ubiquitin-modified nucleosome fluorescence resonance energy transfer probe and its preparation method

Abstract

This invention relates to a ubiquitin-modified nucleosome fluorescence resonance energy transfer probe and its preparation method, belonging to the field of protein synthesis technology. This invention integrates peptide solid-phase synthesis and fragment ligation techniques to construct ubiquitin-modified histones, and labels ubiquitin and DNA with fluorescent molecular pairs capable of forming fluorescence resonance energy transfer. Then, the ubiquitin-modified histones are assembled with three other histones to form an octamer, which is then combined with fluorescently labeled DNA via gradient dialysis to form a complete nucleosome probe. The probe prepared using this method can be specifically hydrolyzed by histone deubiquitinating enzymes, exhibiting substrate characteristics close to physiological states. The activity of histone deubiquitinating enzymes and high-throughput screening of inhibitors can be achieved through changes in fluorescence resonance signal. This preparation method has the advantages of high universality, precise molecular structure, and large-scale production capability.

Description

Technical Field

[0001] This invention relates to the field of protein synthesis technology, and in particular to ubiquitin-modified nucleosome fluorescence resonance energy transfer probes and their preparation methods. Background Technology

[0002] Histone ubiquitination is a core post-translational modification involved in epigenetic regulation, playing a crucial role in almost all DNA-related cellular processes, including replication, transcription, and damage repair. Like many protein post-translational modifications, histone ubiquitination is a dynamic and reversible process, its levels precisely regulated by histone deubiquitinating enzymes, which are therefore essential functional proteases under normal physiological conditions. Studies have shown that dysfunction of histone deubiquitinating enzymes leads to imbalances in histone ubiquitination homeostasis and abnormal changes in the epigenetic modification profile, thus becoming closely related to pathological processes such as tumors, cardiovascular diseases, and neurodegenerative diseases, and representing a highly promising new drug target. Therefore, studying the enzymatic activity of histone deubiquitinating enzymes and discovering their specific inhibitors is of great significance for understanding ubiquitination-mediated epigenetics and disease intervention.

[0003] In recent years, various protein probes with ubiquitin as their backbone have been designed and synthesized for the detection of deubiquitinating enzyme activity and the screening of inhibitors. For example, probes with ubiquitin C-terminus condensed with fluorescent molecules such as coumarin and rhodamine via amide bonds can detect deubiquitinating enzyme activity through fluorescence enhancement after enzymatic hydrolysis. Meanwhile, ubiquitin probes with Lys-TAMRA modified at the C-terminus via isopeptide bonds can detect deubiquitinating enzyme activity through changes in fluorescence polarization after enzymatic hydrolysis. However, these probes lack substrate specificity, limiting their application in the detection of histone deubiquitinating enzyme activity. In fact, histone deubiquitinating enzymes, as a class of deubiquitinating enzymes that specifically target chromatin, typically rely on the overall nucleosome structure for recognition and catalysis, exhibiting significant site specificity, and therefore cannot effectively recognize the aforementioned molecular probes with ubiquitin as their backbone. On the other hand, in the discovery of epigenetic intervention molecules targeting histone post-translational modifications, modified histone short peptides are often used as substrates for high-throughput screening. However, this approach faces the following limitations: First, histone deubiquitinating enzymes may not recognize ubiquitin-modified short peptides, or even if they do, there are significant differences in activity. Second, modified short peptides cannot reflect the widespread cross-interactions between histone modifications. Third, using modified short peptides as screening substrates cannot examine the extensive interaction interface between enzymes and nucleosomes, limiting the interaction to the enzyme's active pocket and the modification itself. This results in the discovered inhibitors often lacking subtype selectivity, which may be one of the important reasons for the low success rate of epigenetic drug development. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a ubiquitin-modified nucleosome fluorescence resonance energy transfer (FRET) probe and its preparation method. This invention constructs ubiquitin-modified histones using peptide solid-phase synthesis and fragment ligation techniques, and labels ubiquitin and DNA with fluorescent molecular pairs capable of forming fluorescence resonance energy transfer. The ubiquitin-modified histones are then assembled with three other histones to form an octamer, which is then combined with fluorescently labeled DNA via gradient dialysis to form a complete nucleosome probe.

[0005] The first objective of this invention is to provide a method for preparing a ubiquitin-modified nucleosome fluorescence resonance energy transfer probe, comprising the following steps:

[0006] Step S1: The first group of proteins is ubiquitinated to obtain ubiquitinated histones;

[0007] Step S2: Label the ubiquitin-modified histone using a donor fluorescent molecule and label the DNA using an acceptor fluorescent molecule to obtain fluorescently labeled ubiquitin-modified histone and fluorescently labeled DNA; or

[0008] The ubiquitin-modified histone was labeled using a receptor fluorescent molecule, and the DNA was labeled using a donor fluorescent molecule to obtain fluorescently labeled ubiquitin-modified histone and fluorescently labeled DNA.

[0009] Step S3: The ubiquitin-modified histone is mixed with the second, third and fourth histones and assembled to obtain a histone octamer;

[0010] Step S4: Mix the histone octamer with the fluorescently labeled DNA to obtain the ubiquitin-modified nucleosome fluorescence resonance energy transfer probe.

[0011] Further, in step S1, the first histone is H2A, H2B, H3, or H4.

[0012] In one embodiment of the present invention, the first histone is H2A.

[0013] Further, in step S1, the lysine residues of the first group of proteins are modified by ubiquitination.

[0014] In one embodiment of the present invention, the lysine residue at position 119 of the first group of proteins is modified by ubiquitination.

[0015] Furthermore, in step S1, the ubiquitination modification of the first group of proteins includes the following steps:

[0016] Step S11: Prepare the first ubiquitin truncated form and the second ubiquitin truncated form respectively, and ligate them to obtain ubiquitin hydrazide;

[0017] Step S12: The amino terminus and carboxyl terminus of the first histone are truncated respectively to obtain the amino-terminal truncated histone and the carboxyl-terminal truncated histone. The ubiquitin hydrazide is then linked to the carboxyl-terminal truncated histone to obtain ubiquitin-modified histone short peptide.

[0018] Step S13: The ubiquitin-modified histone short peptide is linked to the amino-terminal truncated form of the histone to obtain the ubiquitin-modified histone.

[0019] Furthermore, the ubiquitin hydrazide is coupled to the histone carboxyl-terminal truncated form via a prosthetic group-mediated polypeptide hydrazide linker.

[0020] In one embodiment of the present invention, the structural formula of the auxiliary group is: .

[0021] Furthermore, in step S2, the ubiquitin and the fluorescent molecule are linked through a bioorthogonal reaction.

[0022] Furthermore, the ubiquitin is linked to the fluorescent molecule via a thiol-maleimide addition reaction.

[0023] Furthermore, the ubiquitin contains cysteine, which is linked to the fluorescent molecule via a thiol-maleimide addition reaction.

[0024] In one embodiment of the invention, the cysteine ​​is located at the amino terminus of ubiquitin.

[0025] Furthermore, in step S2, the donor fluorescent molecule is Cy3 and the acceptor fluorescent molecule is Cy5.

[0026] A second objective of this invention is to provide ubiquitin-modified nucleosome fluorescence resonance energy transfer probes prepared by any of the above-described preparation methods.

[0027] Furthermore, the structure of the ubiquitin-modified nucleosome fluorescence resonance energy transfer probe is as follows: Figure 1 As shown.

[0028] A third object of the present invention is to provide any of the following applications of the ubiquitin-modified nucleosome fluorescence resonance energy transfer probe described above:

[0029] (1) Application in the detection of histone deubiquitinating enzyme activity;

[0030] (2) Application in screening histone deubiquitinase inhibitors.

[0031] The beneficial effects of this invention are:

[0032] (1) The preparation method of ubiquitin-modified nucleosome fluorescence resonance energy transfer probe provided by the present invention adopts protein chemical synthesis and in vitro nucleosome assembly, which is suitable for ubiquitin modification of any lysine residue of histones and has the advantages of high universality and precise molecular structure.

[0033] (2) The ubiquitin-modified nucleosome fluorescence resonance energy transfer probe provided by the present invention uses the complete nucleosome as the backbone, retains the histone modification spatial conformation, and has substrate characteristics close to the physiological state. This is beneficial to maintain the specific interaction between the enzyme and the substrate, ensures the accurate measurement of enzyme activity, significantly reduces the in vivo and in vitro activity differences caused by ubiquitin or ubiquitin-modified short peptides as substrates, overcomes the defect that traditional ubiquitin probes or short peptide substrates cannot simulate the chromatin environment, and significantly improves the accuracy of enzyme activity detection. Attached Figure Description

[0034] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0035] Figure 1 A schematic diagram of the structure of the ubiquitin-modified nucleosome fluorescence resonance energy transfer probe provided by the present invention;

[0036] Figure 2 The Cy5-labeled K119 ubiquitinated histone H2A (H2AK119Ub) provided by this invention Cy5 Synthesis route diagram;

[0037] Figure 3 The above describes the characterization of ubiquitin truncated form 1 in Example 1 of the present invention, where A is the result of reversed-phase high-performance liquid chromatography and B is the result of electrospray ionization mass spectrometry.

[0038] Figure 4 The above describes the characterization of ubiquitin truncated form 2 in Example 1 of the present invention, where A is the result of reversed-phase high-performance liquid chromatography and B is the result of electrospray ionization mass spectrometry.

[0039] Figure 5 This is a characterization of the histone C-terminal truncated form in Example 1 of the present invention, where A is the result of reversed-phase high-performance liquid chromatography and B is the result of electrospray ionization mass spectrometry.

[0040] Figure 6 The following is a characterization of the histone N-terminal truncated acylhydrazide in Example 2 of the present invention, wherein A is the result of reversed-phase high performance liquid chromatography and B is the result of electrospray ionization mass spectrometry;

[0041] Figure 7 The following is a characterization of ubiquitin hydrazide in Example 3 of the present invention, wherein A is the result of reversed-phase high performance liquid chromatography and B is the result of electrospray ionization mass spectrometry.

[0042] Figure 8 The following is a characterization of the ubiquitin-modified histone short peptide in Example 4 of the present invention, wherein A is the result of reversed-phase high-performance liquid chromatography and B is the result of electrospray ionization mass spectrometry.

[0043] Figure 9 The following is a characterization of ubiquitin-modified histones in Example 5 of the present invention, wherein A is the result of reversed-phase high-performance liquid chromatography and B is the result of electrospray ionization mass spectrometry.

[0044] Figure 10 The following is a characterization of the fluorescently labeled ubiquitin-modified histone in Example 6 of the present invention, wherein A is the result of reversed-phase high-performance liquid chromatography and B is the result of electrospray ionization mass spectrometry.

[0045] Figure 11 The following is a characterization of histone octamer and Cy3-601 DNA in Example 7 of the present invention, wherein A is the molecular size exclusion chromatography and polyacrylamide gel electrophoresis result of octamer, and B is the ion exchange chromatography and agarose gel electrophoresis result of DNA.

[0046] Figure 12 The results of non-denaturing polyacrylamide gel electrophoresis of the ubiquitin-modified nucleosome fluorescence resonance energy transfer probe in Example 7 of this invention;

[0047] Figure 13 The fluorescence detection curves of the histone deubiquitinating enzyme USP16 hydrolyzing ubiquitin-modified nucleosome fluorescence resonance energy transfer probe in Example 8 of the present invention are shown. In the figure, A is the fluorescence intensity corresponding to the enzyme concentration change curve, and B is the fluorescence intensity corresponding to the enzyme digestion time change curve. Detailed Implementation

[0048] 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 and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0049] The ubiquitin-modified nucleosome fluorescence resonance energy transfer probe structure constructed in this invention is as follows: Figure 1 As shown.

[0050] Taking K119 ubiquitinated histone H2A as an example, the chemical synthesis route of the fluorescently labeled ubiquitinated histone constructed in this embodiment of the invention is as follows: Figure 2 As shown. K119 ubiquitinated histone H2A (H2AK119Ub) Cy5The protein was divided into four polypeptide fragments: ubiquitin truncated form 1, ubiquitin truncated form 2, histone C-terminal truncated form, and histone N-terminal truncated hydrazide. The first three were prepared by solid-phase polypeptide synthesis, while the latter was prepared by recombinant expression. Specifically, ubiquitin truncated form 1 had a GC sequence fused to its N-terminus and its thiol group protected with acetamide methyl (Acm); the side chain amino group of the K119 fragment of the histone C-terminal truncated form was coupled with a glycine (aG) cofactor, and the terminal cysteine ​​residue was protected with thiazolidinyl (Thz); the histone N-terminal truncated form was prepared by recombinant expression, with the terminal cysteine ​​residue activated by 2-nitro-5-cyanothiobenzoic acid (NTCB) and then subjected to in-situ ammonolysis to generate the histone N-terminal truncated hydrazide.

[0051] After obtaining four fragments, ubiquitin truncated derivatives 1 and 2 were first linked to a peptide hydrazide and then desulfurized to synthesize ubiquitin hydrazide. Subsequently, ubiquitin hydrazide was linked to the C-terminal truncated histone via a prosthetic group-mediated hydrazide linkage, and the prosthetic group and Thz protecting group were removed to synthesize a ubiquitin-modified histone short peptide. Afterward, it was linked to the N-terminal truncated histone hydrazide and desulfurized to synthesize ubiquitin-modified histone. Finally, the Acm protecting group of cysteine ​​in the ubiquitin-modified histone was removed, and an addition reaction with a maleimide-containing Cy5 group was carried out to obtain H2AK119Ub. Cy5 .

[0052] Example 1: Solid-phase synthesis and purification of ubiquitin and histone truncated derivatives

[0053] Polypeptide hydrazides were prepared using a hydrazine-substituted 2-Cl-Trt-Cl resin via a peptide synthesizer. The specific condensation conditions were: 4.0 equivalents of Fmoc-protected amino acids, 3.8 equivalents of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), and 8.0 equivalents of N,N-diisopropylethylamine (DIEA), with N,N-dimethylformamide (DMF) as the solvent. The Fmoc protecting group was removed using a 20% piperidine / DMF solution. After synthesis, the peptide chains were cleaved and deprotected using a trifluoroacetic acid (TFA) cleavage system (85% TFA, 5% H2O, 5% anisole sulfide, 2.5% phenol, and 2.5% 1,2-ethylenedithiol). Subsequently, TFA was removed by nitrogen, and the crude peptide product was obtained by pre-cooled ether precipitation.

[0054] The obtained crude peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). Phase A consisted of water containing 0.1% TFA, and Phase B consisted of acetonitrile containing 0.1% TFA. The crude peptide was dissolved in an appropriate volume of Phase A, with 30-40 mg of crude product dissolved in 3-4 mL of Phase A being optimal. After complete filtration through a 0.22 μM microporous membrane, the peptide was purified by RP-HPLC and lyophilized to obtain ubiquitin truncated form 1.

[0055] The RP-HPLC and electrospray ionization mass spectrometry (ESI-MS) characterization results of ubiquitin truncated form 1 are as follows: Figure 3 As shown in A and B, the specific structure is: GC(Acm)MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF-NHNH2 (as shown in SEQ ID NO.1), where Acm is an acetamide methyl protecting group.

[0056] Ubiquitin truncated form 2 was prepared according to the above method. The RP-HPLC and ESI-MS characterization results are as follows: Figure 4 As shown in A and B, the specific structure is: CGKQLEDGRTLSDYNIQKESTLHLVLRLRG-NHNH2 (shown in SEQ ID NO.2).

[0057] Histone C-terminal truncated derivatives were prepared according to the above method, and their RP-HPLC and ESI-MS characterization results are as follows: Figure 5 As shown in A and B, the specific structure is: C(Thz)QGGVLPNIQAVLLPKK(aG)TESHHKAKGK (as shown in SEQ ID NO.3), where Thz is a thiazolidinyl protecting group and aG is a glycine prosthetic group condensed with the amino group of the lysine side chain. Figure 2 (As shown).

[0058] Example 2: Synthesis of histone N-terminal truncated somatic hydrazide

[0059] The amino acid sequence of the histones is shown in SEQ ID NO.4, obtained through recombinant expression in *E. coli*. Single colonies of the N-terminal truncated histones were picked and added to 30 mL of LB medium containing 30 μL of ampicillin, and incubated overnight at 37 ℃ and 200 rpm. Subsequently, the bacterial culture was added to LB medium at a ratio of 1:100, along with 500 μL of ampicillin, and incubated at 37 ℃ and 200 rpm for 2-3 hours. The OD of the bacterial culture was then calculated. 600When the pH 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. The bacterial culture is then collected by centrifugation at 4000 rpm for 30 minutes at 4°C. Next, 40-50 mL of lysis buffer (30 mM Tris, 150 mM NaCl, 1 mM EDTA, 10 mM DTT, pH=7.5) is added, and the culture is sonicated on ice (30% power, 3 seconds ON, 6 seconds OFF) for 45 minutes. The lysed culture is then centrifuged at 12000 rpm for 20 minutes at 4°C. The precipitate is collected and 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 was removed. The precipitate was dissolved in 30 mL of denaturing buffer (6.0 M guanidine hydrochloride Gn·HCl, 20 mM Tris, 10 mM DTT, pH=7.5). The supernatant was collected by centrifugation and placed in a dialysis bag. Dialysis was performed overnight in 0.1% ddH2O. After centrifugation to remove the precipitate, the precipitate was purified by semi-preparative RP-HPLC and freeze-dried to obtain the N-terminal truncated histone. The amino acid sequence is shown in SEQ ID NO.5.

[0060] The 160 mg of histone N-terminal truncated derivative obtained above was dissolved in a buffer solution (6.0 M mgn·HCl, 0.1 M Na2HPO4, pH 9.0) to a final volume of 71 mL. NH2NH2, TCEP, and NTCB were added sequentially to final concentrations of 50 mM, 500 μM, and 5 mM, respectively. After mixing, the pH was adjusted back to 9.0, the mixture was sealed with a sealing film, and the reaction was carried out at 37 °C in the dark. After 45 hours, the histone N-terminal truncated derivative acylhydrazide was obtained by RP-HPLC purification. The RP-HPLC and ESI-MS characterization results are shown below. Figure 6 As shown in A and B in the diagram.

[0061] Example 3: Synthesis of ubiquitin hydrazide

[0062] 96.8 mg of ubiquitin truncated form 1 was dissolved in acidic buffer (6.0 M Gn·HCl, 0.1 M NaH2PO4, pH 3.0), sonicated, and then pre-cooled in an ice-salt bath at -15 °C 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-mercaptophenylacetic acid (MPAA) solution (dissolved in neutral buffer 6.0 M Gn·HCl, 0.1 M NaH2PO4, pH 7.0) was added, and the mixture was continuously stirred in an ice-salt bath at -15 °C for 5 minutes. Subsequently, 62.5 mg of ubiquitin truncated form 2 was added, and after complete dissolution, the pH of the system was adjusted to 6.6 with 2 M NaOH. The reaction was allowed to proceed at room temperature for approximately 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. After purification by RP-HPLC and lyophilization, 100.7 mg of the ligation product was obtained.

[0063] The resulting ligation 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 VA-044, a cycloazomididine initiator, was added to a final concentration of 0.1 M. The mixture was then incubated at 37 ℃ for 16 hours. After the reaction, an equal volume of acidic buffer was added and the mixture was stirred at room temperature for 5 minutes. Subsequently, 73.7 mg of ubiquitin hydrazide was obtained by semi-preparative RP-HPLC purification and freeze-drying. The RP-HPLC and ESI-MS characterization results are shown below. Figure 7 As shown in A and B in the diagram.

[0064] Example 4: Synthesis of ubiquitin-modified histone short peptides

[0065] 73.7 mg of ubiquitin hydrazide prepared in Example 3 was dissolved in acidic buffer (6.0 M Gn·HCl, 0.1 M Na2HPO4, pH=3.0), pre-cooled in an ice-salt bath at -15 °C, and then 273.6 μL of 0.2 M NaNO2 solution was added, and the reaction was continued 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 approximately 5.0 with 8.0 M NaOH, followed by the addition of 32.8 mg of the histone C-terminal truncated derivative prepared in Example 2. 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 approximately 6 hours. After the reaction was completed, an equal volume of TCEP solution (0.25 M) was added, and the mixture was stirred at room temperature for 5 minutes. The ligation product was obtained by RP-HPLC purification and freeze-drying.

[0066] The resulting ligation product was dissolved in 10 mL of decoupling reagent (9.5 mL TFA, 125 μL ethylenedithiol, 125 μL anisole sulfide, 250 μL ddH2O) and stirred at room temperature for 3 hours. After the reaction, TFA was removed with a nitrogen stream until the reaction solution was concentrated to approximately 600 μL. 3-4 mL of pre-cooled ether was added to the concentrate for washing and precipitation. The precipitate was collected by centrifugation at 12000 rpm for 5 minutes at 4 °C, and this process was repeated 3-4 times. After drying, the precipitate was dissolved in 0.2 M methoxyamine hydrochloride prepared with acidic buffer (6.0 M Gn·HCl, 0.1 M Na2HPO4, pH=3.0). 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, the product was purified by RP-HPLC, and the molecular weight was confirmed by LC-MS. After freeze-drying, 33.5 mg of ubiquitin-modified histone peptide was obtained. The RP-HPLC and ESI-MS characterization results are shown below. Figure 8 As shown in A and B in the diagram.

[0067] Example 5: Synthesis of ubiquitin-modified histones

[0068] 42.4 mg of the histone N-terminal truncated hydrazide prepared in Example 2 was dissolved in acidic buffer (6.0 M Mn·HCl, 0.2 M Na2HPO4, pH 3.0), pre-cooled in an ice-salt bath at -15 °C, and then 158.12 μL of 0.2 M NaNO2 solution was added. After reacting in the ice-salt bath for 25 minutes, 1.58 mL of 0.3 M MPAA solution was added and reacted for 5 minutes. Subsequently, 33.5 mg of the ubiquitin-modified histone peptide prepared in Example 4 was added, and the pH was adjusted to 6.7 with 2.0 M NaOH for the ligation reaction. After approximately 24 hours, an equal volume of TCEP solution (0.25 M) was added and stirred at room temperature for 5 minutes. The ligation product was obtained by semi-preparative RP-HPLC purification and freeze-drying.

[0069] The obtained ligation product, 34.46 mg, was dissolved in 1.25 mL of desulfurization buffer (6.0 M Gn·HCl, 0.2 M NaH2PO4, 500 mM TCEP, pH 7.5). VA-044 was added to a final concentration of 0.1 M, and the pH was adjusted back to 7.5. The reaction was carried out at 37 °C for 16 hours. After the reaction was complete, an equal volume of acidic buffer was added and the mixture was stirred at room temperature for 5 minutes. Subsequently, the mixture was purified by RP-HPLC and lyophilized to obtain 24.2 mg of ubiquitin-modified histone. The RP-HPLC and ESI-MS characterization results are shown below. Figure 9 As shown in A and B in the diagram.

[0070] Example 6: Fluorescent labeling of ubiquitin-modified histones

[0071] The ubiquitin-modified histone (24.2 mg) prepared in Example 5 was dissolved in 1.06 mL of a mixture of acetic acid and water (1:1 volume ratio), and 2.65 mg of silver acetate was added. The mixture 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, purified by RP-HPLC, and then freeze-dried.

[0072] The lyophilized protein (15 mg) was dissolved in labeling buffer (6.0 M Gn·HCl, 20 mM Tris, 1.0 mMTCEP, pH=7.5), and 1.0 mg of the fluorescent molecule Cy5-maleimide (Cy5-mal) was added. The pH was then adjusted to 7.5, and the reaction was carried out at 37°C in the dark for 2 hours. After purification by RP-HPLC and confirmation by LC-MS, the fluorescently labeled ubiquitin-modified histone H2AK119Ub was obtained after lyophilization. Cy5 The RP-HPLC and ESI-MS characterization results are as follows: Figure 10 As shown in A and B in the diagram.

[0073] Example 7: Preparation of ubiquitin-modified nucleosome fluorescence resonance energy transfer probe

[0074] The fluorescently labeled ubiquitin-modified histones, histone H2B, histone H3, and histone H4 (molar ratio 1:1:1:1) prepared in Example 6 were dissolved separately in pre-chilled denaturing buffer (6.0 M Gn·HCl, 20 mM Tris·HCl, 5.0 mM DTT, pH=7.5) at a concentration of 2 mg / mL and incubated on ice for 30 minutes. The four histone solutions were then mixed and the concentration was adjusted to 1.0 mg / mL with denaturing buffer. The mixture was transferred to a dialysis bag and dialyzed at 4°C with 1.0 L of refolding buffer (2.0 M NaCl, 10 mM Tris·HCl buffer, 1.0 mM EDTA, 5.0 mM 2-ME, pH=7.5) for 12 hours, with the refolding buffer changed twice during the process, for a total of 3 dialyzes. After concentration, the product was purified by size exclusion chromatography, and the histone octamer was obtained after confirmation by polyacrylamide gel electrophoresis (SDS-PAGE). It was then stored at -80 °C (results are shown below). Figure 11 (As shown in A in the diagram).

[0075] Simultaneously, using Wisdom 601 DNA (nucleotide sequence as shown in SEQ ID NO. 6) as a template and a nucleic acid sequence labeled with the fluorescent molecule Cy3 as a primer, large-scale parallel amplification and labeling of 601 DNA were performed under standard PCR conditions. After PCR, the amplification products were combined, purified by ion exchange chromatography, and confirmed by agarose gel electrophoresis to obtain Cy3-601 DNA (results shown in SEQ ID NO. 6). Figure 11 (As shown in B). The primer sequence for the Cy3-labeled primer is as follows:

[0076] F: 5'-Cy3-CTGGAGAATCCCGGTGC-3';

[0077] R: 5'-Cy3-GCACAGGATGTATATATCTGACAC-3'.

[0078] The histone octamer obtained above was mixed with Cy3-601 DNA in a ratio of 1:0.9 (usually 1:0.8-0.9 is acceptable) and 5.0 M NaCl and TE buffer (50 mM Tris, 5.0 mM EDTA) in sequence and placed in a dialysis tube, which was then placed in 250 mL of high-salt buffer (2.0 M NaCl, 50 mM Tris, 5.0 mM EDTA). TE buffer was slowly added dropwise to the high-salt buffer at a flow rate of 1.25 mL / min using a dialysis pump. After the liquid level reached 600 mL, the solution was poured to approximately 250 mL, and TE buffer was continued to be slowly added dropwise to the high-salt buffer at a flow rate of 1 mL / min. After the liquid level reached 1000 mL, the solution was dialyzed against HE buffer (50 mM HEPES, 5.0 mM EDTA) for 4-5 hours to obtain the ubiquitin-modified nucleosome fluorescence resonance energy transfer probe. The results of its non-denaturing polyacrylamide gel electrophoresis are shown below. Figure 12 As shown (including nucleosome controls with single Cy3 or Cy5 labels).

[0079] Example 8: Detection of histone deubiquitinating enzyme activity

[0080] Histone deubiquitinating enzyme USP16 was diluted to concentrations of 0, 0.25, 0.5, 1.0, and 2.0 μM, with three replicates for each group. The solutions were then added to 96-well plates. The ubiquitin-modified nucleosome fluorescence resonance energy transfer probe prepared in Example 7 was added to each well to a final concentration of 500 nM. After incubation at room temperature for 50 minutes, fluorescence intensity was measured using a multi-functional microplate reader. The change in fluorescence intensity corresponding to enzyme concentration was then determined. The experimental results are as follows: Figure 13 As shown in A in the diagram.

[0081] The enzyme concentration was fixed at 2.0 μM and added to each well of a 96-well plate, with three replicates per group. The ubiquitin-modified nucleosome fluorescence resonance energy transfer probe prepared in Example 7 was then added to each well. After incubation at room temperature for 0, 5, 10, 20, and 50 minutes, the fluorescence intensity was measured using a multi-functional microplate reader, and the change curve of fluorescence intensity corresponding to enzymatic digestion time was determined. The experimental results are as follows: Figure 13 As shown in B in Example 8, the ubiquitin-modified nucleosome fluorescence resonance energy transfer probe prepared in Example 8 can be used to detect histone deubiquitinating enzyme activity.

[0082] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a ubiquitin-modified nucleosome fluorescence resonance energy transfer probe, characterized in that, Includes the following steps: Step S1: The first group of proteins is ubiquitinated to obtain ubiquitinated histones; The amino terminus of the ubiquitin is fused with a glycine-cysteine ​​dipeptide sequence, and the thiol group of the cysteine ​​is protected by acetaminomethyl Acm. Step S2: Label the ubiquitin-modified histone with a donor fluorescent molecule and label the DNA with an acceptor fluorescent molecule to obtain fluorescently labeled ubiquitin-modified histone and fluorescently labeled DNA. or The ubiquitin-modified histone was labeled using a receptor fluorescent molecule, and the DNA was labeled using a donor fluorescent molecule to obtain fluorescently labeled ubiquitin-modified histone and fluorescently labeled DNA. The ubiquitin and the fluorescent molecule are linked via a thiol-maleimide addition reaction. Before linking, the acetaminomethylAcm protecting group of the amino-terminal cysteine ​​of ubiquitin needs to be removed. Step S3: The histone with the amino terminus of ubiquitin labeled with fluorescent molecules is mixed with the second, third and fourth histones and assembled to obtain the histone octamer with the amino terminus of ubiquitin labeled with fluorescent molecules. Step S4: Mix the histone octamer with fluorescently labeled amino terminus of ubiquitin with fluorescently labeled DNA to obtain the ubiquitin-modified nucleosome fluorescence resonance energy transfer probe. Step S1, the ubiquitination modification of the first group of proteins includes the following steps: Step S11: Prepare the first ubiquitin truncated form and the second ubiquitin truncated form respectively, and ligate them to obtain ubiquitin hydrazide; Step S12: The amino and carboxyl terms of the first histone are truncated respectively. The amino-terminal and carboxyl-terminal truncated histones are obtained by recombinant expression or solid-phase synthesis. The ubiquitin hydrazide is linked to the carboxyl-terminal truncated histone to obtain ubiquitin-modified histone short peptides. The ubiquitin hydrazide and the carboxyl-terminal truncated histone are linked by a prosthetic group-mediated polypeptide hydrazide. The cysteine ​​residue of the carboxyl-terminal truncated histone is protected with thiazolidinyl Thz. Step S13: The ubiquitin-modified histone short peptide is linked to the amino-terminal truncated histone to obtain the ubiquitin-modified histone. The ubiquitin-modified histone short peptide needs to have its thiazolidinyl (Thz) protecting group removed before linking.

2. The preparation method according to claim 1, characterized in that: In step S1, the first group protein is H2A, H2B, H3, or H4.

3. The preparation method according to claim 1, characterized in that: In step S1, the lysine residues of the first group of proteins are modified by ubiquitination.

4. The preparation method according to claim 1, characterized in that: In step S2, the donor fluorescent molecule is Cy3 and the acceptor fluorescent molecule is Cy5.

5. Any of the following applications of the ubiquitin-modified nucleosome fluorescence resonance energy transfer probe prepared by any of the methods described in claims 1-4: (1) Application in the preparation of reagents for detecting the activity of histone deubiquitinating enzymes; (2) Application in the preparation of reagents for screening histone deubiquitinase inhibitors.

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