YTHDF fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a YTHDF fluorescent probe and a preparation method and application thereof, the chemical formula of the YTHDF fluorescent probe is 5 '-FAM-CCGG (m6A) CUGU, C is cytosine ribonucleotide, G is guanine ribonucleotide, U is uracil ribonucleotide, and m6A is N6-methyladenosine; the compound has a fluorescence characteristic, and has better specific binding to YTHDF; the compound is used as a fluorescence polarization probe, a fluorescence polarization technology is adopted, screening of a YTHDF ligand and evaluation of the affinity level of the ligand can be achieved, a YTHDF micromolecule high-throughput screening scheme established through fluorescence polarization can be used for high-throughput screening of drugs, and the compound has the advantages of being low in price, stable, rapid, efficient and the like.

Description

Technical Field

[0001] The invention relates to a YTHDF fluorescent probe and also to a preparation method and application of the fluorescent probe. Background Art

[0002] N 6 -methyladenosine (m 6 A) is the most common modification in eukaryotic mRNA and noncoding RNA. It was first discovered in the 1970s and was considered a potential regulator of mRNA processing in cells. 6 Methylation can affect neurodevelopment and neurodegenerative diseases, stem cell self-renewal and differentiation, tumor progression and circadian rhythm. This methylation modification is crucial for the post-transcriptional regulation of eukaryotic genes. 6 A exerts its biological effects by affecting the splicing, nuclear export, translation and stability of mRNA in cells. 6 A modification regulation is dynamic and reversible. 6 The abundance and effect of A on RNA are determined by the dynamic interplay between its methyltransferases, binding proteins, and demethylases. 6 A binding protein can recognize and bind m 6 A site, ultimately determines m 6 A modified mRNA fate. These proteins are specific to m 6 The A site binds and recruits various regulatory or functional proteins to control the fate of methylated mRNA.

[0003] Many different types of m have been discovered 6 A reader, in which the YTH domain is m 6 A is the most common "reader" that can be directly connected to m 6 A binds. Proteins containing the YTH domain are involved in the biological processes of RNA splicing, nuclear export, mRNA translation and decay. Proteins containing the YTH domain in human cells are divided into three categories, YTH domain protein 1, YTH domain protein 2 and YTH domain family (YTHDF). The YTHDF protein has three members (YTHDF1, YTHDF2, YTHDF3), which are highly homologous and have similar overall amino acid lengths. Functionally, the YTHDF protein is the main "reader" in the human cytoplasm. The YTHDF protein is expressed through m 6 A modification binds to mRNA and has an important influence on the translation and stability of mRNA. It plays an important role in various physiological processes, including stem cell differentiation, neural development, and immune regulation.

[0004] At present, only a few YTHDF small molecule inhibitors have been reported, such as Ebselen, DC-Y13-27, etc., and they all have the disadvantages of poor inhibitory activity and poor drugability. In addition, no YTHDF small molecule inhibitor has entered the clinical stage so far, and there is still a need to develop new YTHDF small molecule inhibitors. Therefore, in vitro inhibitory activity test methods and high-throughput screening methods are crucial to the development of YTHDF small molecule inhibitors. Accurate, stable and reliable activity test methods are helpful to discover novel lead compounds, guide the structural optimization of lead compounds and obtain candidate drug molecules with better activity, laying the foundation for the development of therapeutic drugs targeting YTHDF.

[0005] The principle of fluorescence polarization assay (FP) is that when small fluorescent labeled molecules (probes) are excited by plane polarized light, the emission light emitted by the free probe is highly depolarized because it flips rapidly. However, when the probe binds to a much larger molecule, its rotation speed slows down, and most of the emitted light is polarized. FP is used to detect interactions between various molecules, such as interactions between proteins, proteins and nucleic acids, and receptors and ligands. FP detection does not require separation of bound ligands and free ligands. This allows ligand binding to be quantified without disturbing the equilibrium, making it suitable for measuring low-affinity interactions. At present, a high-throughput, economical and rapid YTHDF protein screening method has not yet been developed, and compounds suitable as YTHDF fluorescent probes are still in urgent need of development. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a YTHDF fluorescent probe with specific binding and fluorescent properties, and also to provide a preparation method and application of the above-mentioned YTHDF fluorescent probe, so as to provide a basis for the subsequent high-throughput screening of YTHDF-targeted drugs.

[0007] Technical solution: The present invention discloses a YTHDF fluorescent probe, the chemical formula of which is 5'-FAM-CCGG (m 6 A) CUGU, the structure is shown in formula I:

[0008]

[0009] Among them, C is cytosine ribonucleotide, G is guanine ribonucleotide, U is uracil ribonucleotide, m 6 A is N 6 -Methyladenosine.

[0010] The preparation method of the YTHDF fluorescent probe comprises the following steps:

[0011] (1) The target RNA sequence CCGG (m 6 A) CUGU;

[0012] (2) 5'-FAM and RNA sequence CCGG (m 6 A) CUGU undergoes an amide condensation reaction to obtain a crude product;

[0013] (3) Purify the labeled RNA using high performance liquid chromatography or SPPS system to remove unreacted 5'-FAM and byproducts to obtain the YTHDF fluorescent probe.

[0014] Wherein, in step (1), the solid phase peptide synthesis technology adopts a solid phase carrier of controlled pore glass spheres CPG, and the synthesis units are four phosphoramidite monomers of A, U, C, and G;

[0015] Wherein, in step (1), the solid phase peptide synthesis technology includes four reaction cycles of deprotection, condensation, oxidation and capping.

[0016] Wherein, in step (1), after solid phase synthesis is completed, m 6 A modified phosphoramidite monomer, the synthesis column is transferred to a centrifuge tube, ammonia methanol solution is added for aminolysis, and after the aminolysis treatment, a deprotection reagent dimethylethanolamine hydrofluoride is added to remove the tert-butyldimethoxysilyl TBDMS protecting group on RNA.

[0017] Wherein, in step (2), the temperature of the amide condensation reaction is room temperature, and the reaction time is 12 to 24 hours.

[0018] Wherein, in step (2), the organic solvent used in the amide condensation reaction is one or more of tetrahydrofuran, acetonitrile, N,N-dimethylformamide or dimethyl sulfoxide, and the condensing agent used is one or more of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATU, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate HBTU, 1-hydroxybenzotriazole HOBT, N-hydroxy-7-azabenzotriazole HOAT, and benzotriazole hexafluorophosphate-1-oxytris(dimethylamino)phosphorus one or more.

[0019] The above YTHDF fluorescent probe can also be used in the screening of YTHDF protein ligands.

[0020] The application is to mix a YTHDF fluorescent probe, a protein containing a YTH binding domain and a test compound to obtain a mixture; use fluorescence polarization technology to measure the polarization value of the mixture, and confirm that the test compound is a ligand of the YTHDF protein based on the polarization value.

[0021] When the polarization value decreases by more than 25% of the detection window, the compound to be tested can be determined to be a ligand of the YTHDF protein.

[0022] In the application, the buffer solution used is borate borax buffer Na2B4O7-H3BO3, tris(hydroxymethylaminomethane) buffer Tris, phosphate buffer PBS or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer HEPES; preferably Na2B4O7-H3BO3; the pH of the buffer system is 7.4-7.8, preferably 7.4; the ionic strength of the buffer solution is 50-100mM, preferably 50mM Na2B4O7-H3BO3 plus 50mM KCl solution; the probe concentration is 90-120nM, preferably 110nM.

[0023] Principle of the invention: The YTHDF fluorescent probe of the present invention adopts an automated solid-phase synthesis method to synthesize RNA sequences, solving the problems of chemical stability, sequence specificity, purity and yield; a modular synthesis strategy is adopted to accurately control the reaction conditions and yield of each step, reducing the synthesis and difficulty of by-products.

[0024] Experiments show that the probe molecules of the present invention not only maintain the original specific binding ability, but also, compared with 5'-FAM alone, the introduction of RNA sequences optimizes the intramolecular electron transfer process, thereby improving the fluorescence emission efficiency. In addition, the present invention specially selects the Na2B4O7-H3BO3 buffer solution to provide a stable chemical environment; the pH of the system (7.4-7.8) is specially selected to ensure efficient binding of the fluorescent probe to the target protein and inhibit the occurrence of side reactions; the ionic strength range (50-100mM) is specially selected to promote the effective collision of the probe molecule with the target and ensure the stability and uniformity of the system; the probe concentration (90-120nM) is specially selected to fully bind to the protein and avoid interference caused by nonspecific adsorption. In summary, the fluorescent probe 5'-FAM-CCGG (m 6 A) CUGU has fluorescent properties and has good specific binding to YTHDF. The compound is used as a fluorescence polarization probe, and the YTHDF ligand screening and ligand affinity level evaluation can be achieved through a competitive method. The screening method established based on the present invention obtains a fluorescent probe of the YTHDF protein, which provides a basis for the subsequent high-throughput screening of YTHDF-targeted drugs.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The YTHDF fluorescent probe of the present invention has fluorescent characteristics and has good specific binding to YTHDF; (2) The compound is used as a fluorescence polarization probe and the fluorescence polarization technology is adopted to realize the screening of YTHDF ligands and the evaluation of ligand affinity levels. A YTHDF small molecule high-throughput screening scheme established by fluorescence polarization can be used for high-throughput screening of drugs, and has the characteristics of being cheap, stable, rapid, and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The curves of the fluorescence intensity of the YTHDF fluorescent probe of the present invention changing with the concentration in different buffer systems;

[0027] Figure 2 The curve of the fluorescence intensity of the YTHDF fluorescent probe of the present invention in Na2B4O7-H3BO3, pH=7.2 buffer solution changes with the concentration;

[0028] Figure 3 Verification of the statistical indicators of high-throughput screening of the YTHDF fluorescent probe of the present invention;

[0029] Figure 4 The polarization value changes of the YTHDF fluorescent probe of the present invention under different concentrations of DMSO;

[0030] Figure 5 The protein-probe curve of the YTHDF fluorescent probe of the present invention binding to the YTHDF protein; Figure 6 is the effect of DMSO concentration on the experiment; Figure 7 This is the saturation curve of the saturation analysis experiment. DETAILED DESCRIPTION

[0031] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0032] Example 1

[0033] The preparation method of the YTHDF fluorescent probe of the present invention specifically comprises the following steps:

[0034] (1) RNA sequence CCGG (m 6 A) Preparation of CUGU:

[0035] Using solid phase synthesis technology, CPG is used as a solid phase carrier, and four phosphoramidite monomers A, U, C and G are used as synthetic units. In the 3'→5' direction, the target sequence is gradually connected through four reactions of deprotection, condensation, oxidation and capping. During the synthesis process, tert-butyldimethylsilyl chloride is introduced to protect m 6 A modified phosphoramidite monomer. After the synthesis is completed, the synthesis column is transferred to a centrifuge tube, ammonia methanol solution is added, and aminolysis is performed at 100°C for 2 hours to remove the protective group of RNA itself. The RNA after aminolysis is cooled at 0°C for 5 minutes, and then the deprotection reagent dimethylethanolamine hydrofluoride is added, and the deprotection treatment is performed at 70°C for 30 minutes to remove the TBDMS protective group on RNA. After the RNA after deprotection is cooled again, sodium chloride solution and ethanol are added, and it is allowed to stand at -22 to -18°C for 3 hours, and then centrifuged at 11000 to 14000rpm for 2 to 5 minutes and the precipitate is collected to obtain pure RNA. Finally, the purified RNA is quality tested. The concentration of RNA was determined to be 122ng / μL by NanoDrop, OD260 / OD280 was determined to be 2.2 by UV spectrophotometer, and the RNA purity was verified to be 98.2% by high performance liquid chromatography HPLC.

[0036] (2) 5'-FAM-CCGG (m 6 A) Preparation of CUGU:

[0037] Dissolve FAM (100 mg, 0.26 mmol) in 5 mL of DMF solution, add HATU (151 mg, 0.39 mmol), stir at room temperature for 30 min, then add the RNA sequence in step (1), then add TEA (111 μL, 0.79 mmol), and stir at room temperature for 12 h. After the reaction is completed by TLC monitoring, add equal volumes of dichloromethane and water for three times of liquid extraction, combine the organic layers and concentrate under low pressure to remove the solvent to obtain a crude product as an orange-yellow oil;

[0038] (3) Purify the labeled RNA using HPLC or SPPS system to remove unreacted 5'-FAM and byproducts. Finally, a yellow final product (2.5 mg, yield: 16.1%, purity 96.15%) is obtained, which is the YTHDF fluorescent probe of the present invention.

[0039] Example 2

[0040] Expression and purification of YTH domain proteins:

[0041] YTH domain proteins are divided into three types: YTHDF1, YTHDF2 and YTHDF3. They are expressed in Escherichia coli BL21 (DE3) using pET-28a(+), PGEX-6P-1 and pETDuet-1, respectively. Take 50 μL of BL21 competent cells melted on ice, add 4 μL of the target plasmid and mix gently, and let it stand on ice for 25 minutes. Then heat shock in a 42°C water bath for 45 seconds, quickly put it back on ice and let it stand for 2 minutes. Add 700 μL of sterile LB medium (1% tryptone, 0.5% yeast extract and 1% NaCl) without antibiotics to the centrifuge tube, mix well and place it in a shaker at 37°C and 200 rpm for 60 minutes. Take 100 μL and spread it on an LB medium plate containing the corresponding antibiotics (50 mg / L) and grow at 37°C for 12 hours. Pick a monoclonal colony in a small test tube containing about 10 mL of the corresponding antibiotic medium and culture it at 37°C overnight. Take 5 mL of bacterial solution and culture it in a 500 mL bottle at 37°C until the OD value is 0.6-0.8. Induce with 1 mM IPTG at 16°C for 12 h and harvest the bacteria. The protein was extracted and purified by TM pure25 (GE Healthcare, Life Sciences) automatic purification system. YTHDF1 and YTHDF3 were purified and eluted by His column (20mM Tris, 500mM NaCl, 500mM imidazole, pH = 8.0), and YTHDF2 was purified and eluted by GST column (10mM GSH, 50mM Tris, 200mM NaCl, 1mM DTT, pH = 8.0). The extracted protein was dialyzed in PBS for 12h and then placed at -80°C for use. Through the above experiment, 400μL of 30μM YTH domain protein can be obtained for every 1L of E. coli culture medium.

[0042] Example 3

[0043] Establishment of fluorescence polarization probe FP method:

[0044] Fluorescence polarization was measured using a SpectraMax Multi-Mode Microplate Reader (Molecular Devices), and experiments were performed using 384-well, black round-bottom, NBS-surface, polystyrene microplates (Corning).

[0045] (1) Determination of the type of buffer solution: In order to determine the most suitable buffer solution system for the FP method, the fluorescence polarization values ​​of the fluorescent probe (the target molecule prepared in Example 1) at different concentrations (the starting probe concentration was 10 μM, and the concentration was diluted 3 times to 10 concentrations) in different buffer solutions were detected to determine the protein-probe curve (only YTHDF1 was used for condition screening of the protein). The types of buffer solutions include: Na2B4O7-H3BO3, Tris, PBS, and HEPES. The results are shown in Figure 2. Figure 1 As shown, Na2B4O7-H3BO3 with the lowest protein concentration was selected as the basic buffer solution for the FP method.

[0046] (2) Determination of pH of buffer solution: In order to determine the most suitable buffer solution system for the FP method, the fluorescence polarization value of the fluorescent probe (the target molecule prepared in Example 1) under different pH conditions (7.4, 7.6, 7.8, 8.0) was detected, and the probe-protein curve was determined (only YTHDF1 was used for conditional screening of the protein). The results are shown in Table 1. Figure 2 As shown, Na2B4O7-H3BO3 with the most suitable window, pH=7.4 was selected as the buffer system.

[0047] (3) Determination of the ionic strength of the buffer solution: The ionic strength affects the viscosity of the solution and the charge interaction between molecules, thereby affecting the rotation rate and binding kinetics of the fluorescent labeled molecules. First, a series of boric acid borax buffer solutions of different concentrations were prepared (0.05 mol / L, 0.06 mol / L, 0.075 mol / L), and the pH value was kept at 7.4. The fluorescence polarization value of the fluorescent probe (the target molecule prepared in Example 1) was detected in the buffer system of the above concentration, and the probe-protein curve was measured (the protein was only screened using YTHDF1). The result was that the set boric acid borax concentration did not have a significant effect on the experimental results.

[0048] Next, a series of KCl solutions of different concentrations (20mM, 50mM, 100mM, 150mM) were prepared and mixed with 0.05mol / L Na2B4O7-H3BO3 buffer, maintaining pH = 7.4, and measuring the probe-protein curve (only YTHDF1 was used for conditional screening of protein). The results are shown in the figure. Figure 3 As shown, the results obtained show that adding 50mM KCl to the 0.05mol / L Na2B4O7-H3BO3, pH=7.4 buffer solution system can increase the window.

[0049] (4) Determination of probe concentration: In order to determine the optimal probe concentration of the FP method, the fluorescence polarization value of the fluorescent probe (the target compound prepared in Example 1) at different concentrations (the initial probe concentration was 10 μM, and 14 concentrations were diluted by 3-fold concentration gradient) in Na2B4O7-H3BO3 buffer solution was detected to determine the probe curve. The results are shown in Figure 4 As shown, a concentration of 1 nM close to the lower platform was selected as the optimal probe concentration for the FP method.

[0050] (5) High-throughput screening statistical index verification: Z factor is a statistical index for judging whether a test method is suitable for high-throughput screening. The Z factor value is meaningful only when it is in the range of -1<Z≤1. The closer the Z value is to 1, the more reliable the high-throughput test method is. Z factor values ​​between 0.5-1 are acceptable. In order to verify whether the FP method of the present invention is suitable for high-throughput screening, the S / N and Z factor of the FP method are calculated. The results are shown in Figure 2. Figure 5 As shown, the S / N was 32.16 and the Z factor value was 0.72, which indicated that this method was suitable for high-throughput screening.

[0051] (6) Effect of DMSO concentration: The effect of DMSO concentration on the experiment was investigated. The experimental results are as follows: Figure 6 As shown, when the DMSO concentration is lower than 7%, there is basically no significant effect on the FP method.

[0052] (7) Saturation assay: YTHDF1 protein (obtained in Example 2) at different concentrations (initial probe concentration was 25 μM, 3-fold concentration gradient dilution with 10 concentrations) was mixed with the above-determined fluorescence polarization probe and incubated for 30 minutes, and then the fluorescence polarization value of the system was measured.

[0053] The implementation scheme of the saturation experiment in the present invention is as follows: YTHDF1 is diluted with Na2B4O7-H3BO3 buffer, mixed with 1nM fluorescence polarization probe, covered with a plate and incubated with shaking at room temperature for 20 minutes, the fluorescence polarization value is measured, and a saturation curve is drawn for analysis. The results are shown in FIG. Figure 7 As shown, a concentration of 115 nM close to the upper platform was selected as the optimal protein concentration for the FP method.

[0054] Therefore, the YTHDF fluorescent probe of the present invention is used as a tool molecule to establish a YTHDF small molecule high-throughput screening method through the fluorescence polarization (FP) method. This scheme can be used for high-throughput screening of targeted YTHDF ligand compounds, which has the characteristics of being cheap, stable, rapid and efficient.

Claims

1. A YTHDF fluorescent probe, characterized in that The chemical formula of the YTHDF fluorescent probe is 5'-FAM-CCGG (m 6 A) CUGU, the structure is shown in formula I: Among them, C is cytosine ribonucleotide, G is guanine ribonucleotide, U is uracil ribonucleotide, m 6 A is N 6 -Methyladenosine.

2. A method for preparing the YTHDF fluorescent probe according to claim 1, characterized in that: The preparation method comprises the following steps: (1) The target RNA sequence CCGG (m 6 A) CUGU; (2) 5'-FAM and RNA sequence CCGG (m 6 A) CUGU undergoes an amide condensation reaction to obtain a crude product; (3) Purify the labeled RNA using high performance liquid chromatography or SPPS system to remove unreacted 5'-FAM and byproducts to obtain the YTHDF fluorescent probe.

3. The preparation method according to claim 2, characterized in that: In step (1), the solid phase peptide synthesis technology uses a solid phase carrier of controlled pore glass ball CPG, and the synthesis units are four phosphoramidite monomers A, U, C, and G; the solid phase peptide synthesis technology includes four reaction cycles of deprotection, condensation, oxidation and capping.

4. The preparation method according to claim 2, characterized in that: In step (1), after solid phase synthesis is completed, m 6 A modified phosphoramidite monomer, the synthesis column is transferred to a centrifuge tube, ammonia methanol solution is added for aminolysis, and after the aminolysis treatment, a deprotection reagent dimethylethanolamine hydrofluoride is added to remove the tert-butyldimethoxysilyl TBDMS protecting group on RNA.

5. The preparation method according to claim 2, characterized in that: In step (2), the temperature of the amide condensation reaction is room temperature, and the reaction time is 12 to 24 hours.

6. The preparation method according to claim 2, characterized in that: In step (2), the organic solvent used in the amide condensation reaction is one or more of tetrahydrofuran, acetonitrile, N,N-dimethylformamide or dimethyl sulfoxide, and the condensing agent used is one or more of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole, N-hydroxy-7-azabenzotriazole, and benzotriazole hexafluorophosphate-1-oxytris(dimethylamino)phosphine.

7. Use of the YTHDF fluorescent probe according to claim 1 in screening YTHDF protein ligands.

8. The use according to claim 7, characterized in that: The application is to mix the YTHDF fluorescent probe, a protein containing a YTH binding domain and a test compound to obtain a mixture; use fluorescence polarization technology to measure the polarization value of the mixture, and confirm that the test compound is a ligand of the YTHDF protein based on the polarization value.

9. The use according to claim 7, characterized in that: When the polarization value decreases by more than 25% of the detection window, the compound to be tested can be determined to be a ligand of the YTHDF protein.

10. The use according to claim 7, characterized in that: The application adopts a buffer solution of boric acid borax buffer Na2B4O7-H3BO3, tris(hydroxymethylaminomethane) buffer, phosphate buffer or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer; the pH of the buffer system is 7.4-7.8; the ionic strength of the buffer solution is 50-100mM, and the probe concentration is 90-120nM.

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