DNA-ENCODED COMPOUND LIBRARY AND SCREENING METHOD THEREOF

Abstract

A synthesis and screening method of a DNA-encoded compound library. The DNA-encoded compound library consists of a DNA-encoded compound of formula (I). The screening method includes: incubating the DNA-encoded compound library with a protein target, followed by covalent cross-linking to obtain a covalently cross-linked complex; separating the covalently cross-linked complex from members in the library that do not bind to the protein target; and subjecting the covalently cross-linked complex to polymerase chain reaction (PCR) amplification and DNA sequencing.

##STR00001##

Claims

1. A DNA-encoded compound of formula (I): ##STR00021## wherein X is an atomic or molecular scaffold; A.sub.1 is a first moiety comprising a first linker and a first oligonucleotide; A.sub.2 is a second moiety comprising a second linker and a second oligonucleotide; L is a linker moiety comprising at least one group operable for covalent cross-linking; M is a functional moiety comprising at least one structural unit.

2. The DNA-encoded compound of claim 1, wherein X is a carbon atom, a nitrogen atom, a cyclic scaffold or a non-cyclic scaffold.

3. The DNA-encoded compound of claim 2, wherein the DNA-encoded compound is represented by formula (II): ##STR00022## wherein Z.sub.1 is the first oligonucleotide with its 3′ terminus attached to L.sub.1, and Z.sub.2 is the second oligonucleotide with its 5′ terminus attached to L.sub.2; or Z.sub.1 is the first oligonucleotide with its 5′ terminus attached to L.sub.1, and Z.sub.2 is the second oligonucleotide with its 3′ terminus attached to L.sub.2; L.sub.1 is the first linker comprising a first functional group capable of forming a covalent bond with the 3′ terminus or 5′ terminus of Z.sub.1; and L.sub.2 is the second linker comprising a second functional group capable of forming a covalent bond with the 5′ terminus or 3′ terminus of Z.sub.2.

4. The DNA-encoded compound of claim 3, wherein Z.sub.1 and Z.sub.2 are at least partially complementary to each other to form a double-stranded structure; Z.sub.1 and Z.sub.2 each independently have a length of at least 10 bases, and a complementary region of Z.sub.1 and Z.sub.2 has a length of at least 10 bases.

5. The DNA-encoded compound of claim 4, wherein Z.sub.1 and Z.sub.2 each independently has a polymerase chain reaction (PCR) primer sequence.

6. The DNA-encoded compound of claim 3, wherein L.sub.1 and L.sub.2 are independently an alkylene chain or poly(ethylene glycol) chain containing two functional groups, wherein the two functional groups are each independently selected from the group consisting of a phosphate group, an amino group, a hydroxyl group, and a carboxyl group.

7. The DNA-encoded compound of claim 6, wherein L.sub.1 and L.sub.2 are independently ##STR00023## wherein n is an integer selected from 1 to 10.

8. The DNA-encoded compound of claim 3, wherein the at least one group contained in L is a photosensitive group, an electrosensitive group, or other groups capable of forming covalent cross-linking with a protein.

9. The DNA-encoded compound of claim 8, wherein the at least one group contained in L is selected from the group consisting of an acridinyl group, an aryl azido group, a diphenyl ketone group, a sulfonyl fluoride group, an α,β-unsaturated acid group, an α,β-unsaturated ketone group, an α,β-unsaturated ester group, an α,β-unsaturated sulfonyl group, an α-acyl halide group, an epoxy group, an aldehyde group, a cyano group, and a boronic acid group.

10. The DNA-encoded compound of claim 3, wherein L has a structure of:
-S.sub.1-S.sub.2-S.sub.3-; wherein S.sub.1 and S.sub.3 are independently a cyclic or non-cyclic linker formed by carbon atoms, heteroatoms or a combination thereof and carrying at least one functional group, wherein the at least one functional group is each independently selected from the group consisting of a phosphate group, an amino group, a hydroxyl group, a carboxyl group, an aldehyde group, an azido group, an alkynyl group, and a halogen; and S.sub.2 is a linker containing the at least one group operable for covalent cross-linking.

11. The DNA-encoded compound of claim 10, wherein S.sub.1 and S.sub.3 are independently selected from the group consisting of ##STR00024## and a combination thereof, wherein m is a integer selected from 1 to 20.

12. The DNA-encoded compound of claim 10, wherein the at least one group operable for covalent cross-linking is linked to S.sub.1 and S.sub.3, and is selected from the group consisting of: ##STR00025## ##STR00026## wherein R.sub.1 is a carbon or nitrogen atom; and R.sub.2 is hydrogen, an alkyl with or without a heteroatom, or an aryl with or without a heteroatom.

13. The DNA-encoded compound of claim 12, wherein the at least one group operable for covalent cross-linking is no more than 15 atoms away from the functional moiety M.

14. The DNA-encoded compound of claim 3, wherein X is ##STR00027## wherein q is an integer selected from 1 to 10.

15. A DNA-encoded compound library, wherein the DNA-encoded compound library consists of the DNA-encoded compound of claim 1.

16. The DNA-encoded compound library of claim 15, wherein a total of at least 10.sup.2 DNA-encoded compounds are contained in the DNA-encoded compound library.

17. A starting fragment compound for synthesizing a DNA-encoded compound library, wherein the starting fragment compound is represented by formula (III): ##STR00028## wherein X is an atomic or molecular scaffold; Z.sub.1 is a first oligonucleotide with its 3′ terminus attached to L.sub.1, and Z.sub.2 is a second oligonucleotide with its 5′ terminus attached to L.sub.2; or Z.sub.1 is a first oligonucleotide with its 5′ terminus attached to L.sub.1, and Z.sub.2 is a second oligonucleotide with its 3′ terminus attached to L.sub.2; L.sub.1 is a first linker comprising a first functional group capable of forming a covalent bond with the 3′ terminus or 5′ terminus of Z.sub.1; L.sub.2 is a second linker comprising a first functional group capable of forming a covalent bond with the 5′ terminus or 3′ terminus of Z.sub.2; L is a linker moiety comprising at least one group operable for covalent cross-linking; and R is a reactive group linked to a functional moiety.

18. The starting fragment compound of claim 17, wherein X is a carbon atom, a nitrogen atom, a cyclic scaffold or a non-cyclic scaffold; Z.sub.1 and Z.sub.2 are at least partially complementary to each other to form a double-stranded structure; and Z.sub.1 and Z.sub.2 each independently has a length of 5-15 bases; L.sub.1 and L.sub.2 are independently an alkylene chain or poly (ethylene glycol) chain containing two functional groups, wherein the two functional groups are each independently selected from the group consisting of a phosphate group, an amino group, a hydroxyl group, and a carboxyl group; the at least one group contained in L is a photosensitive group, an electrosensitive group, or other groups capable of forming covalent cross-linking with a protein; and R is a phosphate group, an amino group, a hydroxyl group, a carboxyl group, or an aldehyde group.

19. The starting fragment compound of claim 18, wherein X is ##STR00029## wherein q is an integer selected from 1 to 10; L.sub.1 and L.sub.2 are independently ##STR00030##  wherein n is an integer selected from 1 to 10; and L has a structure of -S.sub.1-S.sub.2-S.sub.3-, wherein S.sub.1 and S.sub.3 are independently ##STR00031##  or a combination thereof, or absent; wherein m is an integer selected from 1 to 10; and S.sub.2 is a linker containing the at least one group operable for covalent cross-linking.

20. The starting fragment compound of claim 19, wherein the starting fragment compound is selected from the group consisting of: ##STR00032## wherein Y is ##STR00033##  R.sub.1 is a carbon atom or a nitrogen atom; and R.sub.2 is hydrogen, an alkyl with or without a heteroatom, or an aryl with or without a heteroatom.

21. A screening method for the DNA-encoded compound library of claim 15, comprising: (S1) incubating the DNA-encoded compound library with a protein target, followed by covalent cross-linking to obtain a covalently cross-linked protein-DNA-encoded compound complex; (S2) separating the covalently cross-linked protein-DNA-encoded compound complex from members in the DNA-encoded compound library that do not cross-link with the protein target; and (S3) subjecting the covalently cross-linked protein-DNA-encoded compound complex to polymerase chain reaction (PCR) amplification and sequencing to read DNA sequence information and acquire compound structure information.

22. The screening method of claim 21, wherein in step (S1), the covalent cross-linking is performed by irradiation, heating, electricity, or direct incubation.

23. The screening method of claim 21, wherein in step (S2), the separating is performed through steps of: perform protein immobilization; and eluting the members in the DNA-encoded compound library that do not cross-link with the protein target with an eluent.

24. The screening method of claim 23, wherein the protein immobilization is performed by using magnetic beads.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0058] FIG. 1 schematically depicts a conventional DNA-encoded compound library screening process;

[0059] FIG. 2 schematically shows chemical structures of four DNA-encoded compounds obtained by Example 1;

[0060] FIG. 3 schematically show chemical structures of four compounds in Example 1, whose Kd values with a CAIX target increase in sequence, that is, affinities of the four compounds decrease in sequence; where R1 is a fluorescent group for Kd value determination;

[0061] FIG. 4 schematically depicts a DNA-encoded compound screening process;

[0062] FIG. 5 graphically shows screening recovery results of a DNA-encoded compound in Example 1;

[0063] FIG. 6 graphically shows screening signal results of a DNA-encoded compound library in Example 2;

[0064] FIG. 7 schematically shows a structure of the DNA-encoded compound library constructed in Example 3; and

[0065] FIGS. 8a-8d graphically show screening signal results of a DNA encoded compound library in Example 3.

DETAILED DESCRIPTION OF EMBODIMENTS

[0066] The technical solutions of the present disclosure will be further described below with reference to the accompanying specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present disclosure. The raw materials and equipment used herein are known products, and are purchased from commercially available products.

[0067] DNA-NH.sub.2 or

##STR00017##

in the present disclosure is a DNA structure with a-NH.sub.2 junction formed by single- or double-stranded DNA and a junction group, such as the DNA-NH.sub.2 structure of “compound 1” described in PCT application WO2005058479, and the following DNA structures:

##STR00018##

[0068] where A is adenine, T is thymine, C is cytosine, and G is guanine.

[0069] Other abbreviations used in this application are described as follows. Fmoc represents fluorenylmethoxycarbonyl. DMT-MM represents 2-chloro-4,6-dimethoxy-1,3,5-triazine. DIPEA represents N,N-diisopropylethylamine. DMA represents N,N-dimethylacetamide. HATU represents 2-(7-oxobenzotriazole)-N,N,N′,N′-tetramethyluronium hexafluorophosphate.

Embodiment 1 Synthesis and Screening of a DNA-Encoded Compound

Step (1) Synthesis of a DNA-Encoded Compound

[0070] ##STR00019## [0071] (1) DNA-NH.sub.2 was dissolved in a borate buffer solution (250 mM, pH=9.4) to form a first solution (1 mM). Compound 1 in a DMA solution (50 equiv., 200 mM), HATU in a DMA solution (50 equiv., 400 mM) and DIPEA in a DMA solution (100 equiv., 400 mM) were separately pre-chilled in a −20° C. refrigerator for 5 minutes and mixed to form a mixture solution, and then the mixture solution was stored in a 4° C. refrigerator for 5 min and added to the first solution to form a first reaction mixture, followed by shaking, mixing and standing for reaction at room temperature for 12 h.

[0072] After the reaction was completed, the first reaction mixture was subjected to ethanol precipitation. Specifically, 5M NaCl solution was added to the first reaction mixture, then anhydrous ethanol was added, where the 5M NaCl solution was 10% of a total volume of the first reaction mixture, and the anhydrous ethanol was 3 times the total volume of the first reaction mixture. After uniform mixing by shaking, the first reaction mixture was placed on dry ice and frozen for 2 h, followed by centrifugation at 12,000 rpm for 0.5 h. After that, the supernatant was poured off and the remaining precipitate was dissolved in deionized water to obtain the crude product of compound 2, which was directly used in the subsequent reaction without purification. [0073] (2) The crude product of compound 2 was dissolved in pure water to form a second solution (1 mM). Piperidine was added into the second solution to form a second reaction mixture followed by vortex oscillation and standing for reaction at room temperature for 1-3 h, where the piperidine was 10% of the total volume of the second solution.

[0074] After the reaction was completed, the second reaction mixture was subjected to ethanol precipitation. Specifically, 5M NaCl solution was added to the second reaction mixture, then anhydrous ethanol was added, where the 5M NaCl solution was 10% of a total volume of the second reaction mixture, and the anhydrous ethanol was 3 times the total volume of the second reaction mixture. After uniform mixing by shaking, the second reaction mixture was placed on dry ice and frozen for 2 h, followed by centrifugation at 12,000 rpm for 0.5 h. After that, the supernatant was poured off and the remaining precipitate was dissolved in deionized water to obtain the crude product of compound 3, which was directly used in the subsequent reaction without purification. [0075] (3) The crude product of compound 3 was dissolved in a borate buffer solution (250 mM, pH =9.4) to form a third solution (1 mM), and then sequentially added with a carboxylic acid compound 4 in a DMA solution (100 equiv., 100 mM) and a DMT-MM in deionized water (100 equiv., 100 mM) to form a third reaction mixture. The third reaction mixture was uniformed mixed by vortex oscillation and then standing for reaction at room temperature for 12-16 h.

[0076] After the reaction was completed, the third reaction mixture was subjected to ethanol precipitation. Specifically, 5M NaCl solution was added to the third reaction mixture, then anhydrous ethanol was added, where the 5M NaCl solution was 10% of a total volume of the third reaction mixture, and the anhydrous ethanol was 3 times the total volume of the third reaction mixture. After uniform mixing by shaking, the third reaction mixture was placed on dry ice and frozen for 2 h, followed by centrifugation at 12,000 rpm for 0.5 h. After that, the supernatant was poured off and the remaining precipitate was dissolved in deionized water to obtain the crude product of compound 5, which was purified by preparative chromatography to obtain a purified compound 5. [0077] (4) 100 nmol of the purified compound 5 was dissolved in pure water to obtain a fourth solution (1 mM, 100 μL). Primer 1 in water (166.6 nmol, 1.67 equiv., 2 mM, 83.3 μL), 10 of ligation buffer (66.6 μL), T4 DNA ligase (9.6 μL, 13.97 μg/μL) and deionized water (407 μL) were added into the fourth solution to obtain a fourth reaction mixture followed by vortex oscillation and standing for reaction at 20° C. for 16 h.

[0078] After the reaction was completed, the fourth reaction mixture was subjected to ethanol precipitation. Specifically, 5M NaCl solution was added to the fourth reaction mixture, then anhydrous ethanol was added, where the 5M NaCl solution was 10% of a total volume of the fourth reaction mixture, and the anhydrous ethanol was 3 times the total volume of the fourth reaction mixture. After uniform mixing by shaking, the fourth reaction mixture was placed on dry ice and frozen for 2 h, followed by centrifugation at 12,000 rpm for 0.5 h. After that, the supernatant was poured off and the remaining precipitate was dissolved in deionized water to obtain the crude product.

[0079] The crude product was dissolved in pure water to form a fifth solution (1 mM, 100 μL). The fifth solution was added with ligated DNA fragment 1-DNA fragment 2-DNA fragment 3-primer 2-library ID in water (100 nmol, 1 equiv., 2 mM, 50 μL), 10 of ligation buffer (80 μL), T4 DNA ligase (4.31 μL, 13.97 μg/μL) and deionized water (165 μL) to form a fifth reaction mixture followed by vortex oscillation and standing for reaction at 20° C. for 16 h.

[0080] After the reaction was completed, the fifth reaction mixture was subjected to ethanol precipitation. Specifically, 5M NaCl solution was added to the fifth reaction mixture, then anhydrous ethanol was added, where the 5M NaCl solution was 10% of a total volume of the fifth reaction mixture, and the anhydrous ethanol was 3 times the total volume of the fifth reaction mixture. After uniform mixing by shaking, the fifth reaction mixture was placed on dry ice and frozen for 2 h, followed by centrifugation at 12,000 rpm for 0.5 h. After that, the supernatant was poured off and the remaining precipitate was dissolved in deionized water to obtain the compound 6.

[0081] Four compounds 6 (i.e., compound 6-1, compound 6-2, compound 6-3 and compound 6-4) were synthesized according to the above synthesis method, and their specific structures were shown in FIG. 2.

Step (2) Screening of a DNA-encoded compound

[0082] A DNA-encoded compound (0.2 nM) and 100 pmol of a target protein were added into a screening buffer (the screening buffer consisted of 12.5 mM Tris, 150 mM NaCl, 0.3 mg/mL ssDNA, and 0.05% Tween20, and had a pH of 7.5) having a total volume of 100 μL in a 1.5 mL centrifuge tube. In the meanwhile, a parallel blank control group (no target protein was added) was set up, and each of the target protein group and the blank control group was made for 2 groups. The centrifuge tubes containing reaction mixture were place on a rotary mixer for incubation at 20 rpm and 25° C. for 60 min. Two groups of incubated samples (one for the target protein group and the other for the blank control group) were placed on ice and exposed to 365 nm UV light for the photocrosslinking reaction for 10 min, while the other two groups of samples (one for the target protein group and the other for the blank control group) were placed on ice for 10 min without exposure to the 365 nm UV light.

[0083] Then 25 μL of Ni-charged magnetic beads, which were equilibrated with 250 μL of a screening buffer three times, were put into the UV-lighted or non-lighted samples. Then the centrifuge tubes were placed on the rotary mixer for incubation at 20 rpm and 25° C. for 30 min. By the affinity of the target protein tag with the specific magnetic beads, the target protein and the target protein-bound DNA-encoded complex were separated from the solution with a magnetic holder, and the supernatant was collected.

[0084] The separated magnetic beads were re-suspended with 500 μL of the screening buffer and washed on the rotary mixer at 20 rpm and 25° C. for lmin to wash off the non-specifically bound DNA-encoded compound, and the magnetic holder was used to separate the magnetic beads from the supernatant. These processes were repeated 5 times.

[0085] The washed magnetic beads were re-suspended with 100 μL of an elution buffer (the elution buffer consisted of 12.5 mM Tris and 150 mM NaCl and had a pH of 7.5), and eluted in a metal bath at 95° C. for 10 min. The magnetic beads were separated from the eluted DNA-encoded compound with the magnetic stand. Then, the samples on the magnetic beads were the DNA-encoded compound samples obtained by photocrosslinking, and the elution supernatant was the DNA-encoded compound sample obtained by affinity interaction.

[0086] The elution supernatant and magnetic beads were each diluted 20-fold with a dilution buffer (the dilution buffer consisted of 10 mM Tris and 0.05% Tween20, and had a pH of 8.0), and the DNA-encoded compound was also diluted 100-fold. A sample, a primer, deionized water and a qPCR mix (ABI, A25778) were configured into a 20 μL reaction solution for qPCR testing. The qPCR tests were performed under the following conditions: the pre-denaturation was performed at 95° C. for 10 min; 35 cycles were performed (each cycle included denaturation at 95° C. for 10 s, annealing at 55° C. for 10 s and extension at 72° C. for 10 s); and the signal collection was set at the extension step. After the qPCR tests were completed, the molecular copy number of samples was calculated according to Avogadro's constant and the molecular copy number differences between the samples were analyzed.

[0087] The screening results were shown in FIG. 5. The test results showed that under the screening conditions (non-light conditions) for the conventional DNA-encoded compound library, the DNA recovery ratio was 0.51-1.67 and there was no significant differentiation in the screening signal. By using the DNA-encoded compound library and screening method of the present disclosure, the DNA recovery ratio was 2.27-151.92, the differentiation in the screening signal was significantly increased, and the recovery ratio was in a positive correlation to the compound activity (as shown in FIG. 3).

Embodiment 2 Synthesis of a DNA-Encoded Compound and Screening for a Phosphatase Target

[0088] By using the starting DNA material (compound 3) prepared in Example 1, a DNA-encoded compound library containing 962 compounds was constructed according to the method described in the international patent application WO2005058479, represented by:

##STR00020##

[0089] The screening was performed on the DNA-encoded compound library through the following steps. [0090] (1) The DNA-encoded compound library (5.65 nM) and 250 pmol of a target protein were added into a screening buffer (the screening buffer consisted of 50 mM HEPES, 150 mM NaCl, 0.01% Tween-20, 0.3 mg/mL ssDNA, 10 mM imidazole, and had a pH of 7.4) having a total volume of 100 μL in a 1.5 mL centrifuge tube. In the meanwhile, a parallel blank control group (no target protein was added) was set up, and each of the target protein group and the blank control group was made for 2 groups. The centrifuge tubes containing reaction mixture were placed on a rotary mixer for incubation at 20 rpm and 25° C. for 60 min. Two groups of incubated samples (one for the target protein group and the other one for the blank control group) were placed on ice and exposed to 365 nm UV light for the photocrosslinking reaction for 10 min, while the other two groups of samples (one for the target protein group and the other one for the blank control group) were placed on ice for 10 min without exposure to the 365 nm UV light. [0091] (2) Then 20 μL of Ni-charged magnetic beads, which were equilibrated with 200 μL of a screening buffer three times, were put into the UV-lighted or non-lighted samples. Then the centrifuge tubes were placed on the rotary mixer for incubation at 20 rpm and 25° C. for 30 min. By the affinity of the target protein tag with the specific magnetic beads, the target protein and the target protein-bound DNA-encoded complex were separated from the solution with a magnetic holder, and the supernatant was collected. [0092] (3) The separated magnetic beads were re-suspended with 500 μL of the screening buffer and washed on the rotary mixer at 20 rpm and 25° C. for 1 min to wash off the non-specifically bound DNA-encoded compound, and the magnetic holder was used to separate the magnetic beads from the supernatant. These processes were repeated 5 times. [0093] (4) The washed magnetic beads were re-suspended with 55 μL of an elution buffer (the elution buffer consisted of 50 mM HEPES, 300 mM NaCl and had a pH of 7.4), and eluted in a metal bath at 95° C. for 10 min. The magnetic beads were separated from the eluted DNA-encoded compound with the magnetic stand. Then, the samples on the magnetic beads were the DNA-encoded compound samples obtained by photocrosslinking, and the elution supernatant was the DNA-encoded compound sample obtained by affinity interaction. [0094] (5) The elution supernatant and magnetic beads were each diluted 20-fold with a dilution buffer (the dilution buffer consisted of 10 mM Tris and 0.05% Tween20, and had a pH of 8.0), and the DNA-encoded compound was also diluted 100-fold. A sample, a primer, deionized water and a qPCR mix (ABI, A25778) were configured into a 20 μL reaction solution for qPCR testing. The qPCR tests were performed under the following conditions: the pre-denaturation was performed at 95° C. for 10 min; 35 cycles were performed (each cycle included denaturation at 95° C. for 10 s, annealing at 55° C. for 10 s and extension at 72° C. for 10 s); and the signal collection was set at the extension step. After the qPCR tests were completed, the molecular copy number of samples was calculated according to Avogadro's constant.

[0095] The screening results were shown in FIG. 6, where the S3/S4 Elution on the vertical coordinate represented the screening signal result of the conventional DNA-encoded compound library, and the vertical coordinate value represented the DNA recovery ratio, which showed that the DNA recovery ratios for all compounds were 0-2, with no significant signal differentiation. The S1/S3 Beads on the horizontal coordinate showed the screening results of the DNA-encoded compound library provided in this application, and the vertical coordinate value represented the DNA recovery ratios, which indicated that the DNA recovery ratios of the compounds were 0-60, with significant signal differentiation.

Embodiment 3 Synthesis of a DNA-Encoded Compound and Screening for a Kinase Target

[0096] A DNA-encoded compound library containing 7417 compounds as shown in FIG. 7 was constructed with reference to the method for constructing a DNA-encoded compound library described in Example 1 of the present disclosure and the international patent application WO2005058479, where L was a linker chain different in lengths.

[0097] The kinase target PAK4 was screened using the DNA-encoded compound library provided in this embodiment with reference to the screening method described in Example 2 of this application. The screening groupings were shown in Table 1, where “+” indicated that the condition was present, and “−” indicated that the condition was absent.

TABLE-US-00001 TABLE 1 Screening groupings PAK4 UV Competitive Control protein Irradiation molecule protein Target-free screening − + − − control group PAK4-irradiation group + + − − PAK4 irradiation-free + − − − group PAK4 irradiation group + + + − in the presence of a competitive molecule-added Irradiation group in the − + − + presence of another target

[0098] The results of the screening signals were shown in FIGS. 8a-8d, where the horizontal and vertical coordinates showed the enrichment of the screening signals (molecular enrichment) for the different screening groups, and the values indicated the degree of enrichment. FIGS. 8a-8d showed that the compound library and the screening method of the present disclosure reached a good signal enrichment. In addition, FIG. 8a showed that the molecules were not enriched on materials other than proteins (e.g., magnetic beads). FIG. 8b showed that the enrichment of the molecules under light covalent cross-linking was much higher than the enrichment of the molecules under non-light conditions. FIG. 8c showed that the signal intensity of the molecules was reduced by competitive molecules, indicating that they acted on the correct target pocket. FIG. 8d showed that the molecules did not suffer from severe non-specific binding.

[0099] The experimental results show that by using the DNA encoded compounds/library and screening method of the present disclosure, the binding between the compound and the target can be enhanced, the discrimination of compounds, particularly low affinity compounds, on the screening signal can be improved, producing positive results.

[0100] In summary, the present disclosure provides a method for the synthesis of DNA-encoded compounds and compound libraries, and a screening method thereof. In the present disclosure, the DNA-encoded compound library is incubated with a target site followed by covalent cross-linking to enhance the binding of the compound to the target site. Compared with the conventional affinity screening of DNA-encoded compound libraries, it can improve the discrimination of compounds, particularly low affinity compounds, on the screening signal.