesoporphyrin IX to the long G-quadruplex DNAzyme chain reaction solution obtained in step (4), incubating to form a supramolecular NMM-G-quadruplex structure, performing fluorescence detection, and obtaining the content of FTO in the human breast tissue to be tested according to a linear relationship equation between the known fluorescence intensity and the FTO concentration, wherein the FTO concentration is 1×10 -9 ~ 1×10 -14 M.

[0022] When the content of FTO is detected, the linear relationship equation between the fluorescence intensity and the FTO concentration is F=1877.69lgC+35371.77 (R 2 =0.9994), wherein C is the concentration of FTO, and F is the fluorescence intensity at 614 nm;

[0023] Preferably, the linear relationship equation between the fluorescence intensity and the concentration of AlkB Homolog 5 is F=4730.64lgC+84196.76 (R 2 =0.9826), wherein C is the concentration of AlkB Homolog 5, and F is the fluorescence intensity at 614 nm.

[0024] As preferably, the concentration ratio of the DNA to FTO in step (1) is 2.4:1, and the volume ratio of the DNA to FTO is 1.2:1.

[0025] As preferably, the concentration of the dsDNA product in step (3) is 0.3 μM, and the added amount of DpnII is 5 U.

[0026] As preferably, the concentration ratio of the circular template to dNTP in step (4) is 1:10 nM / μM, and the volume ratio of the circular template to dNTP is 1:1.

[0027] As preferably, the concentration of N-methyl porphyrin IX in step (5) is 50 nM.

[0028] Principle of the present application: The present application designs two ssDNA (i.e. DNA1 and DNA2) and a circular template, in which DNA1 containing sequence 5'-Gm 6 ATC-3' as a specific substrate of FTO, and DNA2 containing sequence 5'-GATC-3' is partially complementary to DNA1. The circular template has two regions, including a primer antisense sequence and two DNA G-quadruplex antisense sequences. In the presence of FTO, it specifically recognizes the 5'-Gm 6 ATC-3' sequence in DNA1 and converts m 6 A into A. Subsequently, the demethylated DNA1 hybridizes with DNA2 to obtain a dsDNA product with DpnII specific recognition sequence 5'-GATC-3' / 3'-CTAG-5'. When DpnII is added, it will cleave the specific site of the demethylated dsDNA product to generate two short dsDNA products. One of the two short dsDNAs unfolds due to the low T m value (33.6℃) compared with the RCA reaction (37℃), resulting in the release of a 13 nt ssDNA and a 24 nt ssDNA. The released 24 nt ssDNA can pair with the circular template as a primer to induce an isothermal RCA reaction to generate a large amount of long ssDNA polymer with repeated G-quadruplex sequences. Since G-quadruplex can specifically bind to NMM (a water-soluble porphyrin that can specifically bind to G-quadruplex DNAzyme to produce a high fluorescence signal), a supramolecular NMM-G-quadruplex structure is formed, resulting in a significantly enhanced fluorescence signal at 614 nm. In contrast, when FTO is absent, neither demethylation of DNA1 nor DpnII-mediated cleavage reaction occurs. Therefore, in this case, neither RCA nor significantly enhanced fluorescence signal is generated.

[0029] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages: 1. The method for label-free detection of FTO in human breast tissue based on RNA demethylation-driven functional supramolecular structure does not have the defects of radioactive labeling, complex equipment, large sample consumption and the like, greatly simplifying the experimental process; 2. The introduction of DpnII can accurately recognize and cut demethylated dsDNA, effectively eliminating background signals and improving detection specificity; 3. DpnII-mediated rolling circle amplification greatly improves the sensitivity of the method; 4. The G-quadruplex-NMM system helps to design a label-free method for measuring FTO, so that no fluorescently labeled DNA / RNA probe is needed; 5. Due to the high specific cutting of DpnII-mediated demethylated DNA, the high amplification efficiency of RCA and the high signal-to-noise ratio of the G-quadruplex-NMM system, the method can realize label-free and sensitive detection of FTO, with a detection limit as low as 3.10 x 10 - 16 M, thereby screening RNA demethylase (such as FTO and ALKBH5) inhibitors and quantitatively measuring the activity of FTO in human cancer cells; 6. The method can distinguish the FTO activity in breast cancer patient tissues and healthy human tissues, and has a broad application prospect in RNA demethylase-related biomedical research and clinical diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 A schematic diagram for label-free detection of FTO in human breast tissue based on RNA demethylation-driven functional supramolecular structure;

[0031] Figure 2 A is agarose gel electrophoresis characterization of RCA product (channel 1 is DNA 1+DNA 2+DpnII+circular template+phi29 DNA polymerase; channel 2 is FTO+DNA 1+DNA 2+DpnII+circular template; channel 3 is FTO+DNA 1+DNA 2+DpnII+phi29 DNA polymerase; channel 4 is FTO+DNA 1+DNA 2+circular template+phi29 DNA polymerase; channel 5 is FTO+DNA 1+DNA 2+DpnII+circular template+phi29 DNA polymerase); Figure 2 B is the fluorescence emission spectrum in the presence and absence of FTO;

[0032] Figure 3 A is the fluorescence emission spectrum of FTO at 580-640 nm at different concentrations; Figure 3 B is a linear relationship between fluorescence intensity and FTO concentration;

[0033] Figure 4Fluorescence intensity of the response of target FTO, BSA, Dam, M.SssI, PNK, MazF and control group;

[0034] Figure 5 A is the fluorescence emission spectrum of ALKBH5 at different concentrations; Figure 5 B is the fluorescence intensity of 1 x 10 -12 -1 x 10 -8 M, the relationship between fluorescence intensity and the logarithm of ALKBH5 concentration;

[0035] Figure 6 A is the initial velocity of the response under the condition of 100 nM FTO and different concentrations of DNA1; Figure 6 B is the initial velocity of the response under the condition of 100 nM ALKBH5 and different concentrations of DNA1;

[0036] Figure 7 A is the chemical structure of different inhibitors (FB23, rhein and IOXI); Figure 7 B is the IC 50 value of FB23; Figure 7 C is the IC 50 value of rhein; Figure 7 D is the IC 50 value of IOXI (the concentrations of FTO and ALKBH5 are 1 nM and 10 nM, respectively);

[0037] Figure 8 A is the change of fluorescence intensity of different concentrations of FB23; Figure 8 B is the change of fluorescence intensity of different concentrations of rhein; Figure 8 C is the relative expression of FTO mRNA after siRNA treatment; Figure 8 D is the fluorescence intensity of siRNA, 10 μM rhein and 100 nM FB23; Figure 8 E is the fluorescence intensity of the response of Hela cells, MDA-MB-231 cells, MCF-7 cells and control group; Figure 8 F is the fluorescence intensity of the response of cytoplasmic extract of HeLa cells after treatment with 20 nM FB23 and 80 nM FB23;

[0038] Figure 9 A is a schematic diagram for detecting the activity of FTO in human breast tissue; Figure 9 B is a heatmap of FTO activity in healthy people (n = 5) and breast cancer patients (n = 5); Figure 9 C is a box plot of fluorescence intensity of healthy people (n = 5) and breast cancer patients (n = 5). DETAILED DESCRIPTION

[0039] The technical solutions of the present application are further described below with reference to the drawings.

[0040] Reagents and materials:

[0041] All HPLC purified oligonucleotides were synthesized by Aikang Biotech Co., Ltd. (Changsha, China); FTO and ALKBH5 were from Active Motif (Carlsbad, CA, USA); DpnII, 10x DpnII buffer solution (100 mM Tris-HCl, 3 M NaCl, 10 mM DTT, 1 mM EDTA, 5 mg / mL BSA, 50% glycerol, pH 7.4), phi29 DNA polymerase and 10x phi29 buffer solution (500 mM Tris-HCl, 100 mM MgCl2, 100 mM (NH4)2SO4, 40 mM DTT, pH 7.5) were purchased from New England Biolabs (Ipswich, MA, USA); Evo M-MLV RT Mix kit, Green Premix Pro Taq HS qPCR kit and dNTP mix solution were from Aikang Biotech Co., Ltd. (Changsha, China); N-methyl mesoporphyrin IX (NMM) was purchased from Cayman Chemical (Ann Arbor, MI, USA); ferrous ammonium sulfate hexahydrate ((NH4)2Fe(SO4)2) and alpha-ketoglutarate (2-OG) were purchased from Sigma-Aldrich (St. Louis, MO, USA); 4,5-dihydroxyanthraquinone-2-carboxylic acid (Rhein), 2-[[2,6-dichloro-4-(3,5-dimethyl-4-isoxazolyl)phenyl]amino]benzoic acid (FB23), 5-carboxy-8-hydroxyquinoline and 8-hydroxy-5-quinolinecarboxylic acid (IOX1) were from MedChemExpress (Shanghai, China); Lipofectamine TM 3000 transfection reagent and Opti- Reduced serum medium was purchased from Thermo Fisher Scientific (MA, USA); human cervical cancer cell line (HeLa cells) and human breast cancer cell lines (MCF-7 cells and MDA-MB-231 cells) were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China); tissue samples of human breast cancer patients and healthy individuals were provided by the Affiliated Hospital of Guangdong Medical University (Guangdong, China), and FTO detection was approved by the Ethics Committee of the Affiliated Hospital of Guangdong Medical University; ultrapure water was prepared by a Millipore filtration system (Millipore, Milford, USA).

[0042] Detection of fluorescence spectra and measurement of actual samples:

[0043] Fluorescence emission spectra were recorded using a FLS-1000 spectrofluorometer (Edinburgh Instruments, UK) at 399 nm excitation, and FTO was quantified using fluorescence intensity at 614 nm. Nuclear extraction kit (Active Motif, Carlsbad, CA) was used to obtain cell lysates and cytoplasmic lysates of human tissues. The resulting extracts were immediately used for FTO detection.

[0044] Example 1 Functional supramolecular structure based on RNA demethylation for label-free detection of FTO in human breast tissues

[0045] As shown in Figure 1 Figure 1, 0.6 μΜ DNA1 (SEQ ID NO. 1: 5'-GTG TAA AGC GGA GTG ATG TTA GGA TCA GTG TCT CGA A-3') (bold A represents: N 6 -methyladenosine (m 6A) mixed with 250 nM FTO in reaction buffer solution (100 μΜ L-ascorbic acid, 150 μΜ (NH4)2Fe(S04)2, 100 μΜ 2-OG, pH 7.5) and reacted for 2 h at 30 °C to give demethylated DNA (SEQ ID NO. 2: 5'-GTG TAA AGC GGA GTG ATG TTA GGA TCA GTG TCT CGA A-3'). Subsequently, mixed with its complementary strand DNA 2 (SEQ ID NO. 3: 5'-ACG ATA CAT CCC ACT GAT CCT AAC ATC CTC-3') in 1 x annealing buffer solution, incubated at 95 °C for 5 min, then slowly cooled to room temperature to generate dsDNA product. Subsequently, the generated dsDNA product was added to 20 μΐ, reaction system containing 5 U DpnII, 1 x DpnII buffer solution, reacted at 37 °C for 60 min, inactivated at 80 °C for 20 min. The generated DNA cleavage product (SEQ ID NO. 4: 5'-GTG TAA AGC GGA GTG ATG TTA G-3') was added to 50 nM circular template (SEQ ID NO. 5: 5'-CCA ACC CAC CCT ACC CAC TTT TTT CTA ACA TCA CTC CGC TTT ACA CTT TTT TCC AAC CCA CCC TAC CCA CTT TTT TTT T-3'), 500 μΜ dNTP, 1 U phi29 DNA polymerase, 1 x phi29 reaction buffer solution for rolling circle amplification (RCA), incubated at 37 °C for 90 min to generate long G-quadruplex DNAzyme strand. Finally, 50 nM NMM was added to the above G-quadruplex DNAzyme reaction solution containing G-quadruplex DNAzyme, incubated at room temperature for 30 min to form supramolecular NMM-G-quadruplex structure, thereby generating amplified fluorescence signal.

[0046] Example 2 Verification of the feasibility of the experiment

[0047] The RCA product was characterized by 1% agarose gel electrophoresis. Among them, Figure 2 M in A is 2000 bp marker. No RCA product band was observed in the absence of FTO (lane 1 in A), indicating that no RCA reaction was initiated. In contrast, when FTO was added, multiple ladder-like super-bright bands of RCA product appeared, with slower migration rate (lane 5 in A). When phi29 DNA polymerase was not added (lane 2 in A), circular template (lane 3 in A), or G-quadruplex DNAzyme (lane 4 in A) was not added, no RCA product band was observed, indicating that the RCA reaction was not initiated. Figure 2 Figure 2 Figure 2 Figure 2 ​​​A, channel 3) and DpnII ( Figure 2 No distinct bands were observed in any of the components (A, channel 4), indicating that these components play a crucial role in RCA. Furthermore, fluorescence measurements were performed in this protocol. Figure 2 B) Without the addition of FTO, only a weak fluorescence signal was detected, indicating that no G-tetrameric DNAzymes were generated. However, the addition of FTO produced an amplified fluorescence signal, indicating that FTO can induce DpnII-mediated RCA to generate a large number of G-tetrameric DNAzymes.

[0048] Example 3 Sensitivity Detection

[0049] Under the conditions of Example 1, the sensitivity of the method at different wavelengths was detected. For example... Figure 3 As shown in Figure A, the measured fluorescence intensity is directly proportional to the concentration of FTO. Furthermore, at 1×10⁻⁶... -9 -1×10 -14 Within the dynamic range of M, fluorescence intensity (F) is linearly related to the logarithm of FTO concentration (C). Figure 3 B). The correction equation is F = 1877.69lgC + 35371.77(R). 2 =0.9994), the limit of detection (LOD) is 3.10×10 -16 M. This method is more sensitive than the hairpin probe fluorescence method (1×10⁻⁶). -8 M) is 8 orders of magnitude higher than that of hybridization chain reaction biosensors (2.273 × 10⁻⁶). -9 M) is 7 orders of magnitude higher than that of the deoxyribonuclease biosensor (5 × 10⁻⁶). -10 The M value is 6 orders of magnitude higher than that of the FRET-based fluorescence method (3.3 × 10⁻⁶). -12 The M value is four orders of magnitude higher than that of the Mazf-based fluorescence method (7.62 × 10⁻⁶). -15 The M) is one order of magnitude higher. The enhanced sensitivity is attributed to: (1) the highly specific cleavage of demethylated DNA mediated by DpnII, (2) the high efficiency of RCA in generating a large number of G-tetramer DNAzymes, and (3) the high signal-to-noise ratio of the G-tetramer-NMM system.

[0050] Example 4: Specific Detection

[0051] The experimental procedure is the same as in Example 1, except that the target is replaced with an interference target. This invention uses bovine serum albumin (BSA), Dam DNA methyltransferase (Dam), M.SssI DNA methyltransferase (M.SssI), polynucleotide kinase (PNK), and MazF mRNA interfering enzyme (MazF) as interferants to verify the selectivity of this method for FTO. BSA is a normally unrelated protein. Dam methylates adenine residues in the 5'-GATC-3' sequence. M.SssI methylates cytosine residues in the 5'-CG-3' sequence. PNK converts the 5'-OH / 3'-PO4 at the end of nucleic acids to 5'-PO4 / 3'-OH. MazF catalyzes the cleavage of unmethylated 5'-ACA-3' RNA sequences. Since DNA 1 demethylation and DpnII-mediated RCA can only be initiated by FTO, the above interferences cannot induce demethylation. Figure 4 As shown, FTO produces a significant fluorescence signal, similar to BSA and Dam (…). Figure 4 The fluorescence signals induced by M.SssI, PNK, MazF and the control group were well distinguished, indicating that this method has good selectivity for FTO.

[0052] Example 5: Detection of Universality

[0053] The experimental procedure was the same as in Example 1. To assess the generality of this method, AlkBHomolog 5 (ALKBH5), a member of the FTO subfamily, was selected as a model. ALKBH5 not only shares high structural and sequence homology with FTO but also exhibits direct DNA / RNA demethylase activity. 1% agarose gel electrophoresis and fluorescence measurements showed that this method can be extended to detect ALKBH5 activity. Different concentrations (0, 1 × 10⁻⁶) were measured using the method described in Example 3. -12 1×10 -11 1×10 -10 1×10 -9 1×10 -8 1×10 -7 2.5×10 -7 The fluorescence intensity produced by M)ALKBH5 ( Figure 5 As the ALKBH5 concentration increased from 0 to 2.5 × 10⁻⁶, the concentration of ALKBH5 increased from 0 to 2.5 × 10⁻⁶. -7 M, fluorescence intensity gradually increases ( Figure 5 A). In 1×10 -12 Up to 1×10 -8 Within the M range, there is a good linear fit between fluorescence intensity (F) and the logarithm of ALKBH5 concentration (C). Figure 5 B). The relevant equation is F = 4730.64lg C + 84196.76(R). 2= 0.9826), LOD is 3.34 x 10 -17 M.

[0054] Example 6 Kinetic analysis

[0055] This example further determined the kinetic parameters of FTO and ALKBH5. The procedure was as follows: different concentrations of DNA 1 substrate were mixed with 100 nM FTO / ALKBH5 in reaction buffer solution (100 μΜ L-ascorbic acid, 150 μΜ (NH4)2Fe(S04)2, 100 μΜ 2-OG, pH 7.5) and reacted at 30 °C for 12 min to obtain the demethylated DNA substrate. The remaining reaction steps were the same as Example 1. As shown in Figure Figure 6 A, the initial velocity (V) of FTO increased accordingly with the increase of DNA 1 concentration. According to the Michaelis-Menten equation, the calculated Vmaxand Kmwere 11.29 s -1 and 354.03 nM, respectively, which were consistent with the results of methylation fluorescence switch probe-based assay (Km= 0.506 ± 0.053 μΜ). Similarly, the initial velocity of ALKBH5 also increased accordingly with the increase of DNA 1 concentration (Figure Figure 6 B). The determined Vmaxand Kmwere 16.15 s -1 and 1113.64 nM, respectively, which were consistent with the Vmaxand Kmvalues determined by methylation switch probe fluorescence method (Km= 2.218 ± 0.28 μΜ). These results indicated that this method could accurately analyze the enzyme kinetics of RNA demethylases (such as FTO and ALKBH5).

[0056] Example 7 Inhibitor analysis

[0057] This example selected a group of structurally diverse small molecules, including FB23, rhein and IOX1, to explore the performance of this method in screening RNA demethylase inhibitors (Figure Figure 7 A). FB23 (Figure Figure 7 B, left panel) and rhein (Figure Figure 7 C, left panel) induced a dose-dependent decrease in the relative activity of FTO, and the IC 50 value of FB23 was 39.31 nM, and the IC 50 value of rhein was 1.13 μΜ, which was consistent with previous studies. In contrast, 100 nM FB23 (Figure Figure 7 B, right panel) and 25 μΜ rhein (Figure Figure 7Figure C (right) shows no effect on the relative activity of ALKBH5, indicating that FB23 and rhein have no inhibitory effect on the demethylation of ALKBH5. These results suggest that FB23 and rhein have higher selectivity for FTO demethylation than ALKBH5. IOX1 is the most potent broad-spectrum inhibitor of the 2-OG oxygenase subfamily, competitively binding to the active site of demethylases. Figure 7 As shown in D, IOX1 can effectively suppress FTO ( Figure 7 D, left figure) and ALKBH5 ( Figure 8 D, right figure), where FTO's IC 50 The value is 1.30 μM, IC of ALKBH5 50 The value was 1.17 μM, consistent with the eutectic structure analysis (3.3 μM, FTO) and radioactivity determination (2.9 μM, ALKBH5), indicating that IOX1 has an inhibitory effect on both FTO and ALKBH5.

[0058] Example 8: Analysis of Actual Samples

[0059] To evaluate the performance of this method in cellular FTO, the inhibitory effects of inhibitors (i.e., FB23 and rhein) on HeLa cell FTO activity were investigated. The experimental procedure was the same as in Example 1, except that the target was replaced with extracted HeLa cells (10,000 cells), which were then incubated with the inhibitors FB23 and rhein. As FB23 ( Figure 8 A) and rhein ( Figure 8 B) As the concentration increased, the fluorescence intensity gradually decreased, indicating that FB23 and rhein effectively inhibited endogenous FTO activity. Notably, the downregulation of endogenous FTO can be achieved by introducing FTO-silencing siRNA into HeLa cells. Twelve hours after transfection, qRT-PCR detected a significant downregulation (75%) of FTO mRNA expression. Figure 8 C). In addition, such as Figure 8 As shown in D, the fluorescence intensity was significantly reduced due to the downregulation of FTO protein, which is the same as the result obtained from treatment with rhein and FB23.

[0060] This method further detected the FTO activity in cytoplasmic extracts from HeLa cells, MDA-MB-231 cells, and MCF-7 cells. FTO is distributed in the nucleus and cytoplasm, while ALKBH5 is distributed in the nucleus of different cancer cells. Figure 8 As shown in Figure E, no obvious fluorescence signal was observed in the control group. However, MDA-MB-231 cells, MCF-7 cells, and HeLa cells all showed high fluorescence signals, consistent with the results of qRT-PCR. Furthermore, as... Figure 9F, Hela cells were treated with 20 nM FB23 and 80 nM FB23, which induced the decrease of fluorescence intensity by 43.6% and 88.9%, respectively, indicating that FB23 effectively inhibited the endogenous FTO activity.

[0061] Example 9 Clinical sample analysis

[0062] The clinical applicability of the proposed method was verified by measuring the FTO activity in 10 tissue samples from healthy people (n = 5) and breast cancer patients (n = 5) (sample pre-treatment: tissue samples were pre-treated by FFPE Total Protein Extraction Kit kit (paraffin-embedded tissue protein extraction kit)). Figure 9 A) The fluorescence intensity generated by breast cancer patient tissues was higher than that generated by healthy human tissues Figure 9 B), indicating that the FTO activity in breast cancer patient tissues was higher than that in healthy human tissues. In addition, the fluorescence intensity of different breast cancer patients differed greatly, indicating that there were differences in the expression of FTO in early and late breast cancer. As shown in ​ C, the scatter plot of FTO showed that there was a significant difference in FTO between breast cancer patients and healthy human tissues, indicating that the overall expression level of FTO in breast cancer patients was significantly higher than that in healthy people. These results show that the method can accurately distinguish between breast cancer patients and healthy people.

Claims

1. A kit characterized in that, DNA1 comprising a nucleotide sequence as shown in SEQ ID NO. 1, DNA2 comprising a nucleotide sequence as shown in SEQ ID NO. 3, DpnII, a circular template comprising a nucleotide sequence as shown in SEQ ID NO. 5, and N-methyl porphyrin IX; the A in GATC in the nucleotide sequence of the DNA1 is replaced by N 6 -methyladenosine.

2. The kit of claim 1, wherein Also include L-ascorbic acid, (NH4)2Fe(SO4)2, 2-ketoglutaric acid, dNTP, phi29 DNA polymerase, 1 × phi29 buffer, 1 × DpnII buffer.

3. Use of the kit according to claim 1 or 2 for detecting the content and / or activity of FTO and / or its subfamily in a sample for non-diagnostic purposes.

4. A method for detecting the content and / or activity of FTO and / or its subfamily in a sample for non-diagnostic purposes, characterized in that, The method comprises the following steps: (1) reacting DNA 1 with a nucleotide sequence as shown in SEQ ID NO. 1 with a sample in a buffer solution to obtain a demethylated DNA; the buffer solution comprises L-ascorbic acid, (NH4)2Fe(SO4)2, α-ketoglutaric acid; A in GATC in the nucleotide sequence of the DNA 1 is N 6 -methyladenosine; (2) Mix the demethylated DNA of step (1) with a complementary strand having a nucleotide sequence as shown in SEQ ID NO. 3, incubate, cool, and generate a dsDNA product; (3) Add the dsDNA product of step (2) to a reaction system containing DpnII, 1 × DpnII buffer solution, inactivate, and obtain a DNA cleavage product; (4) Add the DNA cleavage product of step (3) to a reaction system containing a circular template having a nucleotide sequence as shown in SEQ ID NO. 5, dNTP, phi29 DNA polymerase, 1 × phi29 reaction buffer solution, and perform rolling circle amplification, incubate, and produce a long G-quadruplex DNAzyme strand; (5) adding N-methyl mesoporphyrin IX into the long G-quadruplex DNAzyme chain reaction solution in step (4), incubating to form a supramolecular NMM-G-quadruplex structure, using 614 nm fluorescence intensity for fluorescence detection, and obtaining the FTO and / or subfamily content in the sample to be measured according to the linear relationship equation between the known fluorescence intensity and the FTO concentration, wherein the FTO and / or subfamily content concentration is 1×10 -9 ~1×10 -14 M.

5. The method of claim 4, wherein, When the detection object is FTO, the linear relationship equation between the fluorescence intensity and the FTO concentration is F = 1877.69 lgC + 35371.77, C is the FTO concentration, and F is the fluorescence intensity at 614 nm.

6. The method of claim 4, wherein, When the detection object is the subfamily AlkB Homolog 5 of FTO, the linear relationship equation between the fluorescence intensity and the AlkB Homolog 5 concentration is F = 4730.64 lg C + 84196.76, C is the AlkB Homolog 5 concentration, and F is the fluorescence intensity at 614 nm.

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