A substrate preparation and enzyme activity optimization detection system and kit for PARG enzyme activity homogeneous detection based on TR-FRET technology

Abstract

The application provides a substrate preparation and enzyme activity optimization detection system and kit for PARG enzyme activity homogeneous detection based on TR-FRET technology, the detection system is a PARG enzyme activity homogeneous detection system based on TR-FRET technology by using PARP1 self-PAR action to synthesize PARG substrate and optimizing PARG enzymatic reaction conditions. The PARG enzyme activity homogeneous detection system comprises a reaction unit for synthesizing PARG substrate by using PARP1 self-PAR action, an optimization unit for PARG enzymatic reaction conditions, and a constructed TR-FRET detection system. The application also provides a kit of the detection system. The application efficiently synthesizes natural PARG substrate by using PARP1 self-PAR action, and optimizes PARG enzymatic conditions, so that high signal-to-noise ratio, wide linear range and high-throughput PARG activity quantitative detection are realized.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a detection system and kit for detecting poly(ADP-ribose) hydrolase (PARG) enzyme activity based on time-resolved fluorescence resonance energy transfer (TR-FRET) in molecular biology. The system utilizes PARP1 to synthesize PARG substrates through self-PAR action and optimizes PARG enzymatic reaction conditions. In particular, it relates to a substrate preparation and enzyme activity optimization detection system and kit for homogeneous detection of PARG enzyme activity based on TR-FRET technology. Background Technology

[0002] Poly(ADP-ribose) modification, or PARylation, is a key post-translational modification process involved in DNA damage repair, maintenance of genome stability, and regulation of apoptosis and inflammation. Poly(ADP-ribose) polymerase 1, or PARP1, uses NAD+ to modify PARylation. + Nicotinamide adenine dinucleotide (NADP) acts as a donor, catalyzing the formation of long-chain poly(ADP-ribose) glycosides (PARs) from itself or target proteins. PARG, the poly(ADP-ribose) hydrolase, is the core glycoside hydrolase that specifically hydrolyzes PAR chains, directly reversing the PARylation process. PARG dysfunction is closely related to tumorigenesis, chemotherapy resistance, and neurodegenerative diseases, and has become an important target for the development of antitumor and anti-inflammatory drugs.

[0003] Existing methods for detecting PARG enzyme activity mainly include: radioactive isotope labeling, high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), and conventional fluorescence methods. These methods generally have drawbacks. For example, radioactive methods pose safety risks and are cumbersome to operate; HPLC is time-consuming and has low throughput; ELISA is a heterogeneous detection method requiring multiple washes and has poor repeatability; and conventional fluorescence detection suffers from strong background interference and a low signal-to-noise ratio, making it difficult to meet high-throughput screening requirements.

[0004] Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) technology uses lanthanide rare earth elements as donors and leverages long-lifetime fluorescence and time-resolved detection modes to effectively eliminate background fluorescence interference, achieving homogeneous, wash-free, highly sensitive, and high-throughput detection. It is ideally suited for enzyme activity dynamics and compound screening.

[0005] However, one of the biggest bottlenecks in PARG detection in existing technologies is substrate preparation. PAR chains have complex structures and uneven chain lengths, making chemical synthesis difficult, costly, and unstable, and it is also difficult to mimic the structure of natural PAR in vivo. In addition, the PARG enzymatic reaction is highly sensitive to buffer systems, pH, metal ions, reaction temperature, and time. Existing detection methods lack standardized optimization conditions, resulting in narrow detection windows, poor linearity, and unstable results.

[0006] Currently, there is no publicly available integrated PARG enzyme activity detection scheme that combines PARP1 self-PARation in situ synthesis of PARG substrates, TR-FRET homogeneous signal output, and PARG enzymatic reaction condition system optimization.

[0007] Therefore, developing a PARG detection system that is easy to prepare substrates, has high detection sensitivity, provides stable results, and is suitable for high-throughput screening has significant scientific and industrial value. Summary of the Invention

[0008] The purpose of this invention is to address the problems existing in the prior art by providing a detection system and kit for homogeneous detection of PARG enzyme activity based on TR-FRET technology. This system efficiently synthesizes natural PARG substrates through the self-PAR reaction of PARP1 and systematically optimizes the PARG enzymatic conditions to achieve high signal-to-noise ratio, wide linear range, and high throughput quantitative detection of PARG activity, thereby overcoming the shortcomings of the prior art.

[0009] To achieve the above objectives, the present invention provides a detection system for homogeneous detection of PARG enzyme activity based on TR-FRET technology. The detection system utilizes PARP1 self-PAR reaction to synthesize PARG substrates and optimizes PARG enzymatic reaction conditions. The homogeneous PARG enzyme activity detection system comprises: a reaction unit for synthesizing PARG substrates using PARP1 self-PAR reaction, a unit for optimizing PARG enzymatic reaction conditions, and a constructed TR-FRET detection unit.

[0010] The detection system used in the above-mentioned homogeneous detection of PARG enzyme activity based on TR-FRET technology includes, in which the reaction unit for synthesizing PARG substrates using PARP1 auto-PAR reaction comprises: recombinant PARP1 protein, NAD... + or biotin-NAD + Activated DNA, PARP reaction buffer; reaction conditions: 25℃-37℃, 10-60 minutes; termination method: adding PARP inhibitor, EDTA or heat inactivation; the resulting substrate is PAR-modified PARP1.

[0011] The above-mentioned detection system for homogeneous PARG enzyme activity detection based on TR-FRET technology includes a reaction unit that utilizes PARP1 auto-PARation to synthesize PARG substrates. This unit synthesizes PARG-specific substrates through PARP1 auto-PARation. The process involves substrate synthesis, followed by PARP1 auto-PARation in the presence of activated DNA, to convert NAD+ into NAD+. + Or label NAD + Using it as a substrate, poly(ADP-ribose) chains are synthesized in situ on the PARP1 molecule to form a natural PAR-PARP1 complex, which serves as the optimal substrate for PARG.

[0012] The detection system used for the homogeneous detection of PARG enzyme activity based on TR-FRET technology, wherein the optimization unit for the PARG enzymatic reaction conditions includes: buffer system: 50-100mM Tris-HCl or HEPES, pH 7.0-8.5; metal ions: 0-5mM MgCl2, 0-1mM MnCl2, 0-100mM KCl; protective agent: 0.1-1mg / mL BSA, 1-5mM DTT; reaction temperature: 25℃-37℃; reaction time: 10-120 minutes; substrate concentration to enzyme amount ratio: matching the Km range to ensure the reaction is in the initial rate stage.

[0013] The detection system used in the above-mentioned PARG enzyme activity homogeneous detection based on TR-FRET technology includes a PARG enzyme reaction condition optimization unit, which is obtained by optimizing enzyme activity. By optimizing PARG enzyme reaction conditions including buffer system, pH value, metal ions, reaction temperature, time, substrate / enzyme ratio, and termination method, the detection signal window is maximized and the repeatability is optimized.

[0014] The detection system used for the homogeneous detection of PARG enzyme activity based on TR-FRET technology described above includes the following: Donor: terbium or europium-labeled anti-PAR antibody, anti-His antibody, or anti-Flag antibody; Acceptor: XL665 or APC-labeled streptavidin; Detection mode: homogeneous, no washing; Excitation wavelength: 320-350 nm; Emission wavelength: 615 nm for donor, 665 nm for acceptor; Signal acquisition is performed using time-resolved mode, and the FRET ratio is calculated as 665 nm / 615 nm.

[0015] The aforementioned detection system for homogeneous PARG enzyme activity detection based on TR-FRET technology involves a constructed TR-FRET detection unit that uses a PAR-specific antibody or tag antibody labeled with a lanthanide donor to form an energy transfer pair with an affinity molecule labeled with a receptor. When the PAR chain is intact, a strong TR-FRET signal is generated. However, when PARG hydrolyzes the PAR chain, the energy transfer is disrupted, and the signal decreases. The degree of decrease is positively correlated with PARG enzyme activity, and this is used for detection.

[0016] The aforementioned PARG enzyme activity homogeneous detection system based on TR-FRET technology comprises the following steps: In the reaction unit that utilizes PARP1 self-PARation to synthesize PARG substrates, PAR-PARP1 substrates are prepared through PARP1 self-PARation. These substrates are then mixed with PARG samples or inhibitors, and an optimized enzyme buffer from the PARG enzyme reaction condition optimization unit. The enzymatic reaction is initiated, and then reagents from the constructed TR-FRET detection unit are added. The mixture is incubated at room temperature, and the fluorescence signal is read. The FRET ratio is calculated to quantify PARG enzyme activity, and finally, the PARG detection result is obtained.

[0017] The present invention also provides a kit for the detection system used in the above-mentioned homogeneous detection of PARG enzyme activity based on TR-FRET technology, wherein the kit is a TR-FRET detection kit including substrate preparation and enzyme activity optimization system.

[0018] Compared with existing technologies, the substrate preparation and enzyme activity optimization detection system and kit for PARG enzyme activity homogeneous detection based on TR-FRET technology of the present invention have the following beneficial effects: 1. The substrate is easy to prepare, low in cost, and has a natural structure, requiring no chemical synthesis or complex purification.

[0019] 2. Homogeneous and wash-free, simple to operate, suitable for automated and high-throughput screening.

[0020] 3. The time-resolved fluorescence background is extremely low, the signal-to-noise ratio is high, and the detection sensitivity is significantly improved.

[0021] 4. The PARG enzymatic conditions system was optimized, resulting in stable reactions, a wide linear range, and good reproducibility.

[0022] 5. Can be used for recombinase activity detection, endogenous PARG detection in cell lysates, and inhibitor IC50 detection. 50 That is, the determination of half-inhibitory concentration and high-throughput drug screening. Attached Figure Description

[0023] Figure 1This is a schematic diagram illustrating the principle of TR-FRET detection of PARG enzyme activity according to the present invention.

[0024] Figure 2 This is a diagram illustrating the synthesis and verification of the PARP1 self-PARation substrate of the present invention.

[0025] Figure 3 The figure shows the optimization results of the PARG enzymatic conditions of this invention.

[0026] Figure 4 This is a standard curve of PARG enzyme activity according to the present invention.

[0027] Figure 5 This is a dose-response curve of the PARG inhibitor of the present invention.

[0028] Figure 6 Figures A and B in the diagram show a comparison between the results of this invention and traditional detection methods. Detailed Implementation

[0029] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. These drawings are only for illustrating the embodiments of the present disclosure and are not intended to limit the scope of the disclosure.

[0030] This invention provides a detection system for homogeneous detection of PARG enzyme activity based on TR-FRET technology. This detection system utilizes the self-PAR reaction of PARP1 to synthesize PARG substrates and optimizes the PARG enzymatic reaction conditions. The PARG enzyme activity homogeneous detection system includes: a reaction unit for synthesizing PARG substrates using the self-PAR reaction of PARP1, an optimization unit for PARG enzymatic reaction conditions, and a constructed TR-FRET detection unit.

[0031] Preferably, the reaction unit for synthesizing PARG substrates using PARP1 self-PAR interaction comprises: Recombinant PARP1 protein, NAD + Or Biotin-NAD + Activated DNA, i.e., double-stranded gap DNA, and PARP reaction buffer; The reaction conditions are: 25℃-37℃, 10-60 minutes; Termination methods include: adding a PARP inhibitor, EDTA (ethylenediaminetetraacetic acid), or inactivation by heating; The resulting substrate is PAR-modified PARP1, which has a uniform PAR chain length and a natural structure. It can be efficiently recognized and hydrolyzed by PARG, and therefore can serve as the optimal substrate for PARG.

[0032] This reaction unit, utilizing the auto-PAR reaction of PARP1 to synthesize PARG substrates, synthesizes PARG-specific substrates through the auto-PAR reaction of PARP1. The process involves substrate synthesis, where, in the presence of activated DNA, PARP1 undergoes an auto-PAR reaction to convert NAD+ into NAD+. + Or label NAD + Using it as a substrate, poly(ADP-ribose) chains are synthesized in situ on the PARP1 molecule to form a natural PAR-PARP1 complex, which serves as the optimal substrate for PARG.

[0033] Preferably, the unit for optimizing the PARG enzymatic reaction conditions includes: Buffer system: 50-100mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride) or HEPES (hydroxyethylpiperazine ethanethioic acid), pH 7.0-8.5; Metal ions: 0-5mM MgCl2, 0-1mM MnCl2, 0-100mM KCl; Protectants: 0.1-1 mg / mL BSA (bovine serum albumin), 1-5 mM DTT (dithiothreitol); Reaction temperature: 25℃-37℃; Reaction time: 10-120 minutes; Substrate concentration to enzyme ratio: Match the Km range to ensure the reaction is in the initial rate phase.

[0034] The obtained optimal conditions significantly improve the signal-to-noise ratio, with Z' factor > 0.5, meeting the requirements for high-throughput screening.

[0035] The optimization unit for PARG enzymatic reaction conditions is obtained through enzyme activity optimization. By systematically optimizing PARG enzymatic reaction conditions, including buffer system, pH value, metal ions, reaction temperature, time, substrate / enzyme ratio, termination method, etc., the detection signal window is maximized and repeatability is optimized.

[0036] Preferably, the constructed TR-FRET detection unit includes: Donors: Terbium (Tb) or europium (Eu) labeled anti-PAR antibodies, anti-His antibodies, or anti-Flag antibodies; Receptor: Streptavidin (targeting biotin-NAD) labeled with XL665 fluorescent dye or APC (allocyanin). + ); Detection mode: Homogeneous, no washing; Excitation wavelength: 320-350nm; Emission wavelength: 615nm for donor, 665nm for acceptor; The signal was acquired using time-resolved mode, and the FRET ratio was calculated to be 665nm / 615nm.

[0037] The constructed TR-FRET detection unit is a PAR-specific antibody or tag antibody labeled with a lanthanide donor (e.g., ...). Figure 1 In the middle: europium fluorescent donor SolarEu conjugated with anti-Tag1 monoclonal antibody, i.e., mAb anti-Tag1-SolarEu, and receptor-labeled affinity molecules (e.g. Figure 1 In the middle: Streptavidin-LA (i.e., Streptavidin-Luminescent Acceptor) forms an energy transfer pair; when the PAR chain is intact (e.g., Figure 1 In the PARG enzyme (enzyme + inhibitor pathway), a strong TR-FRET signal is generated, while when PARG hydrolyzes the PAR chain (e.g., ... Figure 1 In the PARG enzyme pathway (where only the PARG enzyme pathway is involved), energy transfer is disrupted, leading to a decrease in signal intensity. The degree of this decrease is positively correlated with PARG enzyme activity, and this is used for detection. The principle of TR-FRET detection of PARG enzyme activity is detailed in [link to relevant documentation]. Figure 1 As shown.

[0038] More preferably, this homogeneous PARG enzyme activity detection system is used in a reaction unit that utilizes PARP1 self-PARation to synthesize PARG substrates, in which PAR-PARP1 substrates are prepared via PARP1 self-PARation (e.g., ...). Figure 1 In the process, a biotin-PARlated substrate is mixed with a PARG sample or inhibitor, along with an optimized enzyme buffer from the PARG enzyme reaction optimization unit. This mixture initiates the enzymatic digestion reaction. Then, reagents from the constructed TR-FRET detection unit are added, and the mixture is incubated at room temperature. The fluorescence signal is then read using a microplate reader, the FRET (fluorescence resonance energy transfer) ratio is calculated, PARG enzyme activity is quantified, and finally, the PARG detection result is obtained.

[0039] The present invention also provides a kit for the detection system used in the homogeneous detection of PARG enzyme activity based on TR-FRET technology. The kit is a TR-FRET detection kit that includes substrate preparation and enzyme activity optimization systems.

[0040] To make the technical problems to be solved, the technical solutions, and the beneficial effects of this application clearer, the following detailed description is provided in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0041] Unless otherwise specified, all techniques or conditions used in the examples are conventional methods, performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. All reagents or instruments used are conventional products that can be purchased through legitimate channels.

[0042] Example 1: Preparation of PARG substrate by self-PARation of PARP1.

[0043] In the presence of activated DNA, PARP1 undergoes auto-PARation to convert NAD+ into NAD+. + Or label NAD + Using PARP1 as a substrate, a poly(ADP-ribose) chain is synthesized in situ on the PARP1 molecule to form a natural PAR-PARP1 complex, which serves as the optimal substrate for PARG, thus obtaining the reaction unit for synthesizing PARG substrates using the self-PAR interaction of PARP1. The specific process is as follows.

[0044] Reaction system (50 μL): 100 mM Tris-HCl pH 8.0, 5 mM MgCl2, 1 mM DTT, 1 μM DNA activator, 200 nM PARP1, 50 μM Biotin-NAD + ; Reaction conditions: 30℃, 30 minutes; Termination: The reaction was terminated by adding 10 μM Olaparib. The obtained PAR-PARP1-biotin was stable when stored at -20°C as a PARG substrate.

[0045] The results of the synthesis verification of the PARP1 PARated substrate are shown in [link to documentation]. Figure 2 As shown.

[0046] Example 2: Optimization of PARG enzymatic reaction conditions.

[0047] By systematically optimizing the PARG enzymatic reaction conditions, including buffer system, pH value, metal ions, reaction temperature, time, substrate / enzyme ratio, and termination method, the detection signal window was maximized and repeatability was optimized, thus obtaining the optimized unit for PARG enzymatic reaction conditions. The specific process is as follows.

[0048] The effects of pH (7.0, 7.5, 8.0, 8.5) and Mg were respectively applied. 2+ The parameters (0, 1, 2, 5 mM), temperature (25℃, 30℃, 37℃), and reaction time (10 min, 30 min, 60 min, 120 min) were optimized. The optimal conditions were determined to be: 50 mM Tris-HCl pH 8.0, 2 mM MgCl2, 0.5 mg / mL BSA, and reaction at 30 °C for 60 minutes. Under these conditions, the signal-to-noise ratio is >10 times, which is significantly improved, and Z'=0.68, meeting the requirements for high-throughput screening.

[0049] The results of the PARG enzymatic optimization are shown in [reference]. Figure 3 As shown.

[0050] Example 3: TR-FRET detection of PARG enzyme activity.

[0051] TR-FRET detection uses PAR-specific antibodies or tag antibodies labeled with lanthanide donors to form energy transfer pairs with affinity molecules labeled with receptors. When the PAR chain is intact, a strong TR-FRET signal is generated. However, when PARG hydrolyzes the PAR chain, the energy transfer is disrupted, and the signal decreases. The degree of decrease is positively correlated with PARG enzyme activity. This is used for detection to obtain the constructed TR-FRET detection unit.

[0052] The TR-FRET detection system for PARG enzyme activity first prepares a PAR-PARP1 substrate using the PARP1 auto-PARation reaction, mixes it with a PARG sample or inhibitor and an optimized enzyme buffer, and initiates the enzymatic digestion reaction. Then, TR-FRET detection reagent is added and incubated at room temperature. Finally, the fluorescence signal is read using a microplate reader, the FRET ratio is calculated, and PARG enzyme activity is quantified. The specific process is as follows.

[0053] Detection system (100 μL): PAR-PARP1-biotin substrate, PARG enzyme, optimized buffer; After the reaction was complete, add: Tb-anti-PAR antibody, SA-XL665; incubate at room temperature for 60 minutes; Detection: Excitation at 340nm, delay at 100μs, readout at 615nm and 665nm; The results showed that the FRET ratio decreased with increasing PARG enzyme activity, exhibiting a good linear relationship. The obtained PARG enzyme activity standard curve can be found here. Figure 4 As shown.

[0054] Example 4: High-throughput screening of PARG inhibitors.

[0055] The KeyTec enzyme activity homogeneous detection system based on TR-FRET technology provided in Examples 1-3 of this invention yielded KeyTec... ® The TR-FRET Human PARG inhibitor screening kit, and the specific procedures of Examples 1-3 of this invention, are used to perform gradient detection of PARG inhibitors.

[0056] The results yielded a typical S-type dose-inhibition curve, IC50 50 The values ​​are accurate and repeatable; the in-segment CV is <5%, meeting the high-throughput screening criteria. The obtained PARG inhibitor dose-response curves are shown below. Figure 5 As shown.

[0057] The PARG enzyme activity homogeneous detection system of this invention is compared with traditional detection methods; the results are shown in [reference needed]. Figure 6 As shown, A represents the present invention, and B represents conventional detection.

[0058] It is evident that the detection system provided by this invention is a high signal-to-noise ratio, wide linear range, and high-throughput PARG activity quantitative detection system.

[0059] Example 5: Detection of endogenous PARG activity in cells.

[0060] HeLa cell lysates were collected and directly added to the detection system for homogeneous detection of PARG enzyme activity based on TR-FRET technology provided in Examples 1-3 of this invention. This system can quantitatively detect changes in endogenous PARG activity, showing that PARG activity changed significantly after drug treatment.

[0061] This invention relates to the expression of recombinant PARP1 and PARG proteins, whose amino acid sequences are publicly known sequences, as shown in SEQ ID NO.1 and SEQ ID NO.2, both of which can be directly obtained from the NCBI database. This invention does not involve the creation of new sequences.

[0062] SEQ ID NO.1, amino acid sequence of recombinant PARP1 protein SEQ ID NO.2, amino acid sequence of PARG recombinant protein MNAGPGCEPCTKRPRWGAATTSPAASDARSFPSRQRRVLDPKDAHVQFRVPPSSPACVPGRAGQHRGSATSLVFKQKTITSWMDTKGIKTAESESLDSKENNNTRIESMMSSVQKDNFYQHNVEKLENVSQLSLDKSPTEKSTQYLNQHQTAAMCKWQNEGKHTEQLLESEPQTVTLVPEQFSNANIDRSPQNDDHSDTDSEENRDNQQFLTTVKLANAKQTTEDEQAREAKSHQKCSKSCDPGEDCASCQQDEIDVVPESPLSDVGSEDVGTGPKNDNKLTRQESCLGNSPPFEKESEPESPMDVDNSKNSCQDSEADEETSPGFDEQEDGSSSQTANKPSRFQARDADIEFRKRYSTKGGEVRLHFQFEGGESRTGMNDLNAKLPGNISSLNVECRNSKQHGKKDSKITDHFMRLPKAEDRRKEQWETKHQRTERKIPKYVPPHLSPDKKWLGTPIEEMRRMPRCGIRLPLLRPSANHTVTIRVDLLRAGEVPKPFPTHYKDLWDNKHVKMPCSEQNLYPVEDENGERTAGSRWELIQTALLNKFTRPQNLKDAILKYNVAYSKKWDFTALIDFWDKVLEEAEAQHLYQSILPDMVKIALCLPNICTQPIPLLKQKMNHSITMSQEQIASLLANAFFCTFPRRNAKMKSEYSSYPDINFNRLFEGRSSRKPEKLKTLFCYFRRVTEKKPTGLVTFTRQSLEDFPEWERCEKPLTRLHVTYEGTIEENGQGMLQVDFANRFVGGGVTSAGLVQEEIRFLINPELIISRLFTEVLDHNECLIITGTEQYSEYTGYAETYRWSRSHEDGSERDDWQRRCTEIVAIDALHFRRYLDQFVPEKMRRELNKAYCGFLRPGVSSENLSAVATGNWGCGAFGGDARLKALIQILAAAAAERDVVYFTFGDSELMRDIYSMHIFLTERKLTVGDVYKLLLRYYNEECRNCSTPGPDIKLYPFIYHAVESCAETADHSGQRTGT The PARG enzyme activity homogeneous detection system and kit based on TR-FRET technology provided by this invention is an integrated PARG enzyme activity detection scheme that combines in-situ synthesis of PARG substrates by PARP1 self-PARation, TR-FRET homogeneous signal output, and PARG enzymatic reaction condition system optimization. The developed substrate preparation is simple, the detection is sensitive, the results are stable, and it is suitable for high-throughput screening of PARG detection, which has important scientific research and industrial value.

[0063] This invention can be used in fields such as PARG enzymology research, anti-tumor drug screening, compound activity evaluation, and clinical sample enzyme activity detection. It can be developed into a commercial detection kit and has broad prospects for scientific research and industrial applications, and is industrially practical.

[0064] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application without departing from the principles of the present invention should be included within the protection scope of this application.

Claims

1. A detection system for homogeneous detection of PARG enzyme activity based on TR-FRET technology, characterized in that, The detection system is a homogeneous detection system for PARG enzyme activity based on TR-FRET technology, which utilizes the self-PAR action of PARP1 to synthesize PARG substrates and optimizes the PARG enzymatic reaction conditions. The homogeneous detection system for PARG enzyme activity includes: a reaction unit for synthesizing PARG substrates using the self-PAR action of PARP1, an optimization unit for PARG enzymatic reaction conditions, and a constructed TR-FRET detection unit. The reaction unit for synthesizing PARG substrates using PARP1 self-PAR interaction includes: Recombinant PARP1 protein, NAD + or biotin-NAD + Activated DNA, PARP reaction buffer; The reaction conditions are: 25℃-37℃, 10-60 minutes; Termination methods include: adding PARP inhibitors, EDTA, or inactivation by heating; The resulting substrate was PAR-modified PARP1; The amino acid sequence of the recombinant PARP1 protein is shown in SEQ ID NO.1; The optimization unit for the PARG enzymatic reaction conditions includes: Buffer system: 50-100mM Tris-HCl or HEPES, pH 7.0-8.5; Metal ions: 0-5mM MgCl2, 0-1mM MnCl2, 0-100mM KCl; Protectants: 0.1-1 mg / mL BSA, 1-5 mM DTT; Reaction temperature: 25℃-37℃; Reaction time: 10-120 minutes; Substrate concentration to enzyme ratio: Match the Km range to ensure the reaction is in the initial rate phase; The optimal conditions were determined to be: 50 mM Tris-HCl pH 8.0, 2 mM MgCl2, 0.5 mg / mL BSA, and reaction at 30℃ for 60 minutes. Under these conditions, the signal-to-noise ratio was >10 times and Z' = 0.68, which met the requirements for high-throughput screening.

2. The detection system for homogeneous detection of PARG enzyme activity based on TR-FRET technology according to claim 1, characterized in that, The reaction unit for synthesizing PARG substrates using PARP1 auto-PARation synthesizes PARG-specific substrates through PARP1 auto-PARation. The process involves substrate synthesis, where PARP1 undergoes auto-PARation in the presence of activated DNA, converting NAD+ into NAD+. + Or label NAD + Using it as a substrate, poly(ADP-ribose) chains are synthesized in situ on the PARP1 molecule to form a natural PAR-PARP1 complex, which serves as the optimal substrate for PARG.

3. The detection system for homogeneous detection of PARG enzyme activity based on TR-FRET technology according to claim 1, characterized in that, The optimization unit for PARG enzymatic reaction conditions is obtained by optimizing enzyme activity. By optimizing PARG enzymatic reaction conditions, including buffer system, pH value, metal ions, reaction temperature, time, substrate / enzyme ratio, and termination method, the detection signal window is maximized and repeatability is optimized.

4. The detection system for homogeneous detection of PARG enzyme activity based on TR-FRET technology according to claim 1, characterized in that, The constructed TR-FRET detection unit includes: Donor: Terbium or europium-labeled anti-PAR antibody, anti-His antibody, or anti-Flag antibody; Receptor: XL665 or APC-tagged streptavidin; Detection mode: Homogeneous, no washing; Excitation wavelength: 320-350nm; Emission wavelength: 615nm for donor, 665nm for acceptor; The signal was acquired using time-resolved mode, and the FRET ratio was calculated to be 665nm / 615nm.

5. The detection system for homogeneous detection of PARG enzyme activity based on TR-FRET technology according to claim 4, characterized in that, The constructed TR-FRET detection unit uses a lanthanide donor-labeled PAR-specific antibody or tag antibody to form an energy transfer pair with a receptor-labeled affinity molecule. When the PAR chain is intact, a strong TR-FRET signal is generated. However, when PARG hydrolyzes the PAR chain, the energy transfer is disrupted, and the signal decreases. The degree of decrease is positively correlated with PARG enzyme activity, and this is used for detection.

6. The detection system for homogeneous detection of PARG enzyme activity based on TR-FRET technology according to claim 1, characterized in that, The homogeneous detection system for PARG enzyme activity involves preparing PAR-PARP1 substrates via a self-PAR reaction in a reaction unit that utilizes PARP1 to synthesize PARG substrates. This PAR-PARP1 substrate is then mixed with a PARG sample or inhibitor, along with an optimized enzyme buffer from a unit optimizing PARG enzyme reaction conditions. The enzymatic reaction is initiated, and reagents from a constructed TR-FRET detection unit are added. The system is incubated at room temperature, and the fluorescence signal is read. The FRET ratio is calculated to quantify PARG enzyme activity, ultimately yielding the PARG detection result.

7. A kit prepared using the detection system for homogeneous detection of PARG enzyme activity based on TR-FRET technology according to any one of claims 1 to 6, characterized in that, The kit is a TR-FRET detection kit that includes substrate preparation and enzyme activity optimization systems.

Images