Small molecule-DNA interaction mapping sequencing method and kit

Abstract

Provided are a small molecule-DNA interaction mapping sequencing method and kit. The method comprises: construction of transposase A coupled and modified by a small molecule compound and biotin; and adding a solution containing the transposase A and magnesium ions into a cell suspension bound to concanavalin A magnetic beads for incubation; after free transposase A is eluted, adding a solution containing transposase B and magnesium ions for continuous incubation, wherein the transposase B is formed by ligating a second adapter at a cleavage activity target site of the transposase; and enriching cleaved DNA fragments and carrying out amplification and sequencing so as to complete construction of a chromatin DNA binding map sequencing library.

Description

A small molecule-DNA interaction map sequencing method and kit

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the Chinese patent application with application number 2023108559060 and application date of July 13, 2023, and claims the priority of the Chinese patent application. The entire content of the Chinese patent application is hereby introduced into this application as a reference. Technical Field

[0003] The present application relates to the field of map sequencing technology, and in particular to a small molecule-DNA interaction map sequencing method and kit. Background Art

[0004] Small molecules that interact with chromatin DNA have significant anti-tumor potential, and many chromatin DNA-interacting molecules have been widely used clinically as drugs, such as Etoposide and Romidepsin (Nat Rev Genet 2014, 15, 783-796.). As research on genomic structure and function becomes increasingly in-depth, strategies to intervene in biological processes and disease states by targeting chromatin DNA or related proteins bound to DNA through small molecules have broad development prospects. To this end, it is crucial to detect the DNA targets of small molecule compounds at the genomic level, because the map of small molecule binding sites to chromatin DNA helps to explain downstream genetic or epigenetic mechanisms.

[0005] The binding sites of proteins and DNA can be detected by mature methods such as chromatin immunoprecipitation sequencing (ChIP-seq) and target shearing and labeling (CUT&Tag). However, the detection of small molecule and DNA binding sites faces huge challenges. The key difficulty is that the dissociation rate constant of small molecule-DNA interaction is usually large, causing the small molecules to detach from the DNA target during the washing step. Several existing small molecule-DNA interaction site analysis methods such as ChIP-seq and CUT&Tag are unable to efficiently enrich target DNA fragments. They are only suitable for small molecules with high affinity and low dissociation rates, and have problems such as high background, weak signal, and large number of cells required. Therefore, the specific genomic targets of many small molecule drugs targeting cellular DNA that have been widely used in clinical practice cannot be clearly defined, which restricts the study of drug action mechanisms and new drug development. Rapid and efficient detection of various small molecules and chromatin DNA binding site maps is an urgent need in this field.

[0006] The process of the classic Chem-seq method is as follows: First, the small molecule compound is biotinylated. Secondly, the biotin-modified small molecule is added to the living cells or cells after in vitro formaldehyde cross-linking and incubated. Then, the chromatin is disrupted by ultrasound, so that the complex of the small molecule compound and the small fragment DNA is released into the solution. Next, magnetic beads with surface modification of streptavidin are added to the solution for incubation to capture the target small molecule compound and the DNA fragment it binds. Then, the captured DNA fragments are purified and eluted. Finally, a DNA library is prepared and second-generation sequencing is performed. Among them, this method is time-consuming and has complex steps. At the same time, the Click-seq method recorded in the Chem-seq method and related technologies (Click chemistry enables preclinical evaluation of targeted epigenetic therapies. Science 2017, 356 (6345), 1397-1401) has a low signal-to-noise ratio, requires a large amount of cells, and has poor reproducibility. The Chem-map method reported in recent literature is: First, the small molecule compound is biotinylated. Next, a biotin-modified small molecule is added to the cell suspension bound to concanavalin A magnetic beads and incubated. A biotin antibody, secondary antibody, and pA / G-Tn5 transposase are then sequentially bound. After adding magnesium ions to activate Tn5 enzyme activity, the DNA at the small molecule binding site is sheared and a sequencing adapter is ligated. After PCR amplification of the DNA fragments, next-generation sequencing is performed. The overall steps are essentially the same as for CUT&Tag, but the required cell number is still around 100,000 cells, and it is only suitable for small molecules with strong binding affinity.

[0007] Therefore, it is urgent to propose a small molecule-DNA interaction map sequencing method with wide adaptability, low cell demand and simple process.

[0008] Summary of the Invention

[0009] The present application aims to solve one of the technical problems in the related art at least to a certain extent.

[0010] To this end, the purpose of this application is to propose a small molecule-DNA interaction map sequencing method and kit. The chromatin DNA binding maps of various small molecules can be detected by this method, which has wide applicability; lower cost, simpler steps, and the fastest library preparation time is only 3 hours.

[0011] According to the first aspect of the present application, a method for sequencing a small molecule-DNA interaction map is proposed, comprising the following steps:

[0012] (1) using a small molecule compound modified with an azide group and biotin to simultaneously modify the 5' end of the linker sequence of the transposase to form a first linker, and the first linker is connected to the cleavage active target site of the transposase to complete the construction of the transposase A modified by coupling the small molecule compound with the biotin;

[0013] (2) adding a solution containing the transposase A and magnesium ions to the cell suspension bound to the concanavalin A magnetic beads and incubating the mixture so that the binding site of the small molecule compound is cut off and the biotin-modified first linker is connected to a specific site of the DNA;

[0014] (3) after eluting the free transposase A, adding a solution containing transposase B and magnesium ions and continuing to incubate to cut the DNA fragments in the open chromatin region of the cell, wherein the transposase B is formed by connecting the second linker to the cleavage active target site of the transposase;

[0015] (4) Enrich the cut DNA fragments and amplify and sequence them to complete the construction of the chromatin DNA binding map sequencing library.

[0016] In some embodiments, in step (1), the method of modifying the small molecule compound with an azide group is: connecting the side chain functional group of the small molecule compound with the azide group, and maintaining a set distance between the azide group and the skeleton of the small molecule compound.

[0017] In some embodiments, at least one linking group is disposed between the azide group and the small molecule compound to maintain a set distance between the two.

[0018] In some embodiments, the small molecule compound is capable of interacting with chromatin DNA, and its side chain functional group includes an amino group, a hydroxyl group, or a carboxyl group.

[0019] In some embodiments, the transposase can specifically bind to double-stranded sequences; the transposase comprises Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn8, Tn9 or Tn10 transposase.

[0020] In some embodiments, in step (1), the first linker is formed by annealing and hybridizing equimolar DBMEA oligonucleotide and ME oligonucleotide; the DBMEA oligonucleotide is obtained by a click chemistry reaction between the small molecule compound modified with the azide group and the DBCO-BMEA oligonucleotide; the sequence of the DBCO-BMEA oligonucleotide is shown in SEQ ID NO.1; the sequence of the ME oligonucleotide is shown in SEQ ID NO.2;

[0021] 5'-DBCO-Biotin dT-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3', SEQ ID NO. 1; wherein the 5' end of the sequence is modified with dibenzocyclooctyne (DBCO), and the first T base at the 5' end is modified with biotin;

[0022] 5'-P-CTGTCTCTTATACACATCT-NH2-3', SEQ ID NO. 2; wherein the 5' end of the sequence is phosphorylated and the 3' end is amino modified.

[0023] In some embodiments, the sequence of the second linker is shown as SEQ ID NO.3;

[0024] MEB oligonucleotide: 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′, SEQ ID NO. 3.

[0025] In some embodiments, step (2) includes the following steps:

[0026] i) capturing cells in the cell suspension using activated Concanavalin A magnetic beads, so that the cells adhere to the Concanavalin A magnetic beads; and

[0027] ii) adding a solution containing digitonin and bovine serum albumin to permeabilize the cells and block non-specific chromatin DNA binding sites in the cells, and then performing the incubation to cleave the small molecule compound binding sites.

[0028] In some embodiments, the DNA fragments cut in step (4) are all located at the binding site of the small molecule compound, and are enriched using streptavidin magnetic beads.

[0029] In some embodiments, the small molecule compound is JQ-1. In some embodiments, the small molecule compound modified with an azide group is a JQ-1-N3 molecule.

[0030] According to two aspects of the present application, a kit for small molecule-DNA interaction map sequencing is proposed, comprising reagents for small molecule-DNA interaction map sequencing using the method described in any of the above embodiments, wherein the reagents include transposase A and transposase B, and further include one or more of a direct sequencing reagent, an annealing buffer, a blocking buffer, or a PCR reagent.

[0031] In some embodiments, the kit for small molecule-DNA interaction map sequencing comprises: transposase A and transposase B and one or more optional sequencing reagents, blocking reagents or PCR reagents, wherein the transposase A comprises a transposase and a first linker, the first linker is connected to the cleavage active target site of the transposase, the first linker comprises the linker sequence of the transposase, a small molecule compound modified with an azide group connected to the 5' end of the linker sequence of the transposase, and biotin; the transposase B comprises the transposase and a second linker, the second linker is connected to the cleavage active target site of the transposase.

[0032] In some embodiments, the side chain functional group of the small molecule compound is connected to the azide group to form the azide-modified small molecule compound, wherein a set distance is maintained between the azide group and the backbone of the small molecule compound.

[0033] In some embodiments, at least one linking group is disposed between the azide group and the small molecule compound to maintain a set distance between the two.

[0034] In some embodiments, the small molecule compound is capable of interacting with chromatin DNA, and the side chain functional group of the small molecule compound includes an amino group, a hydroxyl group, or a carboxyl group.

[0035] In some embodiments, the transposase can specifically bind to double-stranded sequences, and the transposase comprises Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn8, Tn9, or Tn10 transposase.

[0036] In some embodiments, the first linker comprises a DBMEA oligonucleotide and a ME oligonucleotide, the DBMEA oligonucleotide comprises the small molecule compound modified with the azide group and a DBCO-BMEA oligonucleotide, the sequence of the DBCO-BMEA oligonucleotide is shown in SEQ ID NO.1, and the sequence of the ME oligonucleotide is shown in SEQ ID NO.2, the sequence of the SEQ ID NO.1 is: 5'-DBCO-Biotin dT-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3', wherein the 5' end is DBCO modified and the first T base at the 5' end is biotin modified; the sequence of the SEQ ID NO.2 is: 5'-P-CTGTCTCTTATACACATCT-NH2-3', wherein the 5' end is phosphorylated and the 3' end is amino modified.

[0037] In some embodiments, the sequence of the second linker is as shown in SEQ ID NO.3, and the sequence of SEQ ID NO.3 is: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'.

[0038] In some embodiments, the small molecule compound is JQ-1. In some embodiments, the small molecule compound modified with an azide group is a JQ-1-N3 molecule.

[0039] The present invention proposes a small molecule-DNA interaction map sequencing method and kit. It has the following advantages:

[0040] (1) The operation is simple, and the library construction process can be completed in only 3 hours, which is superior to all reported methods;

[0041] (2) High signal-to-noise ratio and strong reproducibility;

[0042] (3) Applicable to small molecules with high and low binding affinity, with a wider range of applications;

[0043] (4) The required cell amount is further reduced, with a minimum of only 100 cells required.

[0044] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the following description of the embodiments in conjunction with the accompanying drawings, in which:

[0046] FIG1 is a schematic diagram of the azide group modification of JQ-1 proposed in one embodiment of the present application;

[0047] FIG2A is a DBMEA-Tn5 transposase linker nucleic acid proposed in one embodiment of the present application;

[0048] FIG2B is a schematic diagram of the MEB-Tn5 transposase linker nucleic acid proposed in one embodiment of the present application;

[0049] FIG3 is a flow chart of a small molecule-DNA interaction map sequencing method proposed in one embodiment of the present application;

[0050] FIG4 is a visualization of the sequencing results of the JQ-1 small molecule and DNA binding map in Example 1 of the present application and the traditional Chem-map method. DETAILED DESCRIPTION

[0051] The following describes in detail embodiments of the present application, examples of which are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. On the contrary, the embodiments of the present application include all changes, modifications, and equivalents that fall within the spirit and scope of the appended claims.

[0052] According to the first aspect of the present application, a small molecule-DNA interaction map sequencing method is proposed as shown in FIG3 , comprising the following steps: (1)-(4).

[0053] (1) using a small molecule compound modified with an azide group and biotin to simultaneously modify the 5' end of the linker sequence of the transposase to form a first linker, and the first linker is connected to the cleavage active target site of the transposase to complete the construction of the transposase A modified by coupling the small molecule compound with biotin;

[0054] (2) adding a solution containing transposase A and magnesium ions to the cell suspension bound to concanavalin A magnetic beads for incubation, so that the binding site of the small molecule compound is cleaved and the biotin-modified first linker is connected to the specific site of the DNA;

[0055] (3) After eluting the free transposase A, a solution containing transposase B and magnesium ions is added and incubated for further treatment to cut the DNA fragments in the open region of the cell's chromatin, wherein transposase B is formed by connecting the second linker to the target site of the transposase's cleavage activity;

[0056] (4) Enrich the cut DNA fragments and amplify and sequence them to complete the construction of the chromatin DNA binding map sequencing library.

[0057] Specifically, in step (1), the small molecule compound is a compound to be tested that can interact with chromatin DNA, wherein as long as the small molecule compound is coupled, it does not affect its binding force with the target and the water solubility of the coupling product, it can be detected by the technology provided in the embodiments of the present application, wherein the side chain functional group of the small molecule compound is connected to the azide group, and a set distance is maintained between the azide group and the skeleton of the small molecule compound, thereby realizing the azide group modification of the side chain functional group of the small molecule compound.

[0058] In some embodiments, to maintain a predetermined distance between the side chain functional groups of the small molecule compound, such as amino, hydroxyl, or carboxyl groups, a suitable linker group should be provided between the azide group and the backbone of the small molecule compound. The number of linkers is typically 1-3.

[0059] In some embodiments, the small molecule compound may be JQ-1, and the small molecule compound modified with an azide group may be a JQ-1-N3 molecule.

[0060] Taking Figure 1 as an example, the JQ-1 molecule and triethylene glycol modified with side chain carboxyl and amino groups and azide were added to 10 volumes of N,N-dimethylformamide solvent in a molar ratio of 1:1. Then, 1.1 equivalents of HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate) and 1.2 equivalents of triethylamine were added to catalyze the reaction. The reaction was carried out at 20 degrees Celsius for 2 hours to obtain the azide-modified JQ-1-N3 molecule.

[0061] In the present application embodiment, the construction of a transposase A modified by coupling a small molecule compound with biotin includes modifying the 5' end of the Tn5 transposase linker nucleic acid with an azide group JQ-1-N3 molecule, and simultaneously modifying the 5' end with biotin; wherein the transposase can specifically bind to a double-stranded sequence, wherein the transposase includes Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn8, Tn9 or Tn10 transposase, and the known transposase is a transposase from a prokaryotic organism and exists in the form of a dimer, for example, Tn5 can specifically bind to the double-stranded sequence AGATGTGTATAAGAGACAG (SEQ ID NO. 4). Under the catalysis of magnesium ions, the Tn5 transposase can cut other double-stranded DNA and connect the linker sequence to the break site of the cut DNA to produce two new DNA fragments.

[0062] In the examples of the present application, JQ-1 is used as a small molecule compound and the transposase is Tn5 transposase as an example to describe the step (1) of the present application, transposase A, in which JQ-1 is coupled to triethylene glycol modified with an amino group and an azide group via a side chain carboxyl group to obtain an azide-modified JQ-1-N3 molecule. As shown in Figures 2A and 2B, the JQ-1-N3 molecule is modified at the 5' end of the Tn5 transposase adapter nucleic acid, and biotin is simultaneously modified at the 5' end to construct a JQ-1 and biotin-coupled Tn5 transposase (Drug & Biotinylated Mosaic-End-Adapter A Chimera Tn5, JQ-1 DBMEA-Tn5 transposase), i.e., transposase A. In this embodiment, the linker nucleic acid of the JQ-1 DBMEA-Tn5 transposase, i.e., the first linker, is formed by annealing and hybridizing equimolar amounts of the JQ-1 DBMEA oligonucleotide and the ME oligonucleotide; the JQ-1 DBMEA oligonucleotide is obtained by a click chemistry reaction between the azide-modified JQ-1-N3 and the DBCO-BMEA oligonucleotide; the sequence of the DBCO-BMEA oligonucleotide is shown in SEQ ID NO. 1; and the sequence of the ME oligonucleotide is shown in SEQ ID NO. 2.

[0063] 5'-DBCO-Biotin dT-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO. 1), wherein the 5' end of the sequence is modified with DBCO, and the first T base at the 5' end is biotin-modified;

[0064] 5'-P-CTGTCTCTTATACACATCT-NH2-3' (SEQ ID NO. 2); wherein the 5' end of the sequence is phosphorylated and the 3' end is amino modified.

[0065] Specifically, transposase A can be JQ-1 DBMEA-Tn5 transposase, and its construction experimental steps are as follows: dissolve the synthetic JQ-1-N3 small molecule in DMSO to obtain a 5 mM solution. Synthesize DBCO-BMEA oligonucleotides and ME oligonucleotides and dissolve them separately in annealing buffer to a final concentration of 100 μM. Add 0.5 μL of 5 mM JQ-1-N3 solution to 10 μL of 100 μM DBCO-BMEA oligonucleotide, mix well, incubate at room temperature for 1 hour, remove small molecules by ultrafiltration, and concentrate by refrigerated centrifugation to obtain 100 μM JQ-1 DBMEA oligonucleotide. Then, mix 10 μL of 100 μM JQ-1 DBMEA oligonucleotide with 10 μL of 100 μM ME oligonucleotide to obtain 20 μL of 50 μM JQ-1 DBMEA-Tn5 transposase linker, i.e., the first linker. The first linker is heated at 95°C for 5 minutes and slowly cooled to anneal. 20 μL of 50 μM JQ-1 DBMEA-Tn5 transposase adapter was mixed with 100 μL of 10 μM Tn5 transposase, mixed thoroughly and incubated at room temperature for 1 h to obtain JQ-1 DBMEA-Tn5 transposase, i.e., transposase A, which was stored at -20°C for future use.

[0066] In some embodiments, step (2) includes the steps of: preparing concanavalin A magnetic beads, for example, diluting 10X Binding Buffer with water to obtain 2 mL of 1X Binding Buffer. Taking 60 μL of concanavalin A magnetic beads, washing them twice with 500 μL of 1X Binding Buffer, and resuspending them in 60 μL of 1X Binding Buffer for later use. In application, the proportion can be adjusted according to the actual sample volume to be tested.

[0067] In this example, cells were collected, washed twice with PBS, and counted; activated concanavalin A magnetic beads were used to capture the cells, allowing them to adhere to the concanavalin A magnetic beads. A solution containing digitonin and bovine serum albumin was then added to incubate the cells to permeabilize the cells and block nonspecific chromatin DNA binding sites in the cells. A solution containing transposase A and magnesium ions was added to the cell suspension bound to the concanavalin A magnetic beads and the cells were incubated at 37°C for 5 minutes to allow the small molecule compound binding site to be cleaved by transposase A, while the biotin-modified first linker was attached to a specific site on the DNA.

[0068] In this process, the role of transposase A is to add the transposase linker sequence and biotin tag to the target site through the cleavage activity of the transposase. Under the condition of a high concentration of 300mM sodium ions, the non-specific binding ability of transposase A to DNA is very weak, and it is unable to indiscriminately cleave the DNA in the open area of ​​chromatin, and no non-specific labeling signal will be generated. Transposase A will target the transposase to the target site on the chromatin based on the specific binding ability of the small molecule compound to the target. Although the binding of small molecule compounds to target DNA is highly dynamic, this method does not require washing unbound small molecule compounds. Instead, while the small molecule compound is acting on the target DNA, it relies on the activity of the transposase to complete DNA shearing and linker connection. This in principle circumvents the removal effect of the washing process in the traditional method on small molecules that have already bound to the target site.

[0069] Upon completion of step (2), the small molecule compound binding site is cleaved, and the biotin-modified first linker is attached to a specific site on the DNA. The cells can be washed to elute free transposase A, and a buffer containing transposase B and magnesium ions is added to the cells. The cells are incubated at 37°C for half an hour to cleave the DNA in the open chromatin region into small fragments.

[0070] In some embodiments, the transposase B is constructed by using the MEB oligonucleotide shown in SEQ ID NO. 3 as the second linker, synthesizing the MEB oligonucleotide and dissolving it in an annealing buffer to a final concentration of 100 μM; wherein the annealing buffer comprises 20 mM Tris·HCl pH=7.5, 1 mM EDTA, and 100 mM NaCl.

[0071] MEB oligonucleotide: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' (SEQ ID NO. 3). Mix 10 μL of 100 μM MEB oligonucleotide with 10 μL of 100 μM ME oligonucleotide to obtain 20 μL of 50 μM MEB-Tn5 transposase adapter, the second adapter. Heat the second adapter at 95°C for 5 minutes and slowly cool to anneal. Mix 20 μL of 50 μM MEB-Tn5 transposase adapter with 100 μL of 10 μM Tn5 transposase, mix thoroughly, and incubate at room temperature for 1 hour to obtain MEB-Tn5 transposase. Store at -20°C until use.

[0072] In the present application embodiment, under the condition of low concentration of sodium ions, the open sites of cell chromatin DNA are randomly sheared with transposase B to obtain small DNA fragments. In the process of steps (2) and (3), the cells are treated respectively with transposase A and transposase B, and primer binding sites for PCR amplification are also added to the target DNA fragments. Since the transposase used in step (2) is a transposase coupled with a biotin-modified adapter when performing enzyme digestion, only the DNA fragments with biotin will be connected near the small molecule compound binding site, so the recovered fragments are all located in the small molecule compound binding site, thereby achieving the goal of the method for specific detection of the small molecule compound binding site map, so after step (3) is completed, only the enrichment product needs to be PCR amplified to achieve the construction of the chromatin DNA binding map sequencing library, which also corresponds to the above-mentioned but mentioned in principle to avoid the removal effect of the washing process in the traditional method on the small molecules already bound to the target site.

[0073] The specific method of step (4) is to enrich the target site DNA fragments by streptavidin magnetic beads. Only those small fragments that are specifically added with biotin tags when transposase A acts can be enriched by magnetic beads, thereby achieving specific capture of the DNA site bound by the small molecule compound. Subsequently, library construction, sequencing (which can be second-generation sequencing, etc.) and data analysis are carried out. Therefore, after completing step (3), it is only necessary to amplify the streptavidin magnetic bead enrichment product to achieve the construction of the chromatin DNA binding map sequencing library.

[0074] According to two aspects of the present application, a kit for small molecule-DNA interaction map sequencing is proposed, including reagents for small molecule-DNA interaction map sequencing using the method in any of the above embodiments, the reagents including transposase A and transposase B, and also including one or more of a direct sequencing reagent, an annealing buffer, a blocking buffer, or a PCR reagent.

[0075] In some embodiments, the kit for small molecule-DNA interaction map sequencing comprises: transposase A and transposase B and one or more optional sequencing reagents, blocking reagents or PCR reagents, wherein the transposase A comprises a transposase and a first linker, the first linker is connected to the cleavage active target site of the transposase, the first linker comprises the linker sequence of the transposase, a small molecule compound modified with an azide group connected to the 5' end of the linker sequence of the transposase, and biotin; the transposase B comprises the transposase and a second linker, the second linker is connected to the cleavage active target site of the transposase.

[0076] In some embodiments, the side chain functional group of the small molecule compound is connected to the azide group to form the azide-modified small molecule compound, wherein a set distance is maintained between the azide group and the backbone of the small molecule compound.

[0077] In some embodiments, at least one linking group is disposed between the azide group and the small molecule compound to maintain a set distance between the two.

[0078] In some embodiments, the small molecule compound is capable of interacting with chromatin DNA, and the side chain functional group of the small molecule compound includes an amino group, a hydroxyl group, or a carboxyl group.

[0079] In some embodiments, the transposase can specifically bind to double-stranded sequences, and the transposase comprises Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn8, Tn9, or Tn10 transposase.

[0080] In some embodiments, the first linker comprises a DBMEA oligonucleotide and a ME oligonucleotide, the DBMEA oligonucleotide comprises the small molecule compound modified with the azide group and a DBCO-BMEA oligonucleotide, the sequence of the DBCO-BMEA oligonucleotide is shown in SEQ ID NO.1, and the sequence of the ME oligonucleotide is shown in SEQ ID NO.2, the sequence of the SEQ ID NO.1 is: 5'-DBCO-Biotin dT-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3', wherein the 5' end is DBCO modified and the first T base at the 5' end is biotin modified; the sequence of the SEQ ID NO.2 is: 5'-P-CTGTCTCTTATACACATCT-NH2-3', wherein the 5' end is phosphorylated and the 3' end is amino modified.

[0081] In some embodiments, the sequence of the second linker is as shown in SEQ ID NO. 3, and the sequence of SEQ ID NO. 3 is: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'.

[0082] In some embodiments, the small molecule compound is JQ-1. In some embodiments, the small molecule compound modified with an azide group is a JQ-1-N3 molecule.

[0083] In some specific embodiments, a kit for small molecule-DNA interaction map sequencing includes 10X Wash Buffer, 10X Binding Buffer, 10X Tn5 Buffer, 25X Blocking Buffer, Transposase A, Transposase B, and Annealing Buffer; the composition and amount of each reagent are exemplified as follows:

[0084] 10X Wash Buffer: 200 mM HEPES-KOH pH = 7.5, 1.5 M NaCl, 5 mM spermidine;

[0085] 10X Binding Buffer: 200mM HEPES-KOH pH=7.9, 100mM KCl, 10mM CaCl2, 10mM MnCl2;

[0086] 10X Tn5 Buffer: 200 mM HEPES-KOH pH = 7.5, 1.5 M NaCl, 5 mM spermidine;

[0087] 25X blocking buffer: 50 mM EDTA pH = 8.0, 2.5% BSA;

[0088] Transposase A (1:300) targeting a specific small molecule target;

[0089] transposase B (1:100);

[0090] Annealing buffer: Tris·HCl pH=7.5 20 mM, EDTA 1 mM, NaCl 100 mM.

[0091] Example 1:

[0092] Using the kit in the above embodiment, the sequencing of the JQ-1 small molecule and DNA binding map in human HEK293FT cells of 6 samples is taken as an example, wherein transposase A is JQ-1 DBMEA-Tn5 transposase and transposase B is MEB-Tn5 transposase, including the following steps:

[0093] (1) Prepare buffer solution

[0094] Prepare Wash Buffer: 750 μL 10X Wash Buffer, 150 μL 50X protease inhibitor, 6600 μL water, and mix well.

[0095] Prepare permeabilization blocking solution: 300 μL Wash Buffer, 3 μL 5% Digitonin, 12 μL 25X blocking buffer;

[0096] Prepare Tn5 Buffer: 750 μL 10X Tn5 Buffer, 150 μL 50X protease inhibitor, 6600 μL water, 15 μL 5% Digitonin, and mix well.

[0097] Prepare JQ-1 DBMEA-Tn5 transposase reaction solution: 300 μL Tn5 Buffer, 3 μL 1 M MgCl2, 1 μL JQ-1 DBMEA-Tn5 transposase, and mix well.

[0098] Prepare MEB-Tn5 transposase reaction solution: 300 μL Wash Buffer, 0.6 μL 5% Digitonin, 3 μL 1 M MgCl2, 6 μL MEB-Tn5 transposase, and mix well.

[0099] (2) Preparation of HEK293FT cells

[0100] HEK293FT cells were cultured to a confluence of 90%. The cells were dissociated with 0.05% trypsin and resuspended in 500 μL of PBS. Centrifuged at 1500 rpm for 2 minutes and the supernatant removed. 500 μL of PBS was added and the cells were centrifuged at 1500 rpm for 2 minutes. The supernatant was removed. This was washed twice. The cells were resuspended in 100 μL of Wash Buffer. The cells were counted to determine the cell concentration.

[0101] (3) Sequencing of the JQ-1 small molecule DNA binding map

[0102] Prepare six 1.5 mL centrifuge tubes and add 5,000–50,000 cells to each tube. Add Wash Buffer to a total volume of 90 μL. Then, add 10 μL of the prepared Concanavalin A magnetic beads to each tube. Mix gently and incubate at room temperature for 10 minutes.

[0103] The tubes were placed on the magnetic stand for 1 min, the supernatant was removed, 50 μL of the prepared permeabilization blocking solution was added to each tube, and the tubes were incubated at room temperature for 20 min.

[0104] The tubes were placed on a magnetic stand for 1 min, the supernatant was removed, 50 μL of the prepared JQ-1 DBMEA-Tn5 transposase reaction solution was added to each tube, and the tubes were incubated at 37°C for 5 min.

[0105] Incubate on a magnetic rack for 1 minute and remove the supernatant. Add 500 μL of the prepared Tn5 buffer to each tube and incubate on a magnetic rack for 1 minute. Remove the supernatant. Repeat the wash cycle twice.

[0106] Add 50 μL of MEB-Tn5 transposase reaction solution to each tube and incubate at 37°C for 30 min.

[0107] Add 1 μL of 10% SDS to each tube and incubate at 55°C for 5 min.

[0108] Place on a magnetic stand for separation, remove the supernatant, transfer to a new 0.2 mL PCR tube, add 2 μL of streptavidin magnetic beads to each tube, and incubate at room temperature for 20 min.

[0109] Incubate on a magnetic rack for 1 minute and remove the supernatant. Add 100 μL of the prepared Wash Buffer to each tube and incubate on a magnetic rack for 1 minute. Remove the supernatant. Repeat the wash cycle once. Resuspend in 35 μL of pure water.

[0110] Add 2.5 μL i5PCR primer, 2.5 μL i7PCR primer, and 10 μL 5X PCR premix to each tube. Perform PCR as follows: 72°C for 3 min, 98°C for 30 s, (98°C for 15 s, 60°C for 20 s, 72°C for 10 s), 16–22 cycles, 72°C for 2 min, and hold at 12°C.

[0111] i5PCR primer: 5'-AATGATACGGCGACCACCGAGATCTACAC[IIIIIIIII]TCGTCGGCAGCGTC-3'(SEQ ID NO.5);

[0112] i7PCR primer: 5'-CAAGCAGAAGACGGCATACGAGAT[IIIIIIII]GTCTCGTGGGCTCGG-3' (SEQ ID NO. 6), where IIIIIIII is an 8-bp index sequence. The experimental results are shown in Figure 4. The sequencing results of the JQ-1 small molecule obtained by the Drug-map method showed a higher signal-to-noise ratio than those obtained by the traditional Chem-map method, which showed a higher background signal.

[0113] It should be noted that, in the description of this application, the terms "first", "second", etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance. In addition, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0114] Any process or method description in a flowchart or otherwise described herein may be understood to represent a module, segment or portion of code comprising one or more executable instructions for implementing the steps of a specific logical function or process, and the scope of the preferred embodiments of the present application includes alternative implementations in which functions may be performed out of the order shown or discussed, including performing functions in a substantially simultaneous manner or in the reverse order depending on the functions involved, which should be understood by those skilled in the art to which the embodiments of the present application belong.

[0115] Throughout this specification, reference to terms such as "one embodiment," "some embodiments," "examples," "specific examples," or "some examples" means that a specific feature, structure, material, or characteristic described in conjunction with that embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0116] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A method for sequencing a small molecule-DNA interaction map, comprising the following steps: (1) using the small molecule compound modified with an azide group and biotin to simultaneously modify the 5' end of the linker sequence of the transposase to form a first linker, and the first linker is connected to the cleavage active target site of the transposase to complete the construction of the transposase A modified by coupling the small molecule compound with the biotin; (2) adding a solution containing the transposase A and magnesium ions to the cell suspension bound to the concanavalin A magnetic beads for incubation, so that the small molecule compound binding site is cleaved, and the first biotin-modified linker is connected to a specific site of the DNA; (3) after eluting the free transposase A, adding a solution containing transposase B and magnesium ions and continuing incubation to cut the DNA fragments in the open chromatin region of the cell, wherein the transposase B is formed by connecting the second linker to the cleavage active target site of the transposase; (4) Enriching the cut DNA fragments and amplifying and sequencing them to complete the construction of the chromatin DNA binding map sequencing library.

2. The method according to claim 1, wherein in step (1), the method of modifying the small molecule compound with an azide group is: connecting the side chain functional group of the small molecule compound with the azide group, and maintaining a set distance between the azide group and the skeleton of the small molecule compound.

3. The method according to claim 2, wherein at least one connecting group is arranged between the azide group and the small molecule compound to maintain a set distance between the two.

4. The method according to claim 2 or 3, wherein: The small molecule compound can interact with chromatin DNA, and its side chain functional group includes amino group, hydroxyl group or carboxyl group.

5. The method according to any one of claims 1 to 4, wherein: The transposase can specifically bind to a double-stranded sequence, and the transposase includes Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn8, Tn9 or Tn10 transposase.

6. The method according to any one of claims 1 to 5, wherein: In step (1), the first linker is formed by annealing and hybridizing equimolar DBMEA oligonucleotide and ME oligonucleotide; the DBMEA oligonucleotide is obtained by click chemistry reaction between the small molecule compound modified with the azide group and the DBCO-BMEA oligonucleotide; the sequence of the DBCO-BMEA oligonucleotide is shown in SEQ ID NO.1; the sequence of the ME oligonucleotide is shown in SEQ ID NO.2; The sequence of SEQ ID NO.1 is: 5'-DBCO-Biotin dT-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3', wherein the 5' end is DBCO modified, and the first T base at the 5' end is biotin modified; The sequence of SEQ ID NO.2 is: 5'-P-CTGTCTCTTATACACATCT-NH2-3', wherein the 5' end is phosphorylated and the 3' end is amino modified.

7. The method according to any one of claims 1 to 6, wherein: The sequence of the second linker is shown in SEQ ID NO.3, The sequence of SEQ ID NO.3 is: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'.

8. The method according to any one of claims 1 to 7, wherein: Step (2) includes: i) using the activated Concanavalin A magnetic beads to capture cells in the cell suspension, so that the cells adhere to the Concanavalin A magnetic beads; and ii) adding a solution containing digitonin and bovine serum albumin to permeabilize the cells and block non-specific chromatin DNA binding sites in the cells, and then performing the incubation to cleave the small molecule compound binding sites.

9. The method according to any one of claims 1 to 8, wherein: The DNA fragments cut in step (4) are all located at the binding site of the small molecule compound, and are enriched using streptavidin magnetic beads.

10. The method according to any one of claims 1 to 9, wherein the small molecule compound is JQ-1, Optionally, the azide group-modified small molecule compound is a JQ-1-N3 molecule.

11. A kit for sequencing small molecule-DNA interaction profiles, comprising: transposase A and transposase B and one or more of optional sequencing reagents, blocking reagents or PCR reagents, The transposase A comprises a transposase and a first linker, wherein the first linker is connected to the cleavage active target site of the transposase, and the first linker comprises a linker sequence of the transposase, an azide group-modified small molecule compound connected to the 5' end of the linker sequence of the transposase, and biotin; The transposase B comprises the transposase and a second linker, wherein the second linker is connected to the cleavage active target site of the transposase.

12. The kit according to claim 11, wherein the side chain functional group of the small molecule compound is connected to the azide group to form a small molecule compound modified with the azide group, wherein a set distance is maintained between the azide group and the skeleton of the small molecule compound.

13. The kit according to claim 12, wherein at least one connecting group is provided between the azide group and the small molecule compound to maintain a set distance between the two. 14 . The kit according to claim 11 , wherein the small molecule compound is capable of interacting with chromatin DNA, and the side chain functional group of the small molecule compound comprises an amino group, a hydroxyl group or a carboxyl group.

15. The kit according to any one of claims 11 to 14, wherein the transposase can specifically bind to double-stranded sequences, and the transposase comprises Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn8, Tn9 or Tn10 transposase.

16. The kit according to any one of claims 11 to 15, wherein the first linker comprises a DBMEA oligonucleotide and a ME oligonucleotide, the DBMEA oligonucleotide comprises the small molecule compound modified with the azide group and a DBCO-BMEA oligonucleotide, the sequence of the DBCO-BMEA oligonucleotide is shown in SEQ ID NO.1, and the sequence of the ME oligonucleotide is shown in SEQ ID NO.2, The sequence of SEQ ID NO.1 is: 5'-DBCO-Biotin dT-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3', wherein the 5' end is DBCO modified, and the first T base at the 5' end is biotin modified; The sequence of SEQ ID NO.2 is: 5'-P-CTGTCTCTTATACACATCT-NH2-3', wherein the 5' end is phosphorylated and the 3' end is amino modified.

17. The kit according to any one of claims 11 to 16, wherein the sequence of the second linker is as shown in SEQ ID NO.3, The sequence of SEQ ID NO.3 is: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'.

18. The kit according to any one of claims 11 to 17, wherein the small molecule compound is JQ-1, Optionally, the azide group-modified small molecule compound is a JQ-1-N3 molecule.

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