WO2019149198A1 - Dna-encoded compound library and compound screening method - Google Patents

Abstract

Provided are a DNA-encoded compound, a compound library, and a compound screening method. Screening by using the provided DNA-encoded compound library eliminates the need to purify or immobilize a target protein, therefore being suitable for screening complex systems such as non-bound proteins, cell transmembrane proteins, and cell lysates. In addition, after the provided DNA-encoded compound is covalently cross-linked with a protein, the protein and a DNA tag portion are completely covalently linked, being able to withstand various separation conditions, such as electrophoretic separation, strong elution conditions, and so on, and the protein and the DNA tag portion not separating due to harsh separation conditions.

Description

DNA coding compound library and compound screening method Technical field

The invention relates to a DNA coding compound library and a compound screening method.

Background technique

In drug discovery, especially in the development of new drugs, high-throughput screening for biological targets is one of the main means of obtaining lead compounds quickly. However, traditional high-throughput screening based on single molecules requires long time, huge equipment investment, limited number of library compounds (millions), and the completion of compound libraries requires decades of accumulation, limiting the discovery efficiency of lead compounds. possibility. In recent years, the DNA coding compound library synthesis technology, combined with combinatorial chemistry and molecular biology techniques, adds a DNA tag to each compound at the molecular level, and can synthesize up to a billion-level compound library in a very short time. Moreover, the compounds can be identified by gene sequencing, which greatly increases the size and synthesis efficiency of the compound library, and becomes the trend of the next-generation compound library screening technology, and has begun to be widely used in the foreign pharmaceutical industry, resulting in many positive effects ( Accounts of Chemical Research, 2014, 47, 1247-1255).

The traditional DNA coding compound library synthesis and coding technology can be well applied to screening, but it also has some application limitations: First, when using the traditional DNA coding compound library for screening, the target protein (protein target) is required. Point) is fixed, however, the spatial structure of some target proteins changes after the immobilization operation, resulting in the selected compounds not binding to the unfixed target protein, thereby limiting the application range of the DNA coding compound library; Second, the traditional DNA coding compound library can only be used to screen for higher purity target proteins. However, due to factors such as technology and protein properties, certain proteins cannot be purified to a purity that can be screened, thereby limiting the range of applications of DNA-encoding compound libraries.

In order to solve the above technical problem, CN107130299A discloses a library of DNA-encoding compounds which can be covalently cross-linked, which introduces a short-stranded DNA having 8 to 12 bases based on a library of conventional single-stranded DNA-encoding compounds, wherein One end of the short-stranded DNA has a photocrosslinking group. The DNA-encoding compound library binds to the target protein through a photo-crosslinking group, and the DNA-encoding compound is covalently bound to the target protein, so that the target protein can be purified and immobilized without expanding, and the application range of the DNA-encoding compound library is expanded. CN107130299A also discloses a screening method based on the above library of DNA encoding compounds, wherein after covalent crosslinking, DNA exonuclease is added to degrade DNA.

However, due to the different nature of different protein targets, when exonuclease is added, exonuclease may degrade the DNA tag of the DNA-encoded compound after covalent cross-linking, thereby limiting its application range. However, if the above-mentioned covalently cross-linked DNA-encoding compound is directly subjected to strong separation or strong elution conditions such as electrophoresis, it may be insufficient because hydrogen bonding between a protein target portion and a DNA tag portion is only a small number of bases. Tolerance results in the separation of the protein target from the DNA tag portion, rendering the DNA tag ineffective.

Therefore, there is a need for a more stable library of covalently cross-linked DNA-encoding compounds that allows target proteins to bind to DNA-encoding compounds more stably, without separation under strong separation and strong elution conditions.

Summary of the invention

In order to solve the above problems and further expand the application range of the DNA coding compound library, the present invention provides a novel DNA coding compound library and a compound screening method.

The present invention first provides a DNA encoding compound having the formula of Formula I:

Wherein X is an atom or a molecular skeleton;

A ₁ is a portion comprising a linking strand and an oligonucleotide;

A ₂ is a portion comprising a linking strand and an oligonucleotide;

M1 is a functional part comprising one or more structural units;

M2 is a moiety comprising one or more operable covalent crosslinks.

Further, X is a molecular skeleton of a carbon atom or a poly atom.

Further, the polyatomic skeleton is a cyclic or acyclic skeleton structure.

其中X ₁、X ₂、X ₃、X ₄分别独立的选自碳、氧、氮、硫。 Further, the acyclic skeleton structure is

Wherein X ₁ , X ₂ , X ₃ and X ₄ are each independently selected from the group consisting of carbon, oxygen, nitrogen and sulfur.

Further, the formula I formula has the structure shown in formula II:

Wherein Z ₁ is an oligonucleotide linked to L ₁ at its 3' end, Z ₂ is an oligonucleotide linked to L ₂ at its 5' end or Z ₁ is linked to L ₁ at its 5' end. Oligonucleotide, Z ₂ is an oligonucleotide linked to L ₂ at its 3'end;

L ₁ is a linking chain comprising a functional group capable of forming a bond with the 3' end or the 5' end of Z ₁ ;

L ₂ is a linking chain comprising a functional group capable of forming a bond with the 5' end or the 3' end of Z ₂ ;

Y ₁ is a functional part;

Y ₂ is a moiety operable to carry out covalent crosslinking;

S ₁ and S ₂ are each independently a linking chain.

Further, Z ₁ and Z _{2 are} complementary to form a double strand.

Further, Z ₁ and Z ₂ are the same length or different.

Further, Z ₁ and Z _{2 each have} a length of 10 or more bases and have 10 or more base pair complementary regions.

Further, both Z ₁ and Z ₂ comprise a PCR primer sequence.

Further, L ₁ and L ₂ are each independently selected from a bifunctional alkylene chain or a difunctional oligodiol chain.

Further, the functional group in L ₁ and L ₂ is selected from a phosphate group, an amino group, a hydroxyl group, and a carboxyl group.

其中，n为1～10的整数。 Further, L ₁ and L ₂ are each independently selected from the group consisting of

Wherein n is an integer of 1 to 10.

Further, S ₁ and S ₂ are each independently selected from a bifunctional alkylene chain or a difunctional oligodiol chain.

Further, the functional group in S ₁ and S ₂ is selected from a carboxyl group, an amino group or an aldehyde group.

其中，m为1～10的整数。 Further, S ₁ , S _{2 are} selected from

Wherein m is an integer of 1 to 10.

Further, Y ₂ contains a photosensitive group, an electrosensitive group or a group which can be covalently crosslinked with a protein.

Further, Y ₂ includes an azide group, a diaziridine group, a sulfonyl fluoride group, a diazo group, a cinnamoyl group, an acrylate group.

Further, Y _{2 is} selected from

The present invention also provides a library of DNA encoding compounds which are composed of the aforementioned DNA encoding compounds.

Further, the DNA encoding a compound library comprising at least ¹⁰⁶ different DNA coding compound.

Further, the DNA encoding a compound library comprising at least ¹⁰⁸ different DNA coding compound.

Further, the library of DNA encoding compounds comprises at least 10 ¹⁰ different DNA encoding compounds.

The invention also provides a screening method for the aforementioned DNA encoding compound library, which comprises the following steps:

a, incubating the compound library with the protein target, and then operating for covalent cross-linking;

b. separating the covalently crosslinked complex of the protein-DNA encoding compound from the uncrosslinked DNA encoding compound;

c. The recovered protein-DNA coding compound is covalently cross-linked to carry out PCR amplification and DNA sequencing, and the DNA sequence information is read to obtain compound structure information.

Further, the covalent crosslinking in the step a is carried out by light, electricity or direct incubation.

Further, in the step b, separation is carried out by electrophoresis.

Further, the step b is carried out by SDS-PAGE.

Further, the separation method in the step b is: immobilizing the protein, and washing off the unfixed substance with an eluent.

Further, the immobilization of the protein is to immobilize the protein using magnetic beads.

The invention also provides a method for screening a cell lysate by the aforementioned DNA encoding compound library, which comprises the following steps:

i. physically break the cells;

Ii, performing centrifugation to separate soluble substances;

Iii. Screening the isolated soluble material using the above library of DNA encoding compounds.

The present invention also provides a method for screening a transmembrane protein by the aforementioned library of DNA encoding compounds, comprising the steps of:

1) physically break the cells;

2), performing the first round of centrifugation, separating soluble substances and cell membranes, and precipitating to remove insoluble substances;

3), performing a second round of centrifugation, and separating and separating the cell membrane;

4) Screening the isolated transmembrane proteins using the above library of DNA encoding compounds.

Screening using the DNA encoding compound library of the present invention does not require purification or immobilization of the target protein, and thus can be applied to screening of complex systems such as non-binding proteins, cell transmembrane proteins, and cell lysates.

After the DNA encoding compound of the present invention is covalently cross-linked with the protein, the protein and the DNA tag portion are all covalently linked, and can withstand various separation conditions, such as electrophoretic separation, strong elution conditions, etc., and the protein and DNA tag portions are not Separation occurs due to harsh separation conditions.

It is apparent that various other modifications, substitutions and changes can be made in the form of the above-described embodiments of the present invention.

The above content of the present invention will be further described in detail below by way of specific embodiments in the form of embodiments. However, the scope of the above-mentioned subject matter of the present invention should not be construed as being limited to the following examples. Any technique implemented based on the above description of the present invention is within the scope of the present invention.

DRAWINGS

Figure 1 shows the results of electrophoretic separation of compounds 4, 5 and 6 after covalent crosslinking with target proteins.

Figure 2 shows the results of electrophoretic separation of compounds 6, 7 and 8 after covalent crosslinking with target proteins.

Detailed ways

Abbreviations: Fmoc for fluorenylmethoxycarbonyl; DMF for N,N-dimethylformamide; DMSO for dimethyl sulfoxide; DMT-MM for 2-chloro-4,6-dimethoxy-1,3, 5-triazine; TEAA means triethylamine acetate, DIEA means N,N-diisopropylethylamine; Boc means t-butoxycarbonyl; DMA means N,N-dimethylacetamide; HATU means 2-(7 - benzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate.

The sequence of the DNA below:

简写为

Abbreviated as

The sequence of the DNA below:

简写为

Abbreviated as

Wherein A is adenine, T is thymine, C is cytosine, and G is guanine;

Post-treatment of chemical reaction or DNase ligation reaction on DNA: 10% reaction solution volume of 5M NaCl aqueous solution and 2-3 volumes of ethanol are added to the reaction solution of DNA reaction or DNase ligation reaction on DNA. . After vortexing, the solution was frozen in a refrigerator at -20 ° C for 1 hour. After the frozen solution was precipitated by high speed centrifugation, the supernatant was removed to obtain a DNA sample.

Example 1. Synthesis of starting DNA material of DNA encoding compound library

Synthesis method one:

Step 1. Synthesis of Compound 1

Compound HUB1 (82 nmol) was dissolved in sodium borate buffer (82 μL, pH=9.4, concentration 250 mM/L) to prepare a starting solution at a concentration of 1 mM/L; then a 4-azido-benzoic acid DMA solution ( 8.2 μmol, concentration 200 mM / L), HATU DMA solution (8.2 μmol, concentration 400 mM / L) and DIPEA DMA solution (8.2 μmol, concentration 400 mM / L) were placed in a -20 ° C refrigerator and cooled, then mixed The mixture was vortexed and thoroughly mixed, and then stored in a refrigerator at 4 ° C for 5 minutes. The above mixture was added to the starting solution of the compound HUB1, thoroughly mixed by vortexing, and reacted at room temperature for 12 hours. 1 μL of the reaction solution was diluted with 100 μL of dd-H ₂ O, and the reaction was monitored by LCMS. After completion of the reaction, work-up was carried out in the same manner as described above to give Compound 1 (77 nm).

Step 2. Synthesis of Compound 4

Compound 1 (77 nmol) was dissolved in a sodium borate buffer (308 μL, pH=9.4, concentration 250 mM/L) to prepare a solution having a concentration of 0.25 mM/L, and the mixture was thoroughly vortexed and then reacted at 90 ° C. hour. 1 μL of the reaction solution was diluted with 100 μL of dd-H ₂ O, and the reaction was monitored by LCMS. After completion of the reaction, work-up was carried out in the same manner as described above to give Compound 4 (73 nm).

Synthesis method two:

Step 1, synthesis of compound 3

Compound HUB (10 μmol) was dissolved in sodium borate buffer (10 ml, pH=9.4, concentration 250 mM/L) to prepare a starting solution at a concentration of 1 mM/L; then a DMA solution of Compound 2 (AOP-Fmoc) was 1000 μmol, concentration 200 mM / L), HATU DMA solution (1000 μmol, concentration 400 mM / L) and DIPEA DMA solution (1000 μmol, concentration 400 mM / L) were placed in a -20 ° C refrigerator and cooled, mixed, the mixture vortex Shake well and mix well, then store in a refrigerator at 4 ° C for 5 minutes. The above mixture was added to the starting solution of the compound HUB, vortexed and thoroughly mixed, and reacted at room temperature for 12 hours. 1 μL of the reaction solution was diluted with 100 μL of dd-H ₂ O, and the reaction was monitored by LCMS. After the reaction was completed, 2 mL of an aqueous solution of sodium chloride (5 M/L) and 60 mL of ethanol were added thereto for 5 minutes, and the mixture was cooled in a refrigerator at -20 ° C for 2-3 hours, and then centrifuged for 40 to 60 minutes. The precipitate was dissolved by adding 10 mL of dd-H ₂ O, and after adding 1 ml of piperidine, it was reacted at room temperature for 1-3 hours, and the reaction was monitored by LCMS. After the reaction was completed, 1 mL of an aqueous solution of sodium chloride (5 M/L) and 60 mL of ethanol were added and shaken for 5 minutes, and then cooled in a refrigerator at -20 ° C for 2-3 hours, and then centrifuged for 40-60 minutes, and the supernatant was separated to obtain a white precipitate. That is, Compound 3 (9 μmol, purity >95%).

Step 2. Synthesis of Compound 4

Compound 3 (1 μmol) was dissolved in sodium borate buffer (1 mL, pH=9.4, concentration 250 mM/L) to prepare a starting solution at a concentration of 1 mM/L; then a solution of p-azidobenzoic acid in DMA (2 μmol, The concentration of 20 mM / L), HATU DMA solution (2 μmol, concentration 20 mM / L) and DIPEA DMA solution (2 μmol, concentration 20 mM / L) were placed in a -20 ° C refrigerator and cooled, mixed, the mixture vortexed Mix well and store in a refrigerator at 4 ° C for 5 minutes. The above mixture was added to the starting solution of Compound 3, thoroughly mixed by vortexing, and reacted at room temperature for 2-4 hours. 1 μL of the reaction solution was diluted with 100 μL of dd-H ₂ O, and the reaction was monitored by LCMS. After the reaction was completed, 0.2 mL of an aqueous solution of sodium chloride (5 M/L) and 10 mL of ethanol were added thereto for 5 minutes, and the mixture was cooled in a refrigerator at -20 ° C for 2-3 hours, and then centrifuged for 40 to 60 minutes to obtain a precipitate. The precipitate was dissolved by adding 1 mL of dd-H ₂ O, and a compound 4 (300 nmol, purity >95%) was obtained by HPLC.

Example 2, DNA coding compound library and screening

Using the starting DNA material prepared in Example 1, a library of DNA encoding compounds of the following structure was constructed:

The above DNA coding compound library is screened by the following steps:

1) incubating the above library of DNA encoding compounds with the target protein ROCK2 in a 100 μL buffer system for 60 minutes, and then reacting at 365 nm for 60 seconds;

2) The reaction solution is heated to 90 ° C to denature the protein, and separated by preparative SDS-PAGE (12-15%), and the corresponding target protein-DNA coding compound covalently crosslinked complex is recovered and extracted by gelatinization. Strip

3) The recovered sample is subjected to PCR amplification and DNA sequencing, and the DNA sequence corresponding to the enriched small molecule compound is read, and then the structural information of the compound is obtained.

Example 3, DNA coding compound synthesis

After screening the structure of the compound by the screening of the DNA-encoding compound library of Example 2, the DNA-free compound was re-synthesized, and its inhibitory activity against ROCK2 was ₅₀ nM. Further, the covalent cross-linking was further verified by synthesizing a DNA-encoding compound as a tool compound.

Step 1: Synthesis of Compound 5

Compound 3 (5 μmol) was dissolved in sodium borate buffer (5 mL, pH=9.4, concentration 250 mM/L) to prepare a starting solution at a concentration of 1 mM/L; then a DMA solution of SM01 (10 μmol, concentration 20 mM/L) ), HATU DMA solution (10 μmol, concentration 20 mM / L) and DIPEA DMA solution (10 μmol, concentration 20 mM / L) were placed in a -20 ° C refrigerator and cooled, mixed, and the mixture was vortexed and thoroughly mixed. It was then stored in a refrigerator at 4 ° C for 5 minutes. The above mixture was added to the starting solution of Compound 3, thoroughly mixed by vortexing, and reacted at room temperature for 2-4 hours. 1 μL of the reaction solution was diluted with 100 uL of dd-H ₂ O, and the reaction was monitored by LCMS. After the reaction was completed, 1 mL of an aqueous solution of sodium chloride (5 M/L) and 50 mL of ethanol were added thereto for 5 minutes, and the mixture was cooled in a refrigerator at -20 ° C for 2-3 hours, and then centrifuged for 40 to 60 minutes to obtain a precipitate. The precipitate was dissolved by adding 5 mL of dd-H ₂ O, and a compound 5 (2 μmol, purity >98%) was obtained by HPLC.

Step 2: Synthesis of Compound 6

Compound 5 (1 μmol) was dissolved in sodium borate buffer (1 mL, pH=9.4, concentration 250 mM/L) to prepare a starting solution at a concentration of 1 mM/L; then a solution of p-azidobenzoic acid in DMA (100 μmol, The concentration of 200 mM / L), HATU DMA solution (100 μmol, concentration 400 mM / L) and DIPEA DMA solution (100 μmol, concentration 400 mM / L) were placed in a -20 ° C refrigerator and cooled, mixed, the mixture vortexed Mix well and store in a refrigerator at 4 ° C for 5 minutes. The above mixture was added to the starting solution of the compound 5, thoroughly mixed by vortexing, and then reacted at room temperature for 2-4 hours. 1 μL of the reaction solution was diluted with 100 μL of dd-H ₂ O, and the reaction was monitored by LCMS. After the reaction was completed, 0.2 mL of an aqueous solution of sodium chloride (5 M/L) and 10 mL of ethanol were added thereto for 5 minutes, and the mixture was cooled in a refrigerator at -20 ° C for 2-3 hours, and then centrifuged for 40 to 60 minutes to obtain a precipitate. The precipitate was Compound 6 (270 nmol, purity >95%).

Step 3: Synthesis of Compound 7

Compound 5 (1 μmol) was dissolved in sodium borate buffer (1 mL, pH=9.4, concentration 250 mM/L) to prepare a starting solution at a concentration of 1 mM/L; then 3-methyl-3H-biguanidin was added. DMA solution of -3-propionic acid (100 μmol, concentration 200 mM / L), HATU DMA solution (100 μmol, concentration 400 mM / L) and DIPEA DMA solution (100 μmol, concentration 400 mM / L) were placed in a -20 ° C refrigerator After cooling, the mixture was vortexed and thoroughly mixed, and then stored in a refrigerator at 4 ° C for 5 minutes. The above mixture was added to the starting solution of the compound 5, thoroughly mixed by vortexing, and then reacted at room temperature for 2-4 hours. 1 μL of the reaction solution was diluted with 100 μL of dd-H ₂ O, and the reaction was monitored by LCMS. After the reaction was completed, 0.2 mL of an aqueous solution of sodium chloride (5 M/L) and 10 mL of ethanol were added thereto for 5 minutes, and the mixture was cooled in a refrigerator at -20 ° C for 2-3 hours, and then centrifuged for 40 to 60 minutes to obtain a precipitate. The precipitate was Compound 7 (310 nmol, purity > 96%).

Step 4: Synthesis of Compound 8

Compound 5 (1 μmol) was dissolved in sodium borate buffer (1 ml, pH=9.4, concentration 250 mM/L) to prepare a starting solution at a concentration of 1 mM/L; then DMA of 4-(fluorosulfonyl)benzoic acid was added. Solution (100 μmol, concentration 200 mM / L), HATU DMA solution (100 μmol, concentration 400 mM / L) and DIPEA DMA solution (100 μmol, concentration 400 mM / L) were placed in a -20 ° C refrigerator and cooled, mixed, the mixture The liquid vortex was thoroughly mixed and then stored in a refrigerator at 4 ° C for 5 minutes. The above mixture was added to the starting solution of the compound 5, thoroughly mixed by vortexing, and then reacted at room temperature for 2-4 hours. 1 μL of the reaction solution was diluted with 100 μL of dd-H ₂ O, and the reaction was monitored by LCMS. After the reaction was completed, 0.2 mL of an aqueous solution of sodium chloride (5 M/L) and 10 mL of ethanol were added thereto for 5 minutes, and the mixture was cooled in a refrigerator at -20 ° C for 2-3 hours, and then centrifuged for 40 to 60 minutes to obtain a precipitate. The precipitate was Compound 8 (250 nmol, purity > 82%).

The beneficial effects of the present invention will be described below by way of test examples.

Test Example 1. Compounds 4, 5, and 6 were covalently crosslinked with a target protein.

1. Test method

(1) Compounds 4, 5 and 6 were each incubated with the target protein for 60 minutes in 25 μL of a buffer system (40 mM PBS, 50 mM NaCl, pH 7.5).

(2) The light source was directly irradiated to the reaction solution for 30 minutes under ultraviolet conditions of 365 nm.

(3) Add 9 μL of 4x loading buffer to the reaction solution, mix by pipetting, and heat in a metal bath at 100 ° C for 10 minutes to completely denature the protein.

(4) A protein-DNA-encoding compound covalently cross-linked complex and an uncrosslinked DNA-encoding compound were separated by SDS-PAGE electrophoresis (120 V, 120 min).

2, test results

The test results are shown in Figure 1.

The above test results show that the DNA encoding compound of the present invention can specifically covalently crosslink with the target protein, and the complex formed by the target protein and the DNA encoding compound does not separate under electrophoresis conditions, but the complex and the target protein alone are It can be separated under electrophoresis.

Test Example 2, Compounds 6, 7 and 8 were covalently crosslinked with a target protein

1. Test method

(1) Compound 6 was incubated with the target protein in a 25 μL buffer system (40 mM PBS, 50 mM NaCl, pH 7.5) for 60 minutes.

(2) A group of light sources were directly exposed to light at 365 nm for 1 minute and 30 minutes. The other set was placed in the dark on ice for 1 minute and 30 minutes.

(3) Add 9 μL of 4x loading buffer to the reaction solution, mix by pipetting, and heat in a metal bath at 100 ° C for 10 minutes to completely denature the protein.

(4) Compound 7 was incubated with the target protein in a 25 μL buffer system (40 mM PBS, 50 mM NaCl, pH 7.5) for 60 minutes.

(5) A group of direct light reaction solutions for 30 minutes and 120 minutes under ultraviolet conditions at 365 nm. The other set was placed in the dark on ice for 30 minutes and 120 minutes.

(6) Add 9 μL of 4x loading buffer to the reaction solution, mix by pipetting, and heat in a metal bath at 100 ° C for 10 minutes to completely denature the protein.

(7) Compound 8 was incubated with the target protein in a 25 μL buffer system (40 mM PBS, 50 mM NaCl, pH 7.5) for 60 minutes.

(8) Store at 4 ° C for 120 minutes.

(9) Add 9 μL of 4x loading buffer to the reaction solution, mix by pipetting, and heat in a metal bath at 100 ° C for 10 minutes to completely denature the protein.

(10) A protein-DNA-encoding compound covalently cross-linked complex and an uncross-linked DNA-encoding compound were separated by SDS-PAGE electrophoresis (120 V, 120 min).

2, test results

The test results are shown in Figure 2.

The above test results indicate that the DNA encoding compound of the present invention can adopt various covalently crosslinkable groups (such as an azide group, a diaziridine group, a sulfonyl fluoride group, etc.) under appropriate conditions. Both can be covalently cross-linked with the target protein, and the complex formed by the target protein and the DNA-encoding compound does not separate under electrophoresis conditions, but the complex can be separated from the target protein alone under electrophoresis.

In summary, screening using the DNA encoding compound library of the present invention does not require purification or immobilization of the target protein, and thus can be applied to screening of complex systems such as non-binding proteins, cell transmembrane proteins, and cell lysates. In addition, after the DNA-encoding compound of the present invention is covalently cross-linked with a protein, the protein and the DNA tag portion are all covalently linked, and can withstand various separation conditions, such as electrophoretic separation, strong elution conditions, etc., protein and DNA tags. Part of it will not separate due to harsh separation conditions.

Claims (30)

A DNA encoding compound characterized in that it has the formula of formula I:

Wherein X is an atom or a molecular skeleton; A ₁ is a portion comprising a linking strand and an oligonucleotide; A ₂ is a portion comprising a linking strand and an oligonucleotide; M1 is a functional part comprising one or more structural units; M2 is a moiety comprising one or more operable covalent crosslinks. The DNA-encoding compound according to claim 1, wherein X is a molecular skeleton of a carbon atom or a poly atom. The DNA-encoding compound according to claim 2, wherein the polyatomic skeleton is a cyclic or acyclic skeleton structure. The DNA encoding compound according to claim 3, wherein the acyclic skeleton structure is

Wherein X ₁ , X ₂ , X ₃ and X ₄ are each independently selected from the group consisting of carbon, oxygen, nitrogen and sulfur. The DNA encoding compound according to any one of claims 1 to 4, wherein the formula I has a structure represented by formula II:

Wherein Z ₁ is an oligonucleotide linked to L ₁ at its 3' end, Z ₂ is an oligonucleotide linked to L ₂ at its 5' end or Z ₁ is linked to L ₁ at its 5' end. Oligonucleotide, Z ₂ is an oligonucleotide linked to L ₂ at its 3'end; L ₁ is a linking chain comprising a functional group capable of forming a bond with the 3' end or the 5' end of Z ₁ ; L ₂ is a linking chain comprising a functional group capable of forming a bond with the 5' end or the 3' end of Z ₂ ; Y ₁ is a functional part; Y ₂ is a moiety operable to carry out covalent crosslinking; S ₁ and S ₂ are each independently a linking chain. The DNA encoding compound according to claim 5, wherein Z ₁ and Z _{2 are} complementary to form a double strand. The DNA encoding compound according to claim 5, wherein Z ₁ and Z ₂ are the same or different in length. The DNA-encoding compound according to claim 6 or 7, wherein each of Z ₁ and Z _{2 has} a length of 10 or more bases and has 10 or more base pair complementary regions. The DNA encoding compound according to claim 5, wherein both Z ₁ and Z ₂ comprise a PCR primer sequence. The DNA-encoding compound according to claim 5, wherein L ₁ and L ₂ are each independently selected from a difunctional alkylene chain or a difunctional oligodiol chain. The DNA-encoding compound according to claim 10, wherein the functional group in L ₁ and L ₂ is selected from the group consisting of a phosphate group, an amino group, a hydroxyl group, and a carboxyl group. The DNA encoding compound according to claim 11, wherein L ₁ and L ₂ are each independently selected from the group consisting of

Wherein n is an integer of 1 to 10. The DNA-encoding compound according to claim 5, wherein S ₁ and S ₂ are each independently selected from a bifunctional alkylene chain or a difunctional oligodiol chain. The DNA-encoding compound according to claim 13, wherein the functional group in S ₁ and S ₂ is selected from a carboxyl group, an amino group or an aldehyde group. The DNA encoding compound according to claim 14, wherein S ₁ and S _{2 are} selected from the group consisting of

Wherein m is an integer of 1 to 10. The DNA-encoding compound according to claim 5, wherein Y ₂ comprises a photosensitive group, an electrosensitive group or a group covalently crosslinkable with the protein. The DNA-encoding compound according to claim 16, wherein Y ₂ comprises an azide group, a diaziridine group, a sulfonyl fluoride group, a diazo group, a cinnamoyl group, or an acrylate group. The DNA encoding compound according to claim 17, wherein Y _{2 is} selected from the group consisting of

A library of DNA-encoding compounds, which is composed of the DNA-encoding compound according to any one of claims 1 to 18. A DNA encoding a library of compounds according to claim 19, wherein: said DNA encoding a compound library comprising at least ¹⁰⁶ different DNA coding compound. A DNA encoding a library of compounds according to claim 19, wherein: said DNA encoding a compound library comprising at least ¹⁰⁸ different DNA coding compound. The DNA-encoding compound library according to claim 19, wherein the DNA-encoding compound library comprises at least 10 ¹⁰ different DNA-encoding compounds. The method for screening a DNA-encoding compound library according to any one of claims 19 to 22, characterized in that it comprises the following steps: a, incubating the compound library with the protein target, and then operating for covalent cross-linking; b. separating the covalently crosslinked complex of the protein-DNA encoding compound from the uncrosslinked DNA encoding compound; c. The recovered protein-DNA coding compound is covalently cross-linked to carry out PCR amplification and DNA sequencing, and the DNA sequence information is read to obtain compound structure information. The screening method according to claim 23, wherein the covalent crosslinking in the step a is carried out by light, electricity or direct incubation. The screening method according to claim 23, wherein the step b is carried out by electrophoresis. The screening method according to claim 25, wherein the step b is carried out by SDS-PAGE. The screening method according to claim 23, wherein the separation method in the step b is: immobilizing the protein, and washing the unfixed substance with an eluent. The screening method according to claim 27, wherein the immobilizing the protein is to immobilize the protein using magnetic beads. A method for screening a cell lysate by the library of DNA-encoding compounds according to any one of claims 19 to 22, characterized in that it comprises the steps of: i. physically break the cells; Ii, performing centrifugation to separate soluble substances; Iii. Screening the isolated soluble material using the above library of DNA encoding compounds. The method for screening a transmembrane protein according to the DNA encoding compound library according to any one of claims 19 to 22, characterized in that it comprises the following steps: 1) physically break the cells; 2), performing the first round of centrifugation, separating soluble substances and cell membranes, and precipitating to remove insoluble substances; 3), performing a second round of centrifugation, and separating and separating the cell membrane; 4) Screening the isolated transmembrane proteins using the above library of DNA encoding compounds.

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