CN116555339B - NHEJ substrate and preparation method and application thereof - Google Patents
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Abstract
The invention relates to the technical field of DNA repair, in particular to a NHEJ substrate and a preparation method and application thereof, wherein the preparation method comprises the following steps: s1, connecting Nanoluc, SV40 and CMV genes to a plasmid vector through enzyme digestion to prepare a dsDNA fragment sequence; s2, after annealing treatment is carried out on the dsDNA fragment sequence prepared in the step S1, connecting one end of a Nanoluc gene in the dsDNA fragment sequence with an N-end long-chain primer and an N-end short-chain primer, and connecting one end of a CMV gene with a C-end long-chain primer and a C-end short-chain primer to prepare a partially double-stranded DNA; s3, connecting the partially double-stranded DNA obtained in the step S2 by DNA ligase to obtain the NHEJ substrate of the double-stranded DNA. The NHEJ substrate prepared by the invention can specifically identify the NHEJ injury repair path in cells, has wide injury repair range and high efficiency, and is beneficial to the screening and research and development of different subsequent medicines.
Description
Technical Field
The invention relates to the technical field of DNA repair, in particular to a NHEJ substrate and a preparation method and application thereof.
Background
DNA repair (dnasairing) is a reaction of cells after they have been damaged to DNA, which may restore the DNA structure as it is, re-performing its original function; however, there are cases where DNA damage is not completely eliminated, but cells are allowed to survive the DNA damage. It is possible that damage that remains without complete repair will manifest itself under certain conditions (e.g., cancerous cells, etc.), but if the cells do not have such repair function, it is not possible to cope with the frequently occurring DNA damage events.
DNA damage can cause serious physiological dysfunction, and thus, different repair methods have been studied for different types of DNA damage. DNA repair in eukaryotes is mainly of 4 types: nucleotide Excision Repair (NER), base Excision Repair (BER), mismatch repair (MMR), and Double Strand Break Repair (DSBR).
NER and BER are collectively called excision repair, and damaged parts can be excised from DNA molecules, and intact DNA double strands are synthesized by using undamaged DNA single strands as templates to complete the repair process. MMR can repair false pairings generated in DNA replication; DSB is a serious injury that results in loss and rearrangement of genomic sequences, and there are mainly three repair pathways:
(1) Homologous Recombination (HR) the activity of HR is limited to the S and G2 phases of the cell cycle. HR requires DNA excision, where nuclear degradation of DSB yields 3' ss DNA overhang, which is initiated by MRE11-RAD50-NBS1 complex (MRN) together with CtIP, followed by prolonged excision catalyzed by EXO1 and DNA 2-BLM. Following excision, the 3' ssdna tail is bound by RPA, followed by activation of RAD51 by the action of BRCA2 and PALB2 (RAD 51 nuclear fiber mediated strand invasion and homology searches on sister chromatids). Finally, the missing nucleotide is filled by copying undamaged staining monomers, and the repair of the minimal change to the original sequence is completed, so that the HR repair efficiency is low and the speed is low.
(2) Polymer theta-mediated end joining (TMEJ): TMEJ is a backup way to repair excised DSB, which is initiated by 5 'to 3' excision factors, involving PARP1, DNA ligase III and polymerase A family enzymes, DNA polymerase θ encoded by PolQ, polθ initiates DNA synthesis with its polymerase domain to fill the gap, and then ligates annealed DSB ends to complete repair. TMEJ is responsible for only a small portion of DNA repair, can complete less than 10% of DNA repair compared to other repair damage pathways, is less efficient, and is not as effective as other repair pathways in experiments where in vitro and cellular DNA repair DSB is deemed to be successful.
(3) Canonical nonhomologous end joining (c-NHEJ) NHEJ is the major repair pathway in mammalian cells, repairing up to 80% of DSB. After Ku70/80 (Ku) heterodimers recognize the cleaved DNA ends, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) stabilizes the DNA ends, initiating a canonical end ligation pathway. The DNA-PK complex phosphorylates various factors to facilitate end ligation by ligase 4 (Lig 4). NHEJ uses additional end processing enzymes such as Artemis, PNKPT1 and X-Pol lambda and Pol mu to closely align and ligate the cleaved DNA ends.
Therefore, it is necessary to develop a substrate for NHEJ repair that is capable of specifically recognizing NHEJ damage repair pathways in cells.
Disclosure of Invention
The invention aims to provide a NHEJ substrate, a preparation method and application thereof, wherein the NHEJ substrate can specifically identify a NHEJ injury repair path in cells, has a wide injury repair range and high efficiency, and is beneficial to the subsequent screening and research and development of different types of medicaments.
In a first aspect of the invention, there is provided a method of preparing a NHEJ substrate comprising the steps of:
s1, connecting Nanoluc, SV40 and CMV genes to a plasmid vector through enzyme digestion to prepare a dsDNA fragment sequence;
s2, after annealing treatment is carried out on the dsDNA fragment sequence prepared in the step S1, connecting one end of a Nanoluc gene in the dsDNA fragment sequence with an N-end long-chain primer and an N-end short-chain primer, and connecting one end of a CMV gene with a C-end long-chain primer and a C-end short-chain primer to prepare a partially double-stranded DNA;
s3, connecting the partially double-stranded DNA obtained in the step S2 by DNA ligase to obtain the NHEJ substrate of the double-stranded DNA.
Preferably, the N-terminal long-chain primer and the C-terminal long-chain primer have 10bp homologous complementary sequences.
Preferably, the N-terminal long-chain primer sequence is shown in SEQ ID NO:1, the sequence of the N-terminal short-chain primer is shown as SEQ ID NO:2, the sequence of the C-terminal long-chain primer is shown as SEQ ID NO:3, the sequence of the C-terminal short-chain primer is shown as SEQ ID NO: 4.
Preferably, the N-terminal long-chain primer and the C-terminal long-chain primer have 20bp homologous complementary sequences.
Preferably, the N-terminal long-chain primer sequence is shown in SEQ ID NO:6, the sequence of the N-terminal short-chain primer is shown as SEQ ID NO:7, the sequence of the C-terminal long-chain primer is shown as SEQ ID NO:8, the sequence of the C-terminal short-chain primer is shown as SEQ ID NO: shown at 9.
Preferably, the dsDNA fragment sequence is set forth in SEQ ID NO: shown at 5.
In a second aspect of the present invention, there is provided a NHEJ substrate obtained by the above method for preparing a NHEJ substrate, wherein the NHEJ substrate has a 10bp or 20bp homologous complementary sequence.
Preferably, the NHEJ substrate sequence is as shown in SEQ ID NO: shown at 10.
Preferably, the NHEJ substrate sequence is as shown in SEQ ID NO: 11.
In a third aspect of the invention, an NHEJ substrate obtained by the above preparation method of an NHEJ substrate or an application of the above NHEJ substrate in DNA repair, drug screening and research and development, and new target screening is provided.
The beneficial effects are that:
the NHEJ substrate obtained by the preparation method of the NHEJ substrate can specifically identify a NHEJ damage repair path in cells, has a wide damage repair range and high efficiency, and is beneficial to the screening and research and development of different subsequent drugs, such as covalent small molecules, protein degradation inducers, RNA drugs and other drugs; the potential application value can deeply explore related targets of the NHEJ repair pathway, can be combined with a screening method of a library, explores new targets of synergistic effect and compensation effect in the pathway, and provides for future research work of combined drug and drug resistance compensation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing comparison of fluorescence signal values of substrates prepared in example I and comparative example of the present invention 5 hours after transfection of cells, wherein DMSO is used as a control, pepossertib is used as an NHEJ repair pathway inhibitor, and ART558 is used as a TMEJ repair pathway inhibitor;
FIG. 2 is a graph showing comparison of fluorescence signal values of substrates prepared in example I and comparative example of the present invention 24 hours after cell transfection, wherein DMSO is used as a control, pepossertib is used as an NHEJ repair pathway inhibitor, and ART558 is used as a TMEJ repair pathway inhibitor;
FIG. 3 is a graph showing comparison of the inhibition of repair of substrates prepared in example I and comparative example of the present invention by compounds 5 hours after cell transfection, wherein DMSO is used as a control, pepossertib is used as an NHEJ repair pathway inhibitor, and ART558 is used as a TMEJ repair pathway inhibitor;
FIG. 4 is a graph showing comparison of the inhibition of repair of substrates prepared in example I and comparative example of the present invention by compounds 24 hours after cell transfection, wherein DMSO is used as a control, pepossertib is used as an NHEJ repair pathway inhibitor, and ART558 is used as a TMEJ repair pathway inhibitor;
FIG. 5 is a graph showing inhibition of repair of substrates prepared according to comparative examples of the present invention by different concentrations of Pepossertib (M3814);
FIG. 6 is a graph showing inhibition of repair of a substrate prepared in example one of the present invention by different concentrations of Pepossertib (M3814);
FIG. 7 is a graph showing inhibition of repair of a substrate prepared in example II by Pepossertib (M3814) at various concentrations;
FIG. 8 is a graph showing that repair of a substrate prepared in the comparative example of the present invention is inhibited by ART558 at various concentrations;
FIG. 9 is a graph showing the inhibition of repair of a substrate prepared in accordance with the example of the present invention by ART558 at various concentrations;
FIG. 10 is a graph showing that repair of a substrate prepared in example II of the present invention is inhibited by ART558 at various concentrations.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms also include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The embodiment provides a preparation method of a NHEJ substrate, which comprises the following steps:
s1, preparation of dsDNA fragment sequences: the Nanoluc, SV40 and CMV genes are connected to a plasmid vector through enzyme digestion to prepare a dsDNA fragment sequence;
specifically, the Nanoluc, SV40 and CMV genes are connected to a pcDNA3.1 vector by enzyme digestion, the vector is amplified, and the amplified plasmid is subjected to double enzyme digestion by XhoI and HindIII to prepare a dsDNA fragment sequence.
S2, preparing a partially double-stranded DNA: after annealing treatment is carried out on the dsDNA fragment sequence prepared in the step S1, one end of a specific Nanoluc gene in the dsDNA fragment sequence is connected with an N-end long-chain primer and an N-end short-chain primer, and one end of a CMV promoter gene is connected with a C-end long-chain primer and a C-end short-chain primer, so that a partially double-stranded DNA is prepared;
s3, preparing a NHEJ detection substrate: and (3) ligating the partially double-stranded DNA prepared in the step (S2) by using T4DNA ligase to prepare a NHEJ substrate of the double-stranded DNA.
In this example, the N-terminal long-chain primer and the C-terminal long-chain primer have 10bp homologous complementary sequences.
In this example, the N-terminal long-chain primer sequence is: TCGAGGACTTGGTCCAGGTTG TAGCCGGCTGTCTGTCGCCAGTCCCCAACGAAATCTTCGAGTGTGAAGACCATGCT (SEQ ID NO: 1), the N-terminal short-chain primer sequence is: GCCGGCTACAAC CTGGACCAAGTCC (SEQ ID NO: 2), the C-terminal long-chain primer sequence is: AGCTTTAA CTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTA TAGGGAGACCCAAGCATGGTCT (SEQ ID NO: 3), the C-terminal short-chain primer sequence is: CCAGTAAGCAGTGGGTTCTCTAGTTAA (SEQ ID NO: 4), wherein the homology and complementarity at the dash-dot line with AGACCATGCT and AGCATGGTCT are 10 bp.
In this example, the dsDNA fragment sequence is set forth in SEQ ID NO: shown at 5.
Example two
The embodiment provides a preparation method of a NHEJ substrate, which comprises the following steps:
s1, preparation of dsDNA fragment sequences: the Nanoluc, SV40 and CMV genes are connected to a plasmid vector through enzyme digestion to prepare a dsDNA fragment sequence;
specifically, the Nanoluc, SV40 and CMV genes are connected to a pcDNA3.1 vector by enzyme digestion, the vector is amplified, and the amplified plasmid is subjected to double enzyme digestion by XhoI and HindIII to prepare a dsDNA fragment sequence.
S2, preparing a partially double-stranded DNA: after annealing treatment is carried out on the dsDNA fragment sequence prepared in the step S1, one end of a specific Nanoluc gene in the dsDNA fragment sequence is connected with an N-end long-chain primer and an N-end short-chain primer, and one end of a CMV promoter gene is connected with a C-end long-chain primer and a C-end short-chain primer, so that a partially double-stranded DNA is prepared;
s3, preparing a NHEJ detection substrate: and (3) ligating the partially double-stranded DNA prepared in the step (S2) by using T4DNA ligase to prepare a NHEJ substrate of the double-stranded DNA.
In this example, the N-terminal long-chain primer and the C-terminal long-chain primer have 20bp homologous complementary sequences.
In this example, the N-terminal long-chain primer sequence is: TCGAGGACTTGGTCCAGGTTG TAGCCGGCTGTCTGTCGCCAGTCCCCAACGAAATCTTCGAGTGTGAAGACCATGCTTGGGT (SEQ ID NO: 6), the N-terminal short-chain primer sequence is: GCCGGCT ACAACCTGGACCAAGTCC (SEQ ID NO: 7), the C-terminal long-chain primer sequence is: AG CTTTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGAC TCACTATAGGGAGACCCAAGCATGGTCTTCACA (SEQ ID NO: 8), the C-terminal short-chain primer sequence is: CCAGTAAGCAGTGGGTTCTCTAGTTAA (SEQ ID NO: 9), wherein TGTGAAGACCATGCTTGGGT and ACCCAAGCATGGTC TTCACA at the dash are 20bp homologous complementary sequences.
In this example, the dsDNA fragment sequence is set forth in SEQ ID NO: shown at 5.
Example III
This example provides a NHEJ substrate obtained by the preparation method of NHEJ substrates of examples one to two, which has a 10bp or 20bp homologous complementary sequence.
In this example, the NHEJ substrate sequence obtained by the preparation method of example one is as set forth in SEQ ID NO: shown at 10.
In this example, the NHEJ substrate sequence obtained by the preparation method of example two is shown in SEQ ID NO: 11.
Comparative example
The present example provides a method for preparing a substrate comprising the steps of:
s1, preparation of dsDNA fragment sequences: the Nanoluc, SV40 and CMV genes are connected to a plasmid vector through enzyme digestion to prepare a dsDNA fragment sequence;
specifically, the Nanoluc, SV40 and CMV genes are connected to a pcDNA3.1 vector by enzyme digestion, the vector is amplified, and the amplified plasmid is subjected to double enzyme digestion by XhoI and HindIII to prepare a dsDNA fragment sequence.
S2, preparing a partially double-stranded DNA: after annealing treatment is carried out on the dsDNA fragment sequence prepared in the step S1, one end of a specific Nanoluc gene in the dsDNA fragment sequence is connected with an N-end long-chain primer and an N-end short-chain primer, and one end of a CMV promoter gene is connected with a C-end long-chain primer and a C-end short-chain primer, so that a partially double-stranded DNA is prepared;
s3, preparing a substrate: and (3) ligating the partially double-stranded DNA prepared in the step (S2) by using T4DNA ligase to prepare a NHEJ substrate of the double-stranded DNA.
In this example, the N-terminal long-chain primer and the C-terminal long-chain primer have 4bp homologous complementary sequences.
In this example, the N-terminal long-chain primer sequence is: TCGAGGACTTGGTCCAGGTTG TAGCCGGCTGTCTGTCGCCAGTCCCCAACGAAATCTTCGAGTGTGAA GACCAT (SEQ ID NO: 12), the N-terminal short-chain primer sequence is: GCCGGCTACAACCT GGACCAAGTCC (SEQ ID NO: 13), the C-terminal long-chain primer sequence is: AGCTTTAAC TAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTAT AGGGAGACCCAAGCATGG (SEQ ID NO: 14), the C-terminal short-chain primer sequence is: c CAGTAAGCAGTGGGTTCTCTAGTTAA (SEQ ID NO: 15), wherein CCAT and ATGG at the dash line are 4bp homologous complements.
In this example, the dsDNA fragment sequence is set forth in SEQ ID NO: shown at 5.
Example IV
The embodiment provides a method for detecting the substrate, which comprises the following steps:
1. preparation work
The Neon transfection system was UV sterilized and E2 and R buffers were warmed to room temperature and the complete medium without antibiotics was warmed up in a 37℃water bath.
b. Setting up a Neon transfection system: the electrode cup was inserted into a Neon transfection system pipette and 3mL of E2 buffer was added to the tube.
2. Cell treatment
a. Cell culture: HEK293T cells were cultured using DMEM medium (complete medium) of 10% heat-inactivated fetal bovine serum and 1% antibiotics.
b. Cell treatment: HEK293T cells grown to 80% -90% in T75 flasks were digested with pancreatin and counted.
Injection to ensure finenessNumber of cell passages<20 and less than 100%, the cell viability was as follows>90% suspension, preparation of cell density of 2-3×10 6 about/mL.
Collection of 2X 10 6 HEK293T cells were isolated with DPBS (Mg-free 2+ And Ca 2+ ) The cells were washed once, centrifuged at 1000rpm for 5min, and the supernatant was discarded. Cells were resuspended with 100. Mu.L of R buffer.
c. Preparing a DNA buffer solution: according to the DNA substrate concentration, 2ug of NHEJ substrate prepared in the first and second examples was taken, 10ug of the substrate prepared in the comparative example was taken, and if the substrate volume was less than 10u L, R buffer was added to prepare 10ul of DNA buffer, respectively.
The resuspended cells were all added to the DNA buffer and mixed well.
3. Cell electroporation
100uL of the cell suspension was mixed with 10uL of the DNA substrate suspension, thereby forming a 110uL total system. Thereafter 100uL was aspirated from it using an electrokinetic gun and added to an electrode cup containing 3mL of E2 buffer, and electroporation was started according to the parameters of Table 1.
TABLE 1
| Pulse voltage(V) | 1600 |
| Pulse width(ms) | 10 |
| Pulse times | 3 |
Placing the transfected experimental cells obtained in the step 3 in a DMEM culture medium without antibiotics, and fully and uniformly mixing.
4. Substrate detection
a. Cell plating: the transformed cells were harvested and seeded at 40000 cells per well in 384 well plates.
b. The gradient diluted Peposertib was added to the cells in step a, and after 24h the Nanoluc signal was read using an microplate reader.
Pepsertib (M3814) is an orally administered small molecule selective DNA-PK inhibitor capable of blocking DNA-PK kinase activity at nanomolar concentrations, inhibiting its function during DNA repair, leading to the continued presence of DNADSB and subsequent cell death. P epoertib often shows synergy with radiotherapy and DSB-induced chemotherapy by preventing the NHEJ repair pathway, radiotherapy or chemotherapy-induced DNADSB in preclinical studies.
c. ART558 after gradient dilution was added to the cells in step a and the signal value of Nanoluc was read 24h later using an enzyme-labeled instrument.
ART558 is a low molecular weight allosteric inhibitor with the advantage of selectively inhibiting POLQ-mediated major DNA repair pathways, not inhibiting other human DNA polymerases (Pol alpha, pol gamma, pol eta and Pol v), not targeting the NHEJ DNA repair pathway. At the same time ART558 was able to inhibit POLQ-mediated DNA DSB repair with nanomolar potency but was not able to be used to inhibit NHEJ, further demonstrating excellent selectivity of molecules.
The substrate detection results were as follows:
(1) The results of the fluorescent signal value 5 hours after transfection of the cells (FIG. 1) and the fluorescent signal value 24 hours after transfection of the cells (FIG. 2) showed that: cells transfected with the substrate with the 10bp homologous complement prepared in example one produced a higher fluorescence signal value than the substrate with the 4bp homologous complement prepared in comparative example, indicating that the substrate prepared in example one had a higher level of repair and was much greater than the substrate with the 4bp homologous complement prepared in comparative example.
(2) 5 hours after transfection of the cells, the repair of the substrate was indicated by inhibition of ART558, pepossertib compounds (FIG. 3): repair of the substrate with the 4bp homologous complement prepared in the comparative example was not inhibited by any compound, indicating that neither NHEJ nor TMEJ was involved in repair of the substrate at this time point; the repair of the substrate with 10bp homologous complement prepared in example one was inhibited by Peposertib but not by ART558, indicating that the repair of the substrate was now dependent on the NHEJ repair pathway and not on the TMEJ repair pathway.
(3) 24 hours after cell transfection, substrate repair was indicated by inhibition of ART558, peposertib compounds (fig. 4): repair of the substrate with the 4bp homologous complement prepared in the comparative example was inhibited by ART558 and not by Peposertib, indicating that at this time point, the TMEJ repair pathway was involved in repair of the substrate; the repair of the substrate with 10bp homologous complement prepared in example I was inhibited by Pepossertib, and was still not inhibited by ART558, indicating that the repair of the substrate was still dependent on the NHEJ repair pathway.
(4) Inhibition of substrate repair by different concentrations of Peposertib (M3814) compounds indicated: as the Peposertib concentration increased, the substrate repair made in the comparative example was not inhibited by Peposertib (fig. 5), indicating that the substrate repair did not rely on the NHEJ pathway; the substrates prepared in example one and example two showed an increase in inhibition with increasing concentration of Peposertib (fig. 6 to 7), indicating that the substrate repair relied on the NHEJ pathway.
(5) The repair of the substrate is indicated by inhibition of the ART558 compound at different concentrations: as the concentration of a RT558 increases, the substrate repair made by the comparative example is inhibited by ART558 (fig. 8), indicating that the substrate repair relies on the TMEJ pathway; the substrates prepared in example one and example two had reduced inhibition with increasing concentrations of ART558 (fig. 9-10), indicating that substrate repair did not rely on the TMEJ pathway.
Example five
The NHEJ substrates obtained by the preparation method of the NHEJ substrates in the first to second embodiments and the NHEJ substrate in the third embodiment can be used for screening and researching and developing different types of medicines, such as covalent small molecules, protein degradation inducers, RNA medicines and other types of medicines; the potential application value can deeply explore related targets of the NHEJ repair pathway, can be combined with a screening method of a library, explores new targets of synergistic effect and compensation effect in the pathway, and provides for future research work of combined drug and drug resistance compensation.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (5)
1. A method for preparing a NHEJ substrate comprising the steps of:
s1, connecting Nanoluc, SV40 and CMV genes to a plasmid vector by enzyme digestion to prepare a dsDNA fragment sequence, wherein the dsDNA fragment sequence is shown in SEQ ID NO:5 is shown in the figure;
s2, after annealing treatment is carried out on the dsDNA fragment sequence prepared in the step S1, connecting one end of a Nanoluc gene in the dsDNA fragment sequence with an N-end long-chain primer and an N-end short-chain primer, and connecting one end of a CMV gene with a C-end long-chain primer and a C-end short-chain primer to prepare a partially double-stranded DNA;
the N-terminal long-chain primer and the C-terminal long-chain primer have 10bp homologous complementary sequences, and the sequence of the N-terminal long-chain primer is shown as SEQ ID NO:1, the sequence of the N-terminal short-chain primer is shown as SEQ ID NO:2, the sequence of the C-terminal long-chain primer is shown as SEQ ID NO:3, the sequence of the C-terminal short-chain primer is shown as SEQ ID NO:4 is shown in the figure;
or alternatively;
the N-terminal long-chain primer and the C-terminal long-chain primer have 20bp homologous complementary sequences, and the sequence of the N-terminal long-chain primer is shown as SEQ ID NO:6, the sequence of the N-terminal short-chain primer is shown as SEQ ID NO:7, the sequence of the C-terminal long-chain primer is shown as SEQ ID NO:8, the sequence of the C-terminal short-chain primer is shown as SEQ ID NO: shown as 9;
s3, connecting the partially double-stranded DNA obtained in the step S2 by DNA ligase to obtain the NHEJ substrate of the double-stranded DNA.
2. A NHEJ substrate obtained by the method of preparation of a NHEJ substrate according to claim 1, which has a 10bp or 20bp homologous complementary sequence.
3. The NHEJ substrate of claim 2, wherein the NHEJ substrate sequence is set forth in SEQ ID NO: shown at 10.
4. The NHEJ substrate of claim 2, wherein the NHEJ substrate sequence is set forth in SEQ ID NO: 11.
5. Use of a NHEJ substrate according to any one of claims 2-4 in drug screening or new target screening.
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| WO2019217785A1 (en) * | 2018-05-10 | 2019-11-14 | St. Jude Children's Research Hospital, Inc. | High-throughput method for characterizing the genome-wide activity of editing nucleases in vitro |
| CN112980890A (en) * | 2021-03-19 | 2021-06-18 | 天津市肿瘤医院（天津医科大学肿瘤医院） | Non-homologous end connection detection system and application thereof |
| CN115786339A (en) * | 2022-10-24 | 2023-03-14 | 北京爱思益普生物科技股份有限公司 | TMEJ detection substrate, preparation method and application thereof |
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| WO2019217785A1 (en) * | 2018-05-10 | 2019-11-14 | St. Jude Children's Research Hospital, Inc. | High-throughput method for characterizing the genome-wide activity of editing nucleases in vitro |
| CN112980890A (en) * | 2021-03-19 | 2021-06-18 | 天津市肿瘤医院（天津医科大学肿瘤医院） | Non-homologous end connection detection system and application thereof |
| CN115786339A (en) * | 2022-10-24 | 2023-03-14 | 北京爱思益普生物科技股份有限公司 | TMEJ detection substrate, preparation method and application thereof |
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