Ubiquitin modified nucleosome fluorescence resonance energy transfer probe and preparation method thereof

Abstract

The invention relates to a ubiquitin modified nucleosome fluorescence resonance energy transfer probe and a preparation method thereof, and belongs to the technical field of protein synthesis. According to the invention, ubiquitin-modified histone is constructed by integrating polypeptide solid-phase synthesis and fragment connection technologies, fluorescent molecular pairs capable of forming fluorescence resonance energy transfer are respectively marked on ubiquitin and DNA, and then the ubiquitin-modified histone and another three histone are assembled into an octamer; and carrying out gradient dialysis on the fluorescence labeled DNA to form a complete nucleosome probe. The probe prepared by the method can be specifically hydrolyzed by histone deubiquitination enzyme, has substrate characteristics close to physiological status, and realizes detection of histone deubiquitination enzyme activity and high-throughput screening of inhibitors through fluorescence resonance signal change. The preparation method has the advantages of high universality, accurate molecular structure and large-scale preparation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein synthesis, and particularly relates to a ubiquitin-modified nucleosome fluorescence resonance energy transfer probe and a preparation method thereof. BACKGROUND

[0002] Histone ubiquitination is one of the core post-translational modifications involved in epigenetic regulation, and plays an important role in almost all DNA-related cellular processes such as replication, transcription, and damage repair. Like many post-translational modifications of proteins, histone ubiquitination is a dynamic and reversible process, and its level is precisely regulated by histone deubiquitinases, which are important functional proteases under normal physiological conditions. Studies have shown that the dysfunction of histone deubiquitinases can lead to imbalance of histone ubiquitination homeostasis and abnormal changes in epigenetic modification patterns, thereby being closely related to pathological processes such as tumors, cardiovascular diseases, and neurodegenerative diseases, and has become a new type of drug target with great potential. Therefore, studying the enzymatic activity of histone deubiquitinases and discovering specific inhibitors are of great significance for understanding ubiquitination-mediated epigenetics and disease intervention.

[0003] In recent years, various ubiquitin-based protein probes have been designed and synthesized to detect the activity of deubiquitinases and screen inhibitors. For example, a probe in which the C-terminal of ubiquitin is condensed with a fluorescent molecule such as coumarin or rhodamine through an amide bond can detect the activity of deubiquitinases through the principle of fluorescence enhancement after enzymatic hydrolysis; and a ubiquitin probe in which the C-terminal is modified with Lys-TAMRA through an isopeptide bond can detect the activity of deubiquitinases through the change in fluorescence polarization after enzymatic hydrolysis. However, these probes lack substrate specificity and have limitations in the detection of the activity of histone deubiquitinases. In fact, as a class of deubiquitinases specifically targeting chromatin, histone deubiquitinases usually rely on the overall structure of nucleosomes for recognition and catalysis, and have significant site specificity, so they cannot effectively recognize the above-mentioned molecular probes with ubiquitin as the backbone. On the other hand, in the discovery of epigenetic intervention molecules targeting histone post-translational modification, modified histone short peptides are usually used as substrates for high-throughput screening, but they face the following limitations: first, histone deubiquitinases may not be able to recognize ubiquitin-modified short peptides, and even if they can recognize them, there is a significant difference in activity; second, modified short peptides cannot reflect the widespread cross-talk between histone modifications; third, using modified short peptides as screening substrates cannot investigate the extensive interaction interface between the enzyme and the nucleosome, but is limited to the interaction between the active pocket of the enzyme and the modification, resulting in the lack of subtype selectivity of the discovered inhibitors, which may be one of the important reasons for the low success rate of epigenetic drug research and development. SUMMARY

[0004] To solve the above technical problems, the present application provides a ubiquitin-modified nucleosome fluorescence resonance energy transfer (FRET) probe and a preparation method thereof. The present application constructs a ubiquitin-modified histone through polypeptide solid-phase synthesis and fragment ligation technology, labels a fluorescence molecule pair capable of forming fluorescence resonance energy transfer on the ubiquitin and DNA respectively, then assembles the ubiquitin-modified histone with another three histones into an octamer, and forms a complete nucleosome probe through gradient dialysis with the fluorescence-labeled DNA.

[0005] The first object of the present application is to provide a preparation method of a ubiquitin-modified nucleosome fluorescence resonance energy transfer probe, comprising the following steps: Step S1, ubiquitinating the first histone to obtain a ubiquitin-modified histone; Step S2, labeling the ubiquitin-modified histone with a donor fluorescence molecule and labeling DNA with an acceptor fluorescence molecule to obtain fluorescence-labeled ubiquitin-modified histone and fluorescence-labeled DNA; or labeling the ubiquitin-modified histone with an acceptor fluorescence molecule and labeling DNA with a donor fluorescence molecule to obtain fluorescence-labeled ubiquitin-modified histone and fluorescence-labeled DNA; Step S3, mixing and assembling the ubiquitin-modified histone with a second histone, a third histone and a fourth histone to obtain a histone octamer; Step S4, mixing the histone octamer with the fluorescence-labeled DNA to obtain the ubiquitin-modified nucleosome fluorescence resonance energy transfer probe.

[0006] Further, in step S1, the first histone is H2A, H2B, H3 or H4.

[0007] In an embodiment of the present application, the first histone is H2A.

[0008] Further, in step S1, the lysine residue of the first histone is ubiquitinated.

[0009] In an embodiment of the present application, the lysine residue at position 119 of the first histone is ubiquitinated.

[0010] Further, in step S1, the ubiquitination of the first histone comprises the following steps: Step S11, respectively preparing a first ubiquitin truncated body and a second ubiquitin truncated body, and connecting to obtain a ubiquitin hydrazine; Step S12, respectively truncating the amino terminal and the carboxyl terminal of the first histone to obtain a histone amino terminal truncated body and a histone carboxyl terminal truncated body, and connecting the ubiquitin hydrazine with the histone carboxyl terminal truncated body to obtain a ubiquitin-modified histone short peptide; Step S13, connecting the ubiquitin-modified histone short peptide with the histone amino-terminal truncation to obtain the ubiquitin-modified histone.

[0011] Further, the ubiquitin hydrazide is coupled with the histone carboxy-terminal truncation through a coenzyme-mediated polypeptide hydrazide connection.

[0012] In an embodiment of the present application, the coenzyme has the following structural formula: .

[0013] Further, in step S2, the ubiquitin is connected with the fluorescent molecule through a bio-orthogonal reaction.

[0014] Further, the ubiquitin is connected with the fluorescent molecule through a thiol-maleimide addition reaction.

[0015] Further, the ubiquitin contains a cysteine, which is connected with the fluorescent molecule through a thiol-maleimide addition reaction.

[0016] In an embodiment of the present application, the cysteine is located at the amino terminal of the ubiquitin.

[0017] Further, in step S2, the donor fluorescent molecule is Cy3 and the acceptor fluorescent molecule is Cy5.

[0018] A second object of the present application is to provide a ubiquitin-modified nucleosome fluorescent resonance energy transfer probe prepared by any of the above preparation methods.

[0019] Further, the ubiquitin-modified nucleosome fluorescent resonance energy transfer probe has the structure as shown in Figure 1 .

[0020] A third object of the present application is to provide any of the following applications of the above ubiquitin-modified nucleosome fluorescent resonance energy transfer probe: (1) application in detection of histone deubiquitinase activity; (2) application in screening of histone deubiquitinase inhibitors.

[0021] Advantages of the present application: (1) The preparation method of the ubiquitin-modified nucleosome fluorescent resonance energy transfer probe provided by the present application adopts protein chemical synthesis and in vitro nucleosome assembly preparation, is suitable for ubiquitin modification of any lysine residue of histone, and has the advantages of high universality and precise molecular structure. (2) The ubiquitin modified nucleosome fluorescence resonance energy transfer probe provided by the application takes a complete nucleosome as a skeleton, retains the spatial conformation of histone modification, has the substrate characteristics close to the physiological state, is beneficial to maintaining the specific interaction of the enzyme and the substrate, ensures the accurate measurement of the enzyme activity, significantly reduces the in vivo and in vitro activity difference caused by the ubiquitin or the ubiquitin modified short peptide as the substrate, overcomes the defect that the traditional ubiquitin probe or the short peptide substrate cannot simulate the chromatin environment, and significantly improves the accuracy of the enzyme activity detection. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to make the content of the application more easily understood, the application will be further described in detail below according to specific embodiments of the application and in conjunction with the drawings, in which: Figure 1 A structure schematic diagram of the ubiquitin modified nucleosome fluorescence resonance energy transfer probe provided by the application; Figure 2 A synthesis route map of the Cy5 fluorescence labeled K119 ubiquitinated histone H2A (H2AK119Ub Cy5 ) provided by the application; Figure 3 Characterization of the ubiquitin truncation body 1 in the embodiment 1 of the application, wherein A is a reversed-phase high performance liquid chromatography result, and B is an electrospray ionization mass spectrometry result; Figure 4 Characterization of the ubiquitin truncation body 2 in the embodiment 1 of the application, wherein A is a reversed-phase high performance liquid chromatography result, and B is an electrospray ionization mass spectrometry result; Figure 5 Characterization of the histone C-terminal truncation body in the embodiment 1 of the application, wherein A is a reversed-phase high performance liquid chromatography result, and B is an electrospray ionization mass spectrometry result; Figure 6 Characterization of the histone N-terminal truncation body hydrazide in the embodiment 2 of the application, wherein A is a reversed-phase high performance liquid chromatography result, and B is an electrospray ionization mass spectrometry result; Figure 7 Characterization of the ubiquitin hydrazide in the embodiment 3 of the application, wherein A is a reversed-phase high performance liquid chromatography result, and B is an electrospray ionization mass spectrometry result; Figure 8 Characterization of the ubiquitin modified histone short peptide in the embodiment 4 of the application, wherein A is a reversed-phase high performance liquid chromatography result, and B is an electrospray ionization mass spectrometry result; Figure 9 Characterization of the ubiquitin modified histone in the embodiment 5 of the application, wherein A is a reversed-phase high performance liquid chromatography result, and B is an electrospray ionization mass spectrometry result; Figure 10 Characterization of the fluorescence labeled ubiquitin modified histone in the embodiment 6 of the application, wherein A is a reversed-phase high performance liquid chromatography result, and B is an electrospray ionization mass spectrometry result; Figure 11 Characterization of the histone octamer and Cy3-601 DNA in Example 7 of the present invention, wherein A is the size exclusion chromatography and polyacrylamide gel electrophoresis results of the octamer, and B is the ion exchange chromatography and agarose gel electrophoresis results of the DNA; Figure 12 This is the result of non-denaturing polyacrylamide gel electrophoresis of the ubiquitin-modified nucleosome fluorescence resonance energy transfer probe in Example 7 of the present invention; Figure 13 This is the fluorescence detection curve of the histone deubiquitinating enzyme USP16 hydrolyzing the ubiquitin-modified nucleosome fluorescence resonance energy transfer probe in Example 8 of the present invention, wherein A is the fluorescence intensity corresponding to the enzyme concentration change curve, and B is the fluorescence intensity corresponding to the enzymatic hydrolysis time change. DETAILED DESCRIPTION

[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0024] The structure of the ubiquitin-modified nucleosome fluorescence resonance energy transfer probe constructed by the present invention is as follows: Figure 1 shown.

[0025] Taking K119 ubiquitinated histone H2A as an example, the chemical synthesis route of the fluorescently labeled ubiquitinated histone constructed in the embodiment of the present invention is as follows: Figure 2 K119 ubiquitinated histone H2A (H2AK119Ub Cy5 ) is divided into four polypeptide fragments, including ubiquitin truncation 1, ubiquitin truncation 2, histone C-terminal truncation, and histone N-terminal truncation hydrazide. The first three are prepared by peptide solid-phase synthesis, while the latter is prepared by recombinant expression. Ubiquitin truncation 1 has a GC sequence fused to its N-terminus, and the sulfhydryl group is protected with an acetamidomethyl (Acm) group. The side chain amino group at K119 of the histone C-terminal truncation is coupled to a prosthetic group, glycine (aG), and the terminal cysteine ​​is protected with a thiazolidine (Thz). The histone N-terminal truncation is prepared by recombinant expression, and the terminal cysteine ​​is activated with 2-nitro-5-thiocyanatobenzoic acid (NTCB). The histone N-terminal truncation hydrazide is generated by in situ aminolysis.

[0026] After obtaining the four fragments, first, ubiquitin thiohydrazide 1 and ubiquitin thiohydrazide 2 were synthesized by polypeptide hydrazide ligation and desulfurization reaction; then, ubiquitin thiohydrazide was synthesized by auxiliary-mediated hydrazide ligation with histone C-terminal thiohydrazide, and removal of the auxiliary and Thz protecting group; thereafter, the hydrazide ligation was performed with the histone N-terminal thiohydrazide, and the desulfurization reaction was performed to synthesize the ubiquitin-modified histone; finally, the Acm protecting group of the cysteine in the ubiquitin-modified histone was removed, and addition reaction with Cy5 containing a maleimide group was performed to obtain H2AK119Ub Cy5 .

[0027] Example 1: Solid-phase synthesis and purification of ubiquitin and histone thiohydrazide The preparation of polypeptide hydrazide was carried out by a polypeptide synthesizer using hydrazine-substituted 2-Cl-Trt-Cl resin. The specific condensation conditions were as follows: 4.0 equivalents of Fmoc-protected amino acid, 3.8 equivalents of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 8.0 equivalents of N,N-diisopropylethylamine (DIEA), with N,N-dimethylformamide (DMF) as the solvent for the condensation reaction. The Fmoc protecting group was removed by treatment with 20% piperidine / DMF solution. After the synthesis was completed, the peptide chain was subjected to resin cleavage and deprotection using a trifluoroacetic acid (TFA) cleavage system (85% TFA, 5% H2O, 5% anisole, 2.5% phenol, and 2.5% 1,2-ethanedithiol). Subsequently, TFA was removed by nitrogen gas, and the crude peptide product was precipitated with pre-cooled ether.

[0028] The obtained crude peptide was purified by reverse-phase high-performance liquid chromatography (RP-HPLC). A phase was water containing 0.1% TFA, and B phase was acetonitrile containing 0.1% TFA. The crude peptide was dissolved in an appropriate volume of A phase, and 30-40 mg of crude product was dissolved in 3-4 mL of A phase. After complete filtration with a 0.22 μM microporous filter, RP-HPLC purification was performed, and the product was obtained after freeze-drying.

[0029] The RP-HPLC and electrospray ionization mass spectrometry (ESI-MS) characterization results of ubiquitin thiohydrazide 1 are shown as A and B in Figure 3 , respectively, and the structure is specifically: GC(Acm)MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF-NHNH2 (SEQ ID NO. 1), wherein Acm is an acetamidemethyl protecting group.

[0030] Ubiquitin thiohydrazide 2 was prepared according to the above method, and the RP-HPLC and ESI-MS characterization results are shown as Figure 4As shown in A and B in , the specific structure is: CGKQLEDGRTLSDYNIQKESTLHLVLRLRG-NHNH2 (shown in SEQ ID NO.2).

[0031] The histone C-terminal truncations were prepared according to the above method, and the RP-HPLC and ESI-MS characterization results were as follows: Figure 5 As shown in A and B in the figure, the specific structure is: C(Thz)QGGVLPNIQAVLLPKK(aG)TESHHKAKGK (shown in SEQ ID NO.3), wherein Thz is a thiazolidine protecting group, and aG is a prosthetic group - glycine condensed with the amino group of the lysine side chain ( Figure 2 shown).

[0032] Example 2: Synthesis of histone N-terminal truncated hydrazide The amino acid sequence of histone is shown in SEQ ID NO.4 and was obtained by recombinant expression in E. coli. A single colony of histone N-terminal truncated product was picked and placed in 30 mL LB medium containing 30 μL ampicillin and cultured overnight at 37°C and 200 rpm in a shaker. Subsequently, the bacterial solution was added to the LB medium at a ratio of 1:100, and 500 μL ampicillin was added at the same time. The culture was cultured at 37°C and 200 rpm for 2-3 hours. When the bacterial solution OD 600When the value is 0.6-0.8, IPTG is added to a final concentration of 0.4 mM to induce protein expression for 12-14 hours, and then the bacteria are centrifuged at 4°C and 4000 rpm for 30 minutes to collect the bacterial solution. Then, 40-50 mL of lysis buffer (30 mM Tris, 150 mM NaCl, 1 mM EDTA, 10 mM DTT, pH=7.5) is added to resuspend the bacteria, and the bacteria are broken by ice-bath ultrasonic crushing (ultrasonic power 30%, 3 seconds ON, 6 seconds OFF) for 45 minutes. The broken bacteria are centrifuged at 4°C and 12000 rpm for 20 minutes to collect the precipitate, which is washed twice with 20 mL of washing buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, 10 mM DTT, 1% Triton X-100, pH=7.5) and once with washing buffer without 1% Triton X-100. After centrifugation at 4°C and 12000 rpm for 20 minutes, the supernatant is removed, and the precipitate is dissolved in 30 mL of denaturing buffer (6.0 M Gn-HCl, 20 mM Tris, 10 mM DTT, pH=7.5), and the supernatant is collected by centrifugation and placed in a dialysis bag for dialysis overnight in 0.1% ddH2O. After centrifugation to remove the precipitate, the histone N-terminal truncation is purified by semi-preparative RP-HPLC, freeze-dried, and obtained. The amino acid sequence is shown in SEQ ID NO. 5.

[0033] The 160 mg of histone N-terminal truncation obtained above is dissolved in a buffer (6.0 M Gn-HCl, 0.1 M Na2HPO4, pH 9.0) with a final volume of 71 mL, and NH2NH2, TCEP, and NTCB are sequentially added to a final concentration of 50 mM, 500 μM, and 5 mM, respectively. After mixing, the pH is adjusted to 9.0 again, the film is sealed, and the reaction is carried out at 37°C in the dark. After 45 hours, the histone N-terminal truncation hydrazide is purified by RP-HPLC, and the RP-HPLC and ESI-MS characterization results are shown as A and B in FIG. 1, respectively. Figure 6

[0034] Example 3: Synthesis of ubiquitin hydrazide ​The 96.8 mg of ubiquitin truncated body 1 was dissolved in an acidic buffer (6.0 M Gn-HCl, 0.1 M NaH2PO4, pH 3.0), after ultrasonic dissolution, it was pre-cooled in an ice-salt bath at -15 ℃ for 5 minutes, 587.4 μL of 0.2 M NaNO2 solution was added, and the reaction was continuously stirred. After 25 minutes, 5.87 mL of 0.2 M p-mercapto phenyl acetic acid (MPAA) solution (dissolved in neutral buffer 6.0 M Gn-HCl, 0.1 M NaH2PO4, pH 7.0) was added, and continuously stirred in an ice-salt bath at -15 ℃ for 5 minutes. Subsequently, 62.5 mg of ubiquitin truncated body 2 was added, and after the protein was completely dissolved, the pH of the system was adjusted to 6.6 with 2 M NaOH, and the reaction was carried out at room temperature for about 6 hours. After the reaction was completed, an equal volume of 0.25 M tris (2-chloroethyl) phosphate (TCEP) solution was added and stirred at room temperature for 5 minutes, and after RP-HPLC purification and freeze-drying, 100.7 mg of the conjugated product was obtained.

[0035] The obtained conjugated product was dissolved in 15.65 mL of desulfurization buffer (6.0 M Gn-HCl, 0.2 M NaH2PO4, 500 mM TCEP, pH = 7.5), and the cyclic azo amidine initiator VA-044 was added to a final concentration of 0.1 M, and the reaction was carried out at 37 ℃ for 16 hours. After the reaction was completed, an equal volume of acidic buffer was added and stirred at room temperature for 5 minutes, and then purified by semi-preparative RP-HPLC and freeze-drying to obtain 73.7 mg of ubiquitin hydrazide, and the RP-HPLC and ESI-MS characterization results thereof are shown as A and B in Figure 7

[0036] Example 4: Synthesis of ubiquitin modified histone short peptide The 73.7 mg of ubiquitin hydrazide prepared in Example 3 was dissolved in an acidic buffer (6.0 M Gn-HCl, 0.1 M Na2HPO4, pH = 3.0), after pre-cooling in an ice-salt bath at -15 ℃, 273.6 μL of 0.2 M NaNO2 solution was added and the reaction was continuously carried out in the ice-salt bath. After 25 minutes, 2.75 mL of 0.2 M MPAA solution was added. After stirring for 2 minutes, the pH of the reaction system was adjusted to about 5.0 with 8.0 M NaOH, then 32.8 mg of the C-terminal truncated body of histone prepared in Example 2 was added, the pH of the system was adjusted to 6.5 with 2.0 M NaOH, and the reaction was carried out at room temperature for about 6 hours. After the reaction was completed, an equal volume of TCEP solution (0.25 M) was added and stirred at room temperature for 5 minutes, and after RP-HPLC purification and freeze-drying, the conjugated product was obtained.

[0037] ​The obtained ligation product was dissolved in 10 mL of a cleavage reagent (9.5 mL of TFA, 125 μL of dithiothreitol, 125 μL of anisole, and 250 μL of ddH2O), and the reaction was stirred at room temperature for 3 hours. After the reaction was completed, the TFA was removed by nitrogen flow until the reaction solution was concentrated to about 600 μL. 3-4 mL of pre-cooled ether was added to the concentrated solution for washing and precipitation, and the precipitate was collected by centrifugation at 12,000 rpm for 5 minutes at 4°C, and the operation was repeated 3-4 times. After the precipitate was air-dried, it was dissolved in 0.2 M methoxyamine hydrochloride prepared with an acidic buffer (6.0 M Gn·HCl, 0.1 M Na2HPO4, pH=3.0), and the pH was adjusted to 4, and the reaction was carried out at room temperature for 2 hours to remove the Thz protecting group. After the reaction was completed, the product was purified by RP-HPLC, and the molecular weight of the product was confirmed by LC-MS, and 33.5 mg of ubiquitin-modified histone short peptide was obtained by freeze-drying, and the RP-HPLC and ESI-MS characterization results thereof are shown as A and B in FIG. 1, respectively. Figure 8

[0038] Example 5: Synthesis of ubiquitin-modified histone The 42.4 mg of the hydrazide of the N-terminal truncated histone prepared in Example 2 was dissolved in an acidic buffer (6.0 M Gn·HCl, 0.2 M Na2HPO4, pH 3.0), and after being pre-cooled in an ice-salt bath at -15°C, 158.12 μL of a 0.2 M NaNO2 solution was added. After the ice-salt bath reaction for 25 minutes, 1.58 mL of a 0.3 M MPAA solution was added and reacted for 5 minutes. Then, 33.5 mg of the ubiquitin-modified histone short peptide prepared in Example 4 was added, and the pH was adjusted to 6.7 with 2.0 M NaOH for ligation reaction. After about 24 hours, an equal volume of a TCEP solution (0.25 M) was added and stirred at room temperature for 5 minutes, and the ligation product was purified by semi-preparative RP-HPLC and freeze-dried.

[0039] The obtained ligation product 34.46 mg was dissolved in 1.25 mL of a desulfurization buffer (6.0 M Gn·HCl, 0.2 M NaH2PO4, 500 mM TCEP, pH 7.5), and VA-044 was added to a final concentration of 0.1 M, and the pH was adjusted to 7.5 again, and the reaction was carried out at 37°C for 16 hours. After the reaction was completed, an equal volume of an acidic buffer was added and stirred at room temperature for 5 minutes, and then purified by RP-HPLC and freeze-dried to obtain 24.2 mg of ubiquitin-modified histone, and the RP-HPLC and ESI-MS characterization results thereof are shown as A and B in FIG. 2, respectively. Figure 9

[0040] Example 6: Fluorescent labeling of ubiquitin-modified histone ​​The ubiquitin-modified histone prepared in Example 5 (24.2 mg) was dissolved in a mixed solution of acetic acid and water (volume ratio of 1:1), 2.65 mg of silver acetate was added, and the reaction was stirred at room temperature for 12 hours. After the reaction was completed, an equal volume of 1.0 M DTT solution was added, and the mixture was stirred at room temperature for 5 minutes. The supernatant was collected by centrifugation, and the precipitate was washed 3-4 times. The supernatants were combined and purified by RP-HPLC and freeze-dried.

[0041] The freeze-dried protein (15 mg) was dissolved in a labeling buffer (6.0 M Gn-HCl, 20 mM Tris, 1.0 mM TCEP, pH=7.5), 1.0 mg of fluorescent molecule Cy5-maleimide (Cy5-mal) was added, and then the pH was adjusted to 7.5. The reaction was carried out at 37°C for 2 hours in the dark, and the product was purified by RP-HPLC and confirmed by LC-MS. After freeze-drying, the fluorescently labeled ubiquitin-modified histone H2AK119Ub was obtained. Cy5 The RP-HPLC and ESI-MS characterization results of Figure 10 are shown in A and B, respectively.

[0042] Example 7: Preparation of a ubiquitin-modified nucleosome FRET probe The fluorescently labeled ubiquitin-modified histone, histone H2B, histone H3, and histone H4 prepared in Example 6 (molar ratio of 1:1:1:1) were each dissolved in pre-cooled denaturing buffer (6.0 M Gn-HCl, 20 mM Tris-HCl, 5.0 mM DTT, pH=7.5) to a concentration of 2 mg / mL, and the solutions were placed on ice for 30 minutes. The four histone solutions were mixed and the concentration was adjusted to 1.0 mg / mL with denaturing buffer. The mixture was transferred to a dialysis bag and dialyzed against 1.0 L of renaturation buffer (2.0 M NaCl, 10 mM Tris-HCl buffer, 1.0 mM EDTA, 5.0 mM 2-ME, pH=7.5) at 4°C for 12 hours, with the buffer being replaced twice during dialysis. After concentration, the sample was purified by size exclusion chromatography, and the histone octamer was obtained by SDS-PAGE and stored at -80°C (results shown in Figure 11 A of FIG. 6).

[0043] Meanwhile, Wisdom 601 DNA (nucleotide sequence shown in SEQ ID NO. 6) was used as a template, and a Cy3-labeled nucleic acid sequence was used as a primer. The 601 DNA was amplified and labeled under conventional PCR conditions. After the PCR was completed, the amplification products were combined and purified by ion exchange chromatography. The Cy3-601 DNA was obtained by agarose gel electrophoresis (results shown in Cy5 FIG. 7).Figure 11 The Cy3-labeled primer sequence is as follows: F: 5'-Cy3-CTGGAGAATCCCGGTGC-3'; R: 5'-Cy3-GCACAGGATGTATATATCTGACAC-3'.

[0044] The obtained histone octamer and Cy3-601 DNA were mixed in a ratio of 1:0.9 (generally, 1:0.8-0.9) and 5.0 M NaCl, TE buffer (50 mM Tris, 5.0 mM EDTA) in sequence, and then placed in a dialysis tube and in 250 mL high-salt buffer (2.0 M NaCl, 50 mM Tris, 5.0 mM EDTA). The TE buffer was slowly dripped into the high-salt buffer at a flow rate of 1.25 mL / min. After the liquid level rose to 600 mL, the solution was poured to about 250 mL, and the TE buffer was slowly dripped into the high-salt buffer at a flow rate of 1 mL / min. After the liquid level rose to 1000 mL, the solution was dialyzed for 4-5 hours with HE buffer (50 mM HEPES, 5.0 mM EDTA) to obtain the ubiquitin-modified nucleosome fluorescence resonance energy transfer probe. The non-denaturing polyacrylamide gel electrophoresis result of the ubiquitin-modified nucleosome fluorescence resonance energy transfer probe is shown in FIG. 2B (which includes a Cy3 or Cy5 single-labeled nucleosome control). Figure 12

[0045] Example 8: Detection of the activity of histone deubiquitinase The histone deubiquitinase USP16 was diluted to a concentration of 0, 0.25, 0.5, 1.0, and 2.0 μM, respectively, and each group was set with three duplicate wells. The prepared ubiquitin-modified nucleosome fluorescence resonance energy transfer probe of Example 7 was added to each well to a final concentration of 500 nM. The fluorescence intensity value was measured by a multifunctional enzyme label instrument after incubation at room temperature for 50 minutes, and the change curve of the fluorescence intensity corresponding to the enzyme concentration was determined. The experimental results are shown in FIG. 3A. Figure 13

[0046] The enzyme concentration was fixed at 2.0 μM, and the prepared ubiquitin-modified nucleosome fluorescence resonance energy transfer probe of Example 7 was added to each well. The fluorescence intensity value was measured by a multifunctional enzyme label instrument after incubation at room temperature for 0, 5, 10, 20, and 50 minutes, respectively, and the change curve of the fluorescence intensity corresponding to the enzyme digestion time was determined. The experimental results are shown in FIG. 3B. It can be seen that the prepared ubiquitin-modified nucleosome fluorescence resonance energy transfer probe of Example 8 can be applied to detect the activity of histone deubiquitinase. Figure 13

[0047] ​​​Obviously, the above embodiments are merely example for clearly illustrating, and are not limitation to the embodiments. For ordinary skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments need not and can not be exhausted. The obvious changes or variations derived therefrom are still within the scope of the present invention.

Claims

1. A method for preparing a ubiquitin-modified nucleosome fluorescence resonance energy transfer probe, characterized in that: The following steps are involved: Step S1, performing ubiquitination modification on the first histone to obtain ubiquitin-modified histone; Step S2, using a donor fluorescent molecule to label the ubiquitin-modified histone, and using an acceptor fluorescent molecule to label DNA, to obtain fluorescently labeled ubiquitin-modified histone and fluorescently labeled DNA; or Using an acceptor fluorescent molecule to label the ubiquitin-modified histone, and using a donor fluorescent molecule to label DNA, to obtain fluorescently labeled ubiquitin-modified histone and fluorescently labeled DNA; Step S3, mixing the ubiquitin-modified histone with the second histone, the third histone, and the fourth histone to assemble a histone octamer; Step S4: mixing the histone octamer with DNA labeled with a fluorescent molecule to obtain the ubiquitin-modified nucleosome fluorescence resonance energy transfer probe.

2. The preparation method according to claim 1, wherein: In step S1, the first histone is H2A, H2B, H3 or H4.

3. The preparation method according to claim 1, wherein: In step S1, the lysine residues of the first histone are ubiquitinated.

4. The preparation method according to claim 1, wherein: In step S1, ubiquitination modification of the first group of proteins includes the following steps: Step S11, preparing a first ubiquitin truncation and a second ubiquitin truncation respectively, and connecting them to obtain ubiquitin hydrazide; Step S12, truncating the amino terminus and carboxyl terminus of the first histone, respectively, obtaining a histone amino terminus truncation and a histone carboxyl terminus truncation by recombinant expression or solid phase synthesis, and linking the ubiquitin hydrazide to the histone carboxyl terminus truncation to obtain a ubiquitin-modified histone short peptide; Step S13: Connecting the ubiquitin-modified histone short peptide to the histone amino-terminal truncation to obtain the ubiquitin-modified histone.

5. The preparation method according to claim 4, characterized in that: The ubiquitin hydrazide is linked to the histone carboxyl terminal truncation through a prosthetic group-mediated polypeptide hydrazide.

6. The preparation method according to claim 1, wherein: In step S2, the ubiquitin is linked to the fluorescent molecule through a bioorthogonal reaction.

7. The preparation method according to claim 6, characterized in that: The ubiquitin is linked to the fluorescent molecule via a thiol-maleimide addition reaction.

8. The preparation method according to claim 1, wherein: In step S2, the donor fluorescent molecule is Cy3, and the acceptor fluorescent molecule is Cy5.

9. The ubiquitin-modified nucleosome fluorescence resonance energy transfer probe prepared by the preparation method according to any one of claims 1 to 8.

10. Any of the following uses of the ubiquitin-modified nucleosome fluorescence resonance energy transfer probe according to claim 9: (1) Application in the detection of histone deubiquitinase activity; (2) Application in screening of histone deubiquitinase inhibitors.

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