CN112941634A - Method for screening DNA coding compound library capable of simultaneously combining multiple biological targets - Google Patents
Method for screening DNA coding compound library capable of simultaneously combining multiple biological targets Download PDFInfo
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Abstract
The invention discloses a method for screening compounds simultaneously combined with a plurality of biological targets through a DNA coding compound library. The invention also discloses a method for screening a compound simultaneously combining the E3 ubiquitin ligase and a target protein through a DNA coding compound library. The method further widens the application range of the DNA coding compound library, and is suitable for screening various biological targets.
Description
Technical Field
The invention belongs to the field of drug screening, and particularly relates to a method for screening a compound capable of simultaneously combining multiple biological targets through a DNA coding compound library.
Background
In the field of new drug development, high-throughput screening for biological targets is one of the main means for rapidly obtaining lead compounds. However, traditional high throughput screening based on single molecules requires long time, large equipment investment, limited number of library compounds (millions), and the building of compound libraries requires decades of accumulation, limiting the efficiency and possibility of discovery of lead compounds. The recently developed DNA coding compound library technologies (WO2005058479, WO2018166532, CN103882532) combine the technologies of combinatorial chemistry and molecular biology, and each compound is labeled with a DNA tag on the molecular level, so that a compound library up to hundred million levels can be synthesized in a very short time, and the compound can be identified by a gene sequencing method, so that the size and synthesis efficiency of the compound library are greatly increased, and the technology becomes the trend of the next generation compound library screening technology. DNA-encoded compound library technology is beginning to be widely used in the pharmaceutical industry and produces many positive effects (Accounts of Chemical Research,2014,47, 1247-.
ProTAC (protein TArgeting Chimera) is a bifunctional small molecule, one end of which is combined with a ligand of a target protein, and the other end of which is combined with a ligand of E3 ubiquitin ligase and connected through a section of chain. The target protein and the E3 enzyme can be brought into proximity in vivo, so that the target protein is labeled with ubiquitin and then degraded by the ubiquitin-proteasome pathway. There has been recent report on molecular glue (molecular glue) of E3 ubiquitin ligase, i.e. smaller molecules can simultaneously bind E3 ubiquitin ligase and target protein to realize degradation of target protein.
Based on the development of new drug targeting technologies, there is a need to screen compounds that can simultaneously bind two or more targets, such as PROTAC protein degradation targeting chimeras or molecular glue. Therefore, a novel compound screening technology for a DNA coding compound library needs to be developed to improve the application value of the DNA coding compound library technology.
Disclosure of Invention
The present invention provides a method for screening a library of DNA-encoded compounds for compounds that bind multiple biological targets simultaneously, comprising the steps of:
a. labeling n biological targets with one of n different complementary labels, respectively;
b. simultaneously incubating the biological target with the label n and the DNA coding compound library;
c. adding the other of the 1 st complementary tags, removing unbound molecules, and dissociating from the biological target to obtain a DNA-encoding compound;
d. replacing the library of DNA encoding compounds in step b with the DNA encoding compounds obtained by dissociation, replacing the other party of the 1 st complementary tag with the other party of the 2 nd complementary tag in step c, and repeating steps b and c until the other party of the nth complementary tag is added in step c;
wherein n is 2, 3, 4 or 5.
Further, the biological target is protein, RNA, DNA.
Further, one of the complementary tags is a biotin tag, a His tag, a GST tag, an MBP tag, a FLAG tag, a C-Myc tag, a SUMO tag, an SNAP tag, one of complementary DNAs, or one of complementary RNAs.
The other complementary tag is avidin, streptomycin, nickel, glutathione, polysaccharide resin, anti-FLAG, anti-C-Myc, SUMO protease, Benzylguanine (BG), the other complementary DNA, or the other complementary RNA.
Further, after adding the other of the 1 st to n th complementary tags in step c and step d, unbound molecules are removed by physical, chemical or biological means.
The invention also provides a method for screening a compound simultaneously binding E3 ubiquitin ligase and a target protein through a DNA coding compound library, which comprises the following steps:
a. labeling the E3 ubiquitin ligase with one of the 1 st complementary tags, labeling the target protein with one of the 2 nd complementary tags, the 1 st complementary tag being different from the 2 nd complementary tag;
b. incubating a pool of DNA-encoding compounds simultaneously with E3 ubiquitin ligase from the party bearing the 1 st complementary tag and the target protein from the party bearing the 2 nd complementary tag;
c. adding the other of the 1 st complementary tags, removing unbound molecules, and dissociating from the biological target to obtain a DNA-encoding compound;
d. c, incubating the target protein of the party with the 1 st complementary label, the E3 ubiquitin ligase and the party with the 2 nd complementary label with the DNA coding compound obtained by dissociation in the step c;
e. the other of the 2 nd complementary tags is added and the unbound molecules are removed and the DNA-encoded compound is dissociated from the biological target.
Furthermore, one of the 1 st complementary tag is a His tag, and the other of the 1 st complementary tag is nickel.
Further, one of the 2 nd complementary tags is a GST tag, and the other of the 2 nd complementary tags is glutathione.
Further, the other of the 1 st complementary tag and the other of the 2 nd complementary tag are immobilized on a solid support; preferably, the solid phase carrier is a magnetic bead.
Further, the incubation in the steps b and d is carried out in a screening buffer solution, and the screening buffer solution contains salmon sperm DNA; further, the screening buffer was 1)20mM phosphate buffer containing 150mM sodium chloride, 0.3mg/ml salmon sperm DNA, 0.1% tween-20, 10mM imidazole (for nickel-coated magnetic beads only), pH 7.0, or 2)50mM HEPES ((4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer containing 150mM sodium chloride, 0.3mg/ml salmon sperm DNA, 0.1% tween-20, 10mM imidazole (for nickel-coated magnetic beads only), pH 7.0.
Further, nickel-coated magnetic beads were added in step c, and unbound molecules were removed by centrifugation.
Further, adding an elution buffer solution after the step c, and repeatedly eluting for 5-10 times; specifically, the elution buffer was 1)20mM phosphate buffer containing 150mM sodium chloride, 0.3mg/ml salmon sperm DNA, 0.1% tween-20, 10mM imidazole (for nickel-coated magnetic beads only), pH 7.0, or 2)50mM HEPES buffer containing 150mM sodium chloride, 0.3mg/ml salmon sperm DNA, 0.1% tween-20, 10mM imidazole (for nickel-coated magnetic beads only), pH 7.
Further, after removing the unbound molecules in the step e, adding an elution buffer solution, and repeatedly eluting for 3-5 times;
further, the steps c and e are dissociated from the biological target by heating to obtain the DNA coding compound. Preferably, the heating temperature is 85-95 ℃. Preferably, the thermal dissociation time is 10 to 20 minutes.
Further, the dissociation is performed in a dissociation buffer; specifically, the dissociation buffer is 1) a 20mM phosphate buffer comprising 150mM sodium chloride at pH 5.5, or 2) a 10-50mM MES (2- (N-morpholino) ethanesulfonic acid) buffer comprising 150mM sodium chloride at pH 6, or 3) a 20mM phosphate buffer comprising 150mM sodium chloride at pH 7.0; or 4)50mM HEPES buffer containing 150mM sodium chloride at pH 7.0.
Further, glutathione-coated magnetic beads are added in the step e, and unbound molecules are removed by centrifugation.
Further, qPCR quantification is performed on the number of DNA molecules after the DNA coding compound is obtained from the biological target by dissociation in the step e.
Further, if the qPCR quantification result has a super molecular numberOver 108The dissociated DNA-encoding compound is used as a new round of DNA-encoding compound library, and the screening is repeated until the number of molecules is reduced to 107～108。
Further, sequencing the DNA coding compound obtained by dissociation, and decoding the sequencing result to obtain an enrichment signal.
In some embodiments of the invention, the E3 ubiquitin ligase is CRBN, VHL, MDM2, IAPs, DCAF15, DCAF16, RNF4 and RNF 114.
The method can also be used for screening molecules which can simultaneously have affinity with tissue specific protein and therapeutic target protein, and can be used for targeted medication, reducing toxic and side effects, improving the drug absorption of target tissues, enhancing the bioavailability of drugs and the like. The method can also be used for screening the drug molecules with double targets acting simultaneously, and can be used for treating diseases with double Mechanisms (Dual Mechanisms). Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 shows the structure of 6 positive compounds with different DNA tags added to a library of DNA encoding compounds in example 1 of the present invention;
FIG. 2 shows the signal intensity of 6 positive compounds in example 1 of the present invention in different samples screened with a library of DNA encoding compounds;
FIG. 3 is the structure of the proteolytic degradation agent corresponding to dBET1 and 6 DNA-encoded positive compounds in example 1 of the present invention;
FIG. 4 shows that dBET1 and 6 protein-degrading agents are at MV4 in example 1 of the present invention; 11 picture of BRD4 protein degradation assay in cells;
FIG. 5 is a schematic diagram showing the structure of a library of DNA-encoded compounds of the E3 ligase with respect to CRBN in example 2 of the present invention;
FIG. 6 is a signal diagram of the compounds obtained by screening the proteins CRBN and BRD4 in example 2 of the invention, and a comparison of Tanimoto similarity of the structures of 4 representative compounds (R)2、R3Expressed in a specific reagent code);
FIG. 7 shows that the screening of CRBN and BRD4 proteins in example 2 of the present invention resulted in the identification of 4 representative PROTAC molecules at MV 4; 11 cells for BRD4 protein degradation assay results;
FIG. 8 is a graph of the comparison of Compound 75-NX-1 of example 2 of the present invention and its corresponding Compound 57-NX-1 without thalidomide and Linker to MV 4; 11 results of cell proliferation inhibition assay;
Detailed Description
Example 1 screening of Compounds that bind to both CRBN and BRD4
Libraries of DNA-encoding compounds were constructed according to the method described in WO 2006135786. A positive control compound with a DNA tag as shown in FIG. 1 was additionally added to the library of DNA encoding compounds. The protein CRBN-His tag/DDB1 used in example 1 was expressed internally by HitGen and the protein BRD4-GST tag was from Active Motif (cat 81855).
The screening buffer used in this example was 1) a 20mM phosphate buffer containing 150mM sodium chloride, 0.3mg/ml salmon sperm DNA, 0.1% tween-20, 10mM imidazole (for nickel coated magnetic beads only), pH 7.0; or 2)50mM HEPES ((4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer containing 150mM sodium chloride, 0.3mg/ml salmon sperm DNA, 0.1% tween-20, 10mM imidazole (for nickel-coated magnetic beads only), pH 7.0.
The elution buffer used in this example was 1) a 20mM phosphate buffer containing 150mM sodium chloride, 0.3mg/ml salmon sperm DNA, 0.1% tween-20, 10mM imidazole (for nickel-coated magnetic beads only), pH 7.0; or 2)50mM HEPES buffer containing 150mM sodium chloride, 0.3mg/ml salmon sperm DNA, 0.1% tween-20, 10mM imidazole (for nickel coated magnetic beads only), pH 7.
The dissociation buffer used in this example was 1) a 20mM phosphate buffer containing 150mM sodium chloride at pH 5.5; or 2)10-50mM MES (2- (N-morpholino) ethanesulfonic acid) buffer comprising 150mM sodium chloride, pH 6; or 3)20mM phosphate buffer containing 150mM sodium chloride at pH 7.0; or 4)50mM HEPES buffer containing 150mM sodium chloride at pH 7.0.
The libraries of target proteins and DNA-encoding compounds were incubated in screening buffer according to the following protocol.
| Group | Target proteins | |
| 1 | CRBN/DDB1 Complex (250pmol) | |
| 2 | BRD4(250pmol) | |
| 3 | CRBN/DDB1 Complex/BRD 4 (250pmol each) | |
| 4 | Nickel-coated magnetic bead and glutathione-coated magnetic bead |
For cohorts 1, 2 and 4, the screening solutions were incubated in closed centrifuge tubes for 1 hour on a low speed shaker at room temperature. Then, nickel-coated magnetic beads were added to group 1, glutathione-coated magnetic beads were added to group 2, and incubation was performed at room temperature for 30 min. Centrifuge for 250g x 3min and remove the supernatant containing unbound molecules. Adding 200-500 mu L of elution buffer solution for incubation for 1min, further performing centrifugal elution, and repeating for 3-5 times. Adding 50-100 mu L of dissociation buffer solution, heating and dissociating for 10-20 minutes at 85-95 ℃, centrifuging and collecting supernatant. qPCR quantitating the number of DNA molecules in the supernatant, e.g., the number of molecules exceeds108Using the supernatant as a new round of DNA coding compound library, adjusting pH, desalting with a purification column to remove additional buffer solution components, and repeating the screening until the number of molecules is reduced to 107～108. The sequence was determined using Illumina NovaSeq6000 or HiSeq2500 and its standard reagents. And further analyzing and decoding the sequencing result to obtain each group of enrichment signal distribution maps.
For group 3, the screening solution was incubated in a closed centrifuge tube for 1 hour on a low speed shaker at room temperature. Nickel coated magnetic beads were then added and incubated at room temperature for 30 min. Centrifuge for 250g x 3min and remove the supernatant containing unbound molecules. Adding 200-500 mu L of elution buffer solution for incubation for 1min, further performing centrifugal elution, and repeating for 5-10 times. Adding 50-100 mu L of dissociation buffer solution, heating and dissociating for 10-20 minutes at 85-95 ℃, centrifuging and collecting supernatant. After pH adjustment of the supernatant and desalting of the column to remove additional buffer components, the supernatant was used as a new round of DNA-encoding compound library, and repeated incubation with CRBN/DDB1 complex/BRD 4 for 1 hour at room temperature in a low speed shaker. Glutathione coated magnetic beads were added and incubated at room temperature for 30 min. Centrifuge for 250g x 3min and remove the supernatant containing unbound molecules. Adding 50-100 mu L of elution buffer solution for incubation for 1min, further performing centrifugal elution, and repeating for 3-5 times. Adding 50-100 mu L of elution buffer solution, heating and dissociating for 10-20 minutes at 85-95 ℃, centrifuging and collecting supernatant. qPCR quantitating the number of DNA molecules in the supernatant, e.g., greater than 108The supernatant was used as a new round of DNA-encoding compound library, and the screening was repeated again until the number of molecules decreased to 107～108. The sequence was determined using Illumina NovaSeq6000 or HiSeq2500 and its standard reagents. And further analyzing and decoding the sequencing result to obtain each group of enrichment signal distribution maps.
Figure 2 shows the signal intensity (Sequence Count) of 6 positive compounds in each of the 4 groups screened. Results in groups 1 and 2 b these 6 compounds are directed to the binding of CRBN and BRD4 proteins, respectively, but it cannot be stated that these compounds bind the optimal combination of both CRBN and BRD4 proteins. The 3 rd panel showed that Conjugate-4 was apparently the best compound for binding both proteins at the same time, with a chain length identical to dBET 1. The correspondence of 6 compounds in fig. 1 to the screening results was further verified by the PROTAC molecules corresponding to these 6 compounds (formamide structures without DNA tag but containing linker DNA, structures of dBET1 and 6 PROTAC molecules are shown in fig. 3), using the method of Western Blot for MV 4; 11 (cell line sensitive to BRD4 protein degradation) BRD4 protein degradation was assessed and the results are shown in figure 4. The protein degradation effect of PROTAC-4 in FIG. 4 was optimal, consistent with the signal of Conjugate-4 in screening group 3.
Example 2 screening of Compounds that bind to both CRBN and BRD4 novel protein degradation Agents
Libraries of DNA-encoding compounds as in figure 5 were constructed according to the method described in WO2006135786 and then screened. The protein CRBN-His tag/DDB1 used in this example was expressed internally by HitGen and the protein BRD4-GST tag was from Active Motif (cat 81855).
The screening buffer used in this example was 1) a 20mM phosphate buffer containing 150mM sodium chloride, 0.3mg/ml salmon sperm DNA, 0.1% tween-20, 10mM imidazole (for nickel coated magnetic beads only), pH 7.0; or 2)50mM HEPES ((4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer containing 150mM sodium chloride, 0.3mg/ml salmon sperm DNA, 0.1% tween-20, 10mM imidazole (for nickel-coated magnetic beads only), pH 7.0.
The elution buffer used in this example was 1) a 20mM phosphate buffer containing 150mM sodium chloride, 0.3mg/ml salmon sperm DNA, 0.1% tween-20, 10mM imidazole (for nickel-coated magnetic beads only), pH 7.0; or 2)50mM HEPES buffer containing 150mM sodium chloride, 0.3mg/ml salmon sperm DNA, 0.1% tween-20, 10mM imidazole (for nickel coated magnetic beads only), pH 7.
The dissociation buffer used in this example was 1) a 20mM phosphate buffer containing 150mM sodium chloride at pH 5.5; or 2)10-50mM MES (2- (N-morpholino) ethanesulfonic acid) buffer comprising 150mM sodium chloride, pH 6; or 3)20mM phosphate buffer containing 150mM sodium chloride at pH 7.0; or 4)50mM HEPES buffer containing 150mM sodium chloride at pH 7.0.
The libraries of target proteins and DNA-encoding compounds were incubated in screening buffer according to the following protocol.
| Group | Target proteins | |
| 1 | CRBN/DDB1 Complex (250pmol) | |
| 2 | BRD4(250pmol) | |
| 3 | CRBN/DDB1 Complex/BRD 4 (250pmol each) | |
| 4 | Nickel-coated magnetic bead and glutathione-coated magnetic bead |
For cohorts 1, 2 and 4, the screening solutions were incubated in closed centrifuge tubes for 1 hour on a low speed shaker at room temperature. Then, nickel-coated magnetic beads were added to group 1, glutathione-coated magnetic beads were added to group 2, and incubation was performed at room temperature for 30 min. Centrifuge for 250g x 3min and remove the supernatant containing unbound molecules. Adding 200-500 mu L of elution buffer solution for incubation for 1min, further performing centrifugal elution, and repeating for 3-5 times. Adding 50-100 mu L of dissociation buffer solution, heating and dissociating for 10-20 minutes at 85-95 ℃, centrifuging and collecting supernatant. qPCR quantitating the number of DNA molecules in the supernatant, e.g., greater than 108Using the supernatant as a new round of DNA coding compound library, adjusting pH, desalting with a purification column to remove additional buffer solution components, and repeating the screening until the number of molecules is reduced to 107～108. The detection is carried out by using Illumina NovaSeq6000 or HiSeq2500 and matched standard reagents thereofAnd (4) sequencing. And further analyzing and decoding the sequencing result to obtain each group of enrichment signal distribution maps.
For group 3, the screening solution was incubated in a closed centrifuge tube for 1 hour on a low speed shaker at room temperature. Nickel coated magnetic beads were then added and incubated at room temperature for 30 min. Centrifuge for 250g x 3min and remove the supernatant containing unbound molecules. Adding 200-500 mu L of elution buffer solution for incubation for 1min, further performing centrifugal elution, and repeating for 5-10 times. Adding 50-100 mu L of dissociation buffer solution, heating and dissociating for 10-20 minutes at 85-95 ℃, centrifuging and collecting supernatant. After pH adjustment of the supernatant and desalting of the column to remove additional buffer components, the supernatant was used as a new round of DNA-encoding compound library, and repeated incubation with CRBN/DDB1 complex/BRD 4 for 1 hour at room temperature in a low speed shaker. Glutathione coated magnetic beads were added and incubated at room temperature for 30 min. Centrifuge for 250g x 3min and remove the supernatant containing unbound molecules. Adding 50-100 mu L of elution buffer solution for incubation for 1min, further performing centrifugal elution, and repeating for 3-5 times. Adding 50-100 mu L of elution buffer solution, heating and dissociating for 10-20 minutes at 85-95 ℃, centrifuging and collecting supernatant. qPCR quantitating the number of DNA molecules in the supernatant, e.g., greater than 108The supernatant was used as a new round of DNA-encoding compound library, and the screening was repeated again until the number of molecules decreased to 107～108. The sequence was determined using Illumina NovaSeq6000 or HiSeq2500 and its standard reagents. And further analyzing and decoding the sequencing result to obtain each group of enrichment signal distribution maps.
FIG. 6 shows the enrichment of the compound library of group 3 in example 2 for both CRBN and BRD4 proteins, as was the case for the signal in example 1, which is positively correlated with the stability of library compound molecules in binding to CRBN and BRD 4. Compounds with different signal intensities in group 3 were selected for verification, and the selected compounds are shown in figure 6 corresponding to Sequence Count. Tanimoto similarity (less similar closer to 0 and more similar closer to 1) of four representative compounds to the BRD4 inhibitor JQ1 (the moiety dBET1 binds to BRD 4) shows that these compounds are novel structures screened through DNA-encoded compound libraries. Targeting MV4 by synthesizing 4 PROTAC molecules (75-NX-1, 78-NX-1, 123-NX-1, 127-NX-1, fig. 6); 11 cells BRD4 protein degradation assay. The results show that the strength of the screening signal is positively correlated with the protein degradation (FIG. 7). MV4 was further performed on compound 75-NX-1 and its compound 57-NX-1, which did not contain thalidomide and Linker moieties; 11 cell proliferation inhibition assay. The results show that the PROTAC molecules have significantly enhanced cellular activity compared to the inhibitors.
In conclusion, the method can screen compounds which are simultaneously combined with E3 ubiquitin ligase and target protein through a DNA coding compound library, discover a novel protein degradation agent (PROTAC), sequence the protein degradation capability of the compounds and embody good commercial application value. The method further widens the application range of the DNA coding compound library, has higher screening efficiency and accuracy, and is suitable for screening various biological targets simultaneously combined.
Claims (15)
1. A method of screening a library of DNA-encoded compounds for compounds that bind multiple biological targets simultaneously, comprising the steps of:
a. labeling n biological targets with one of n different complementary labels, respectively;
b. simultaneously incubating the biological target with the label n and the DNA coding compound library;
c. adding the other of the 1 st complementary tags, removing unbound molecules, and dissociating from the biological target to obtain a DNA-encoding compound;
d. replacing the library of DNA encoding compounds in step b with the DNA encoding compounds obtained by dissociation, replacing the other party of the 1 st complementary tag with the other party of the 2 nd complementary tag in step c, and repeating steps b and c until the other party of the nth complementary tag is added in step c;
wherein n is 2, 3, 4 or 5.
2. The method of claim 1, wherein: the biological target is protein, RNA and DNA.
3. The method of claim 1, wherein: one of the complementary tags is a biotin tag, a His tag, a GST tag, an MBP tag, a FLAG tag, a C-Myc tag, a SUMO tag, an SNAP tag, one of complementary DNAs, and one of complementary RNAs.
4. The method of claim 1, wherein: the other complementary label is avidin, streptomycin, nickel, glutathione, polysaccharide resin, anti-FLAG, anti-C-Myc, SUMO protease, Benzylguanine (BG), the other complementary DNA, and the other complementary RNA.
5. The method of claim 1, wherein: and c, adding the other one of the 1 st to n th complementary tags in the step d, and removing unbound molecules by a physical, chemical or biological method.
6. A method of screening a library of DNA-encoded compounds for compounds that bind both E3 ubiquitin ligase and a target protein comprising the steps of:
a. labeling the E3 ubiquitin ligase with one of the 1 st complementary tags, labeling the target protein with one of the 2 nd complementary tags, the 1 st complementary tag being different from the 2 nd complementary tag;
b. incubating a pool of DNA-encoding compounds simultaneously with E3 ubiquitin ligase from the party bearing the 1 st complementary tag and the target protein from the party bearing the 2 nd complementary tag;
c. adding the other of the 1 st complementary tags, removing unbound molecules, and dissociating from the biological target to obtain a DNA-encoding compound;
d. c, incubating the target protein of the party with the 1 st complementary label, the E3 ubiquitin ligase and the party with the 2 nd complementary label with the DNA coding compound obtained by dissociation in the step c;
e. the other of the 2 nd complementary tags is added and the unbound molecules are removed and the DNA-encoded compound is dissociated from the biological target.
7. The method of claim 6, wherein: one of the 1 st complementary tag is a His tag and the other of the 1 st complementary tag is nickel.
8. The method of claim 6, wherein: one of the 2 nd complementary tags is a GST tag, and the other of the 2 nd complementary tags is glutathione.
9. The method of claim 6, wherein: the other side of the 1 st complementary label and the other side of the 2 nd complementary label are fixed on a solid phase carrier; preferably, the solid phase carrier is a magnetic bead.
10. The method of claim 6, wherein: the incubation in the steps b and d is carried out in a screening buffer solution, and the screening buffer solution contains salmon sperm DNA.
11. The method of claim 6, wherein: in step c, nickel-coated magnetic beads are added and unbound molecules are removed by centrifugation.
12. The method of claim 6, wherein: the step c and the step e are dissociated from the biological target by heating to obtain a DNA coding compound; preferably, the heating temperature is 85-95 ℃.
13. The method of claim 6, wherein: and e, adding glutathione-coated magnetic beads, and removing unbound molecules by centrifugation.
14. The method of claim 6, wherein: and e, performing qPCR quantification on the number of DNA molecules after the DNA coding compound is obtained by dissociation from the biological target.
15. The method of claim 6, wherein: and e, sequencing the DNA coding compound obtained by dissociation in the step e, and decoding a sequencing result to obtain an enrichment signal.
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