WO2019157693A1 - Dna编码分子库及应用广的化合物筛选方法 - Google Patents

Dna编码分子库及应用广的化合物筛选方法 Download PDF

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Publication number
WO2019157693A1
WO2019157693A1 PCT/CN2018/076803 CN2018076803W WO2019157693A1 WO 2019157693 A1 WO2019157693 A1 WO 2019157693A1 CN 2018076803 W CN2018076803 W CN 2018076803W WO 2019157693 A1 WO2019157693 A1 WO 2019157693A1
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dna
compound
molecular library
screening
protein
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PCT/CN2018/076803
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English (en)
French (fr)
Inventor
韩珂珩
托斯特·迪恩
赵劲
黄湧
周海鹏
李笑宇
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深圳劲宇生物科技有限公司
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Priority to PCT/CN2018/076803 priority Critical patent/WO2019157693A1/zh
Publication of WO2019157693A1 publication Critical patent/WO2019157693A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B70/00—Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes

Definitions

  • the invention belongs to the technical field of biochemistry, and particularly relates to a DNA coding molecular library and a screening method for compound application widely used.
  • the DNA-encoded molecular library enables high-throughput screening of millions or even billions of scales in a very small system.
  • the screening results can be decoded and analyzed by PCR amplification and DNA sequencing to obtain lead compounds for further drug development.
  • DNA coding molecular libraries have been widely recognized and applied in the field of new drug research and development, and become an important supporting technology in the development of new drugs.
  • the DNA-encoding molecular library is used for drug screening.
  • Most of the targets used are purified proteins. After the protein target is modified, it is immobilized on a solid phase such as magnetic beads, and then incubated with the molecular library. Small molecules that cannot bind to the target protein are eluted, separated from small molecules bound to the protein target, and then eluted, PCR amplified, and DNA sequenced by the bound small molecule under protein denaturation conditions. The coding sequence is thus read out to obtain the chemical structure of the small molecule bound to the target.
  • the use of purified, immobilized protein targets limits the range of applications of DNA-encoding molecular libraries.
  • the object of the present invention is to overcome the above-mentioned deficiencies of the prior art, and to provide a DNA encoding molecular library and a widely used compound screening method, aiming at solving the existing DNA encoding molecular library for drug screening, and can only be applied to purification and immobilization.
  • the protein target which limits the technical problem of the range of applications of DNA-encoding molecular libraries.
  • the invention provides a DNA encoding molecular library comprising a DNA tag, the DNA tag comprising a first primer region and a second primer region, wherein the first primer region is linked with a compound at one end, and the DNA encoding molecular library further comprises Short-stranded DNA and splint DNA, one end of the short-stranded DNA is ligated to the first primer region, the other end of the short-stranded DNA is joined to the splint DNA by base pairing, and the splint DNA is The DNA tag is bound by base pairing; the short-stranded DNA is linked to a photocrosslinking group at one end of the compound.
  • Another aspect of the present invention provides a method for screening a compound encoding a DNA encoding molecule, comprising the steps of:
  • the DNA coding molecule library is added to the DNA ligase for ligation, and then incubated with the protein target and then treated with light;
  • the light-treated product is subjected to electrophoretic separation to collect a DNA tag bound to the protein target;
  • the DNA tag bound to the protein target is subjected to DNA sequencing to select the corresponding compound.
  • the DNA encoding molecular library provided by the present invention introduces a unique short-chain DNA (defined as PC-DNA in the present specification) and splint DNA, and the splint DNA is combined with short-stranded DNA and DNA tags by base complementary pairing, respectively.
  • short-stranded DNA can be ligated to a DNA tag
  • the PC-DNA has a sequence that is complementary to a base of a PCR primer region to which one end of the compound is ligated to the DNA tag.
  • the PC-DNA can also return the PCR primer region bound to the tag to form a double-strand-macro-issued hairpin structure;
  • all DNA tags have the same PCR primer region at both ends, so only one PC-DNA is required to bind to the PCR primer region on all DNA tags.
  • a DNA tag that binds to the protein target can be selected, and the DNA tag is subjected to DNA sequencing to obtain sequence information, thereby screening for binding to the protein target. compound of.
  • the method for screening a compound using the DNA encoding molecular library of the present invention completely eliminates the requirement for purification and immobilization of a protein target in the screening of a conventional DNA encoding molecular library.
  • the method no longer relies on physical elution to separate compounds that bind to the target and not bind to the target, nor does it rely on enzymatic degradation, but uses PC-DNA to achieve ligand-induced photocrosslinking and electrophoresis from the system.
  • the compound that does not bind to the target is separated and removed, and the DNA tag bound to the protein target is sequenced to select the corresponding compound, and therefore, in principle, the method can be applied to any protein target.
  • the method has been proved to be capable of screening DNA coding molecular libraries of various complex systems such as non-loaded proteins, protein complexes, living cell surface membrane proteins, cell lysates, etc., and can be directly applied to membrane proteins and protein complexes.
  • Drug targets such as living cells, pathological tissues, and other existing screening methods that cannot be applied.
  • FIG. 1 is a schematic flow chart of screening a compound using a DNA encoding molecular library in the present invention
  • Example 2 is a schematic diagram showing screening of a desthiobiotin compound in Example 1 of the present invention; wherein a) is a screening process, and b) is a sequencing result;
  • FIG. 3 is a schematic diagram showing screening of a GLCBS compound in Example 2 of the present invention; wherein a) is a screening process, and b) is a sequencing result;
  • Example 4 is a schematic diagram showing the screening of a compound having a molecular library of 4800 different macrocyclic polypeptides in Example 3 of the present invention, which comprises a label sequence with GLCBS as a positive control in addition to a large number of background sequences; , a) is the screening process, and b) is the enrichment result.
  • an embodiment of the present invention provides a DNA encoding molecular library comprising a DNA tag, the DNA tag comprising a first primer region and a second primer region, wherein the first primer region is ligated with a compound at one end, and the DNA coding
  • the molecular library further includes short-stranded DNA and one of the splint DNA, one end of the short-stranded DNA is bound to the first primer region, and the other end of the short-stranded DNA is coupled to the splint DNA by base pairing, and the splint
  • the DNA is bound to the DNA tag by base pairing; the short-stranded DNA is linked to a photocrosslinking group at one end of the compound.
  • a DNA molecule ie, a DNA tag
  • a DNA tag is used as a barcode to encode a compound in a molecular library, that is, each compound is added with a DNA tag (the same compound corresponds to a unique base sequence).
  • a unique short-chain DNA and splint DNA are introduced, and the splint DNA is combined with the short-stranded DNA and the DNA tag, respectively, to form a short-stranded DNA and DNA.
  • PC-DNA has a sequence complementary to the base of the PCR primer region (ie, the first primer region) to which one end of the compound is linked to the DNA tag
  • the PC-DNA after the PC-DNA is ligated to the DNA tag by the help of splint DNA, it can also return the PCR primer region bound to the tag to form a double-strand-macro-issued hairpin structure; meanwhile, due to the DNA-encoding molecular library, All DNA tags have the same PCR primer region at both ends, so only one PC-DNA is required to bind the PCR primer region on all DNA tags.
  • the photocrosslinking group includes at least one of phenyl azide, benzophenone, and propyl acridine.
  • the photocrosslinking group is capable of cross-linking with a protein target to select a DNA tag corresponding to the compound that binds to the protein target; and if the compound does not bind to the protein target, the protein target is cross-linked with the PC-DNA Union cannot happen.
  • photocrosslinking groups of the present invention there are many photocrosslinking groups of the present invention, and photocrosslinking groups having the same function are within the scope of the present invention, and are not limited thereto.
  • the photocrosslinking group is attached to the first to third bases of the short-stranded DNA near one end of the compound, that is, the light
  • a cross-linking group is attached to the short-stranded DNA at a position close to the first to third bases of one end of the compound.
  • the photocrosslinking group is capable of better crosslinking reaction with the protein target.
  • the photocrosslinking group is attached to the first base of the short stranded DNA near one end of the compound ( That is, the terminal base of the short-stranded DNA sequence).
  • the splint DNA and the DNA tag are complementary paired by 7-10 bases, and the splint DNA and the short-chain DNA pass 7-10 bases.
  • Complementary pairing In the range of the base, the DNA tag and the short-stranded DNA can be more firmly bound to the splint DNA, which is more advantageous for the subsequent DNA ligase. Still more preferably, the short-stranded DNA has 20-25 bases.
  • the short-stranded DNA in the base range provides the number of bases complementary to the splint DNA and the number of bases complementary to the first primer region on the DNA tag (in one embodiment of the invention) 10 bases).
  • the splint DNA and the second primer region on the DNA tag are bound by base pairing, such that one end of the DNA tag and one end of the short-stranded DNA are very close by the help of the splint DNA, and it is better under the action of DNA ligase. Connected to a complete DNA strand.
  • the embodiment of the present invention further provides a screening method for a compound of a DNA encoding molecular library, and the flow thereof is as shown in FIG. 2, and includes the following steps:
  • the DNA encoding molecular library of the embodiment of the present invention is first added with DNA ligase for ligation; the PC-DNA and the DNA tag are connected by the help of the DNA splint. Together, DNA ligase is added to linearly link the DNA tag and PC-DNA to form a strong and stable intact DNA strand through the action of DNA ligase, thus cross-linking with the protein target during subsequent electrophoresis purification.
  • the PC-DNA is not separated from the corresponding DNA tag to screen for the corresponding compound.
  • the DNA encoding molecular library unique to the embodiment of the present invention is treated with DNA ligase, and then incubated with the protein target, and a part of the compound can bind to the target, so that the photocrosslinking group carried on the PC-DNA is at the protein target. In the vicinity, under light conditions, the photocrosslinking group can covalently capture the protein target, and if the compound does not bind to the protein target, the cross-linking of the protein target with the PC-DNA cannot occur; and the protein target is not
  • the cross-linked compound due to its small molecular weight, is separated from the compound cross-linked with the protein target under electrophoresis conditions, thereby achieving separation from the "target-DNA" conjugate, which is obtained by separation.
  • the DNA tag on the target-DNA" conjugate is subjected to DNA sequencing to read the chemical structure of the selected compound.
  • the method is applicable to a DNA-encoding molecular library with a small molecule compound attached to any end, and is not limited by the coding direction.
  • the existing DNA-encoding molecular library can be used for screening by adding short-stranded DNA and splint DNA, and no molecular library is needed. The label is redesigned or rebuilt.
  • the above-mentioned method for screening compounds of DNA-encoding molecular libraries completely eliminates the requirement for purification and immobilization of protein targets in the screening of traditional DNA-encoding molecular libraries, which no longer relies on physical elution to separate and bind target sites.
  • Compounds, rather than relying on enzymatic degradation, use PC-DNA to achieve ligand-induced photocrosslinking, and electrophoretic separation of compounds that do not bind to the target, and DNA tags that bind to protein targets. Sequencing to screen out the corresponding compounds, therefore, in principle, the method can be applied to any protein target.
  • the method has been experimentally proven to be useful for the screening of DNA-encoding molecular libraries of various complex systems such as non-loaded proteins, protein complexes, living cell surface membrane proteins, and cell lysates.
  • the protein target may be a purified protein and/or a non-purified protein, the protein target may also be a modified protein or a non-modified protein, and the protein target may also be a immobilized protein and/or a non-solid protein.
  • Loading protein In principle, the method can be applied to any protein target, and after culturing a DNA-encoding molecular library having PC-DNA with a non-loaded, unmodified protein target, a part of the compound in the DNA encoding molecular library can Protein target binding allows the photocrosslinking group on the PC-DNA to be in the vicinity of the protein target. Under light conditions, the photocrosslinking group can crosslink with the protein target, and if the small molecule does not When the protein target binds, cross-linking of the protein target with PC-DNA cannot occur.
  • the conditions of the illumination processing are: a wavelength of 365 nm and a time of 30 s.
  • the photocrosslinking group can be made to perform photocrosslinking reaction better with the protein target.
  • the electrophoretic separation was carried out by 12% SDS-PAGE gel electrophoresis.
  • the solution system after amplification and extension of the DNA polymer is directly subjected to 12% SDS-PAGE gel electrophoresis analysis. Since the molecular weight of the "target-DNA" conjugate is high, it will be above the protein target itself on the gel; The molecular library compound that is not cross-linked with the protein target, because it has a smaller molecular weight, is at the bottom of the gel, thus achieving separation from the "target-DNA" conjugate.
  • the separated tape is subjected to gelatinization, extraction, and precipitation to obtain a purified "target-DNA”. More preferably, the isolated "target-DNA” band is excised from the gel, soaked overnight in IX PBS (phosphate buffer), and then precipitated with ethanol to obtain purified "target-DNA”.
  • IX PBS phosphate buffer
  • the PC-DNA and the corresponding DNA tag are linked into a complete DNA strand by the DNA ligase, thereby forming a double-strand-macro-issued hairpin structure, thereby making "
  • the target-DNA" conjugate is more stable and does not destroy the structure under strong denaturation conditions such as SDS-PAGE gel electrophoresis; the gel electrophoresis separation method can better remove compounds that cannot bind to the protein target, and reduce The background makes the screening more efficient.
  • the DNA tag bound to the protein target can be PCR amplified before DNA sequencing, and then DNA sequencing, the chemical structure of the selected compound can be read. PCR amplification and re-sequencing are performed first, and the sequencing effect is more accurate.
  • the DNA encoding molecular library (which is added with PC-DNA and double-stranded hairpin structure) Label DNA) was treated with DNA ligase, then incubated with the target protein avidin, light treatment (light conditions: 365 nm, 30 s), gel separation, gelation, extraction concentration, ethanol precipitation, and obtained "avidin-DNA”.
  • the target protein avidin light treatment (light conditions: 365 nm, 30 s)
  • gel separation gelation
  • extraction concentration ethanol precipitation
  • a DNA strand with a small molecule GLCBS attached to the 5-terminal end of the DNA tag (DNA-3 as shown in Figure 3a).
  • GLCBS is a known, specifically capable of binding to CA-II.
  • a small molecule, a DNA strand with a small molecule GLCBS attached to it, is encoded with a "GCTT”.
  • TCCC another coding region on the DNA strand without any small molecule
  • the two DNA strands are mixed at a ratio of 1:10, 1:100, and then the DNA tag is ligated to a PC-DNA sequence having a photocrosslinking group under the action of splint DNA and DNA ligase, and Form a double-chain-large ring hairpin structure.
  • a PC-DNA sequence having a photocrosslinking group under the action of splint DNA and DNA ligase, and Form a double-chain-large ring hairpin structure.
  • the extract was concentrated and ethanol precipitated to obtain a "CAII-DNA" sample. After the sample was subjected to PCR amplification, Sanger sequencing was performed, and the sequencing result is shown in Fig. 3b.
  • the coding region in the sequence of the mixture is essentially a sequence of TCCC, reflecting excess DNA-4 in the system.
  • the results of Sanger sequencing showed that the sequence in the coding region became a GCTT sequence, demonstrating that DNA-3 was selectively enriched by CA-II target protein, and further The feasibility of the DNA coding molecule library for compound screening experiments was demonstrated.
  • FIG. 1 A compound screening method applied to a truly chemically diverse DNA-encoding molecular library, the entire process is shown in FIG.
  • the DNA-encoding library contains: 4800 different macrocyclic polypeptides, each of which is encoded by a specific DNA sequence, and we include a sequence with GLCBS as a positive control ( Figure 4a Show).
  • DNA sequencing to decode Due to the chemical diversity and sequence diversity of the library, we used a second-generation high-throughput sequencer to sequence the molecular library.
  • the final data is shown in Figure 4b as a scatter plot. It can be seen from Fig. 4b that the positive control GLCBS is enriched nearly 60 times, and other compounds in the molecular library are not substantially enriched due to the lack of chemical structure combined with CA-II.

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Abstract

一种DNA编码分子库，包括DNA标签，该标签包括第一引物区和第二引物区，第一引物区一端连接有化合物，DNA编码分子库还包括短链DNA和夹板DNA，短链DNA的一端与第一引物区结合，短链DNA的另一端与夹板DNA通过碱基配对结合，且夹板DNA与DNA标签通过碱基配对结合；短链DNA在靠近化合物的一端连接有光交联基团。一种DNA编码分子库的化合物筛选方法：将上述DNA编码分子库中加入DNA连接酶进行连接处理，然后与蛋白质靶点孵育后光照处理；将光照处理后的产物进行电泳分离，收集与蛋白质靶点结合的DNA标签并进行DNA测序，以筛选出对应的化合物。该方法够用于任何蛋白质靶点的DNA编码分子库的筛选。

Description

DNA编码分子库及应用广的化合物筛选方法 技术领域
本发明属于生物化学技术领域，具体涉及一种DNA编码分子库及应用广的化合物筛选方法。
 背景技术
当代药物研发中，针对疾病的药物靶点，通过构建大型的候选药物分子库，进行高通量、大规模筛选是新药研发中不可或缺的手段。当今世界上主要的制药公司均拥有大型的分子库和大规模的筛选平台用于新药研发。然而，传统的分子库和筛选平台成本高昂、技术门槛高、管理运行复杂，严重制约高通量筛选的发展和应用。近5年来，DNA编码分子库技术逐渐发展起来，成为药物研发中的新兴筛选方法。在DNA编码分子库中，每一个化合物与一个特异性的DNA链相连接，成为一个特异的条形码，实现对化合物的特异性编码。DNA编码分子库能够在极小的体系中，实现千万乃至上亿级的高通量筛选。筛选结果可以通过PCR扩增和DNA测序进行解码分析，以获得先导化合物用于进一步药物研发。近年来，DNA编码分子库已经得到新药研发领域中的广泛认可和应用，成为新药研发中的一种重要支撑技术。
使用DNA编码分子库进行药物筛选，所使用的靶点大多为纯化后的蛋白质，蛋白质靶点经修饰后，固载在磁珠之类的固相之上，再与分子库进行孵育。不能与靶点蛋白结合的小分子被洗脱，与结合在蛋白靶点上的小分子相分离，再在蛋白质变性条件下，对结合的小分子进行洗脱、PCR扩增，以及DNA测序，从而读出编码序列，获得与靶点结合的小分子的化学结构。然而，使用纯化、固载的蛋白靶点限制了DNA编码分子库的应用范围，很多其它类型的药物靶点，例如膜蛋白、蛋白质复合体、活细胞、病理组织等，由于较难或无法纯化和固载，并不能够用于DNA编码分子库的筛选，成为本领域中的一个瓶颈问题。
 技术问题
本发明的目的在于克服现有技术的上述不足，提供一种DNA编码分子库及应用广的化合物筛选方法，旨在解决现有DNA编码分子库进行药物筛选时，只能应用于纯化、固载的蛋白靶点，从而限制了DNA编码分子库的应用范围的技术问题。
 技术解决方案
为实现上述发明目的，本发明采用的技术方案如下：
本发明一方面提供一种DNA编码分子库，包括DNA标签，所述DNA标签包括第一引物区和第二引物区，所述第一引物区一端连接有化合物，所述DNA编码分子库还包括短链DNA和夹板DNA，所述短链DNA的一端与所述第一引物区结合，所述短链DNA的另一端与所述夹板DNA通过碱基配对结合，且所述夹板DNA与所述DNA标签通过碱基配对结合；所述短链DNA在靠近所述化合物的一端连接有光交联基团。
本发明另一方面提供一种DNA编码分子库的化合物筛选方法，包括如下步骤：
将上述DNA编码分子库中加入DNA连接酶进行连接处理，然后与蛋白质靶点孵育后光照处理；
将所述光照处理后的产物进行电泳分离，收集与所述蛋白质靶点结合的DNA标签；
将与所述蛋白质靶点结合的DNA标签进行DNA测序，以筛选出对应的化合物。
 有益效果
本发明提供的DNA编码分子库中，引入了特有的短链DNA（本说明书中定义为PC-DNA）和夹板DNA，夹板DNA分别与短链DNA、DNA标签均通过碱基互补配对相结合，在其协助下，可使短链DNA与DNA标签连接，而PC-DNA中具有能够与DNA标签上连接有化合物一端的PCR引物区碱基互补的序列。这样通过夹板DNA协助使PC-DNA和DNA标签连接成链之后，PC-DNA还能够返回结合在标签上的PCR引物区，形成一个双链-大环的发卡结构；同时，因DNA编码分子库中，所有的DNA标签在两个末端具有相同的PCR引物区，所以仅需一种PC-DNA，就能够结合在所有DNA标签上的PCR引物区。这样，当光交联基团与蛋白质靶点发生交联反应后，可选出与蛋白质靶点结合的DNA标签，对该DNA标签进行DNA测序获取其序列信息，就可筛选与蛋白质靶点结合的化合物。
本发明提供的用上述本发明的DNA编码分子库进行化合物筛选的方法，彻底摆脱了传统DNA编码分子库筛选中对蛋白质靶点纯化和固载的要求。本方法不再依赖于物理洗脱来分离结合靶点和不结合靶点的化合物，也不依赖于酶降解，而是利用PC-DNA来实现配体诱导的光交联，以及从体系中电泳分离去除不与靶点结合的化合物，对与蛋白质靶点结合的DNA标签进行测序以筛选出对应的化合物，因此，从原理上讲，本方法能够应用于任何蛋白质靶点。本方法已经通过实验证明能够用于非固载蛋白质、蛋白质复合体、活细胞表面膜蛋白、细胞裂解液等多种复杂体系的DNA编码分子库的筛选，能够直接应用于膜蛋白、蛋白质复合体、活细胞、病理组织等其它现有筛选方法无法应用的药物靶点。
 附图说明
图1为本发明中利用DNA编码分子库进行化合物筛选的流程示意图；
图2为本发明实施例1中对desthiobiotin化合物进行筛选的示意图；其中，a）是筛选流程，b）是测序结果；
图3为本发明实施例2中对GLCBS化合物进行筛选的示意图；其中，a）是筛选流程，b）是测序结果；
图4为本发明实施例3中具有4800个不同大环多肽的分子库进行化合物筛选的示意图，该分子库除了大量的背景序列之外，包含了一个带有GLCBS的标签序列做阳性对照；其中，a）是筛选流程，b）是富集结果。
 本发明的实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合附图和实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
一方面，本发明实施例提供了一种DNA编码分子库，包括DNA标签，所述DNA标签包括第一引物区和第二引物区，所述第一引物区一端连接有化合物，所述DNA编码分子库还包括短链DNA和夹板DNA，所述短链DNA的一端与所述第一引物区结合，所述短链DNA的另一端与所述夹板DNA通过碱基配对结合，且所述夹板DNA与所述DNA标签通过碱基配对结合；所述短链DNA在靠近所述化合物的一端连接有光交联基团。
DNA编码分子库中是把DNA分子（即DNA标签）作为一种条形码，对分子库中的化合物进行编码，即每个化合物加上一个DNA标签（相同的化合物对应唯一碱基序列）。而本发明实施例提供的DNA编码分子库中，引入了特有的短链DNA和夹板DNA，夹板DNA分别与短链DNA、DNA标签均具有碱基互补配对相结合，可使短链DNA与DNA标签连接（后续通过DNA连接酶作用形成完整的DNA链），而PC-DNA中具有能够与所述DNA标签上连接有化合物的一端的PCR引物区（即第一引物区）碱基互补的序列，这样PC-DNA通过夹板DNA协助下和DNA标签连接成链之后，还能够返回结合在标签上的PCR引物区，形成一个双链-大环的发卡结构；同时，因DNA编码分子库中，所有的DNA标签在两个末端具有相同的PCR引物区，所以仅需一种PC-DNA，就能够结合在所有DNA标签上的PCR引物区。这样，当光交联基团与蛋白质靶点发生交联反应后，可选出与蛋白质靶点结合的DNA标签，对该DNA标签进行DNA测序获取其序列信息，就可筛选与蛋白质靶点结合的化合物。
进一步地，在本发明实施例的DNA编码分子库中，光交联基团包括苯基叠氮、二苯甲酮、丙基吖啶中的至少一种。光交联基团能够与蛋白质靶点发生交联反应以选出能与蛋白质靶点结合的化合物对应的DNA标签；而如果化合物不与蛋白质靶点结合，则蛋白质靶点与PC-DNA的交联不能发生。当然本发明的光交联基团很多种，具有相同功能的光交联基团都在本发明保护范围内，不局限于此几种。
进一步地，在本发明实施例的DNA编码分子库中，所述光交联基团连接在靠近所述化合物一端的所述短链DNA的第1-3个碱基内上，即所述光交联基团与所述短链DNA连接，连接位置在靠近所述化合物一端的第1-3个碱基范围内。在该范围内，光交联基团能够与蛋白质靶点更好地发生交联反应，在一优选实施例中，光交联基团连接在短链DNA靠近化合物一端的第1个碱基（即短链DNA序列的末端碱基）。
进一步地，在本发明实施例的DNA编码分子库中，所述夹板DNA与所述DNA标签通过7-10个碱基互补配对，所述夹板DNA与所述短链DNA通过7-10个碱基互补配对。在该碱基范围内可使DNA标签和短链DNA更加牢固地结合夹板DNA，更有利于后续DNA连接酶的作用。更进一步优选地，短链DNA具有20-25个碱基。该碱基范围内的短链DNA即可提供与夹板DNA互补配对连接的碱基数，又可提供和DNA标签上第一引物区互补配对连接的碱基数（在本发明一实施例中为10个碱基）。更进一步地，所述夹板DNA与所述DNA标签上的第二引物区通过碱基配对结合，这样DNA标签一端和短链DNA一端在夹板DNA协助下非常靠近，在DNA连接酶作用下更好地连接成完整的DNA链。
另一方面，本发明实施例还提供了一种DNA编码分子库的化合物筛选方法，其流程如图2所示，包括如下步骤：
S01：将上述DNA编码分子库中加入DNA连接酶进行连接处理，然后与蛋白质靶点孵育后光照处理；
S02：将上述光照处理后的产物进行电泳分离，收集与所述蛋白质靶点结合的DNA标签；
S03：将与所述蛋白质靶点结合的DNA标签进行DNA测序，以筛选出对应的化合物。
本发明实施例提供的DNA编码分子库的化合物筛选方法中，先将本发明实施例的DNA编码分子库中加入DNA连接酶进行连接处理；PC-DNA和DNA标签是通过DNA夹板的协助连在一起的，加入DNA连接酶进行处理，可通过DNA连接酶的作用将DNA标签和PC-DNA线性连接形成一条牢固、稳定的完整DNA链，这样后续电泳纯化过程中与蛋白质靶点发生交联反应连接PC-DNA与对应的DNA标签就不会分开，从而筛选对应的化合物。将本发明实施例特有的DNA编码分子库用DNA连接酶处理后，再与蛋白质靶点孵育，一部分化合物能够与靶点结合，使得PC-DNA上所带的光交联基团处于蛋白质靶点附近，在光照条件下，光交联基团能够共价捕获蛋白质靶点，而如果化合物不与蛋白质靶点结合，则蛋白质靶点与PC-DNA的交联不能发生；而没有和蛋白质靶点交联的化合物，由于具有较小的分子量，而在电泳条件下，与和蛋白质靶点交联的化合物相分离，因此实现了与“靶点-DNA”偶合物的分离，将分离获得的“靶点-DNA”偶合物上的DNA标签进行DNA测序，即可读出被选择的化合物的化学结构。本方法适用于任意一端连有小分子化合物的DNA编码分子库，不受编码方向的限制，同时利用现有的DNA编码分子库加入短链DNA和夹板DNA就可进行筛选，不需要分子库的标签重新设计或重建。
上述DNA编码分子库的化合物筛选方法，彻底摆脱了传统DNA编码分子库筛选中对蛋白质靶点纯化和固载的要求，其不再依赖于物理洗脱来分离结合靶点和不结合靶点的化合物，也不依赖于酶降解，而是利用PC-DNA来实现配体诱导的光交联，以及从体系中电泳分离去除不与靶点结合的化合物，对与蛋白质靶点结合的DNA标签进行测序以筛选出对应的化合物，因此，从原理上讲，本方法能够应用于任何蛋白质靶点。本方法已经通过实验证明能够用于非固载蛋白质、蛋白质复合体、活细胞表面膜蛋白、细胞裂解液等多种复杂体系的DNA编码分子库的筛选。
进一步地，在上述步骤S01中，蛋白质靶点可以为纯化蛋白质和/或非纯化蛋白质，蛋白质靶点还可以为修饰蛋白质或非修饰蛋白质，蛋白质靶点还可以为固载蛋白质和/或非固载蛋白质。从原理上讲，本方法能够应用于任何蛋白质靶点，将具有PC-DNA的DNA编码分子库与非固载、无修饰的蛋白质靶点孵育之后，该DNA编码分子库中的一部分化合物能够与蛋白质靶点结合，使得PC-DNA上所带的光交联基团处于蛋白质靶点附近，在光照条件下，光交联基团能够与蛋白质靶点发生交联反应，而如果小分子不与蛋白质靶点结合，则蛋白质靶点与PC-DNA的交联不能发生。
进一步地，在上述步骤S01中，所述光照处理的条件为：波长365nm，时间30s。在该光波长和光照时间条件下，可使光交联基团能够与蛋白质靶点更好地进行光交联反应。
进一步地，在上述步骤S02中，电泳分离为12%SDS-PAGE凝胶电泳分离。将DNA聚合物扩增延伸之后的溶液体系直接进行12%SDS-PAGE凝胶电泳分析，由于“靶点-DNA”偶合物分子量较高，在凝胶上将会处于蛋白质靶点本身之上；而没有和蛋白质靶点交联的分子库化合物，由于具有较小的分子量，处于凝胶的最下方，因此实现了与“靶点-DNA”偶合物的分离。
更进一步优选地，分离后的胶带，通过切胶、萃取、沉淀，获得纯化的“靶点-DNA”。更优选地，将分离后的“靶点-DNA”条带从胶上切下来，用1X PBS（磷酸盐缓冲液）浸泡过夜萃取，然后利用乙醇沉淀来获得纯化的“靶点-DNA”。
上述过程中，因蛋白质与PC-DNA之间是共价连接，而PC-DNA与对应的DNA标签通过DNA连接酶连成完整的DNA链，进而形成双链-大环的发卡结构，使得“靶点-DNA”偶合物在经过SDS-PAGE凝胶电泳这样强变性条件时更稳定且不被破坏结构；经过凝胶电泳分离的方法能够更好的去除不能和蛋白质靶点结合的化合物，降低背景，使得筛选效率更高。
最后，进一步地，在上述步骤S03中，将与蛋白质靶点结合的DNA标签进行DNA测序前可先PCR扩增，然后DNA测序，即可读出被选择的化合物化学结构。先进行PCR扩展再测序，测序效果更准确。
本发明先后进行过多次试验，现举一部分试验结果作为参考对发明进行进一步详细描述，下面结合具体实施例进行详细说明。
实施例1：
通过模型实验，验证我们提出的DNA编码分子库，含有DNA标签和PC-DNA形成的发卡结构，进行化合物筛选，该策略整个过程如图2所示。
我们首先选取了一个desthiobiotin的小分子化合物（结构式如图2a所示），将连接到一个DNA链上（即DNA标签），这个DNA链中有一个TTT的序列对之进行编码（如图2a所示的DNA-1）；desthiobiotin是一个已知的，能够与avidin蛋白质选择性结合，并具有高结合力的配体。与此相对应，另一个DNA链上面不带有任何小分子化合物，在相应的编码区域是一个“DDD”的混合序列（如图2a所示的DNA-2），其中D代表A，C，G三种碱基中的任意一种。
我们将这两种DNA以1:1000的比例进行混合之后，按照上文说明书所提出的：首先将DNA编码分子库（该分子库中加入有形成双链-大环发卡结构的PC-DNA和标签DNA）用DNA连接酶处理，然后与靶点蛋白avidin孵育相结合、光照处理（光照条件为：365 nm，30s）、凝胶分离、切胶、萃取浓缩、乙醇沉淀，获得“avidin-DNA”样品，对该样品经过PCR扩增之后，进行Sanger测序，测序结果如图2b所示。
从图2b中可知“，在筛选之前，1:1000的混合物的序列中，编码区仅仅能够看到DDD的混合序列，反映了体系中1000倍过量的DNA-2。然而在筛选之后（即经过图1中的流程之后），Sanger测序的结果显示，在编码区的序列变为了TTT序列，即证明了DNA-1被avidin靶点蛋白选择性的富集。本数据在原理上验证了我们所提出方法的可行性。
实施例2：
在用上述实施例1初步验证了我们的方法之后，我们进一步进行了实际的化合物筛选试验，整个过程如图3所示。。
如图3a所示，我们选取了DNA标签的5端连接有小分子GLCBS的DNA链（如图3a所示的DNA-3），GLCBS是一个已知的，能够与CA-II特异性结合的小分子，5端连接有小分子GLCBS的DNA链上带有一个“GCTT”对之进行编码。相对应的，另一条不带有任何小分子的DNA链上编码区域为“TCCC”（如图3a所示的DNA-4）。
将这两条DNA链按照1:10，1:100的比例混合，然后在夹板DNA和DNA连接酶的作用下，使DNA标签连接上一段带有光交联基团的PC-DNA序列，而且形成一个双链-大环的发卡结构。我们将用DNA酶连接处理形成的具有双链-大环结构的稳定DNA与靶点蛋白CA-II孵育相结合、光照处理（光照条件为：365 nm，30s）、凝胶分离、切胶、萃取浓缩、乙醇沉淀，获得“CAII-DNA”样品，对该样品经过PCR扩增之后，进行Sanger测序，测序结果如图3b所示。
从图3b可知：在筛选之前，混合物的序列中，编码区基本上都是TCCC的序列，反映了体系中过量的DNA-4。然而在筛选之后（即经过图1中的流程之后），Sanger测序的结果显示在编码区的序列变为了GCTT序列，证明了DNA-3被CA-II靶点蛋白选择性的富集，也进一步证明了该DNA编码分子库进行化合物筛选实验的可行性。
实施例3：
一种应用于真正的具有化学多样性的DNA编码分子库的化合物筛选方法，整个过程如图4所示。
该DNA编码分子库中：具有4800个不同大环多肽，其中每一个大环多肽均由一个特异的DNA序列所编码，同时我们在包含了一个带有GLCBS的序列作为阳性对照（如图4a所示）。此具有4801个化合物的分子库，有形成发卡结构的PC-DNA和标签DNA，按照上述实施例2的方法，对非固载无修饰的蛋白质靶点CA-II的筛选之后，进行PCR扩增和DNA测序来解码。由于本分子库的化学多样性和序列多样性，我们采用了第二代高通量测序仪来完成分子库的测序，最终数据以散点图的方式显示在图4b之中。从图4b可知：阳性对照GLCBS被富集了近60倍，而分子库中的其它化合物，由于缺乏和CA-II相结合的化学结构，基本没有被富集。
我们提供的数据从简单的模型分子库开始，到具有化学多样性的真正DNA编码分子库，较为系统地验证了所提出的新筛选方法（带光交联基团的PC-DNA连接、形成双链-大环的发卡结构、与靶点孵育、光交联）针对于非固载、无修饰蛋白质靶点筛选的可行性，本方法从原理上适用于各种DNA编码分子库，并不受分子库原始合成方法的限制，具有良好的普适性。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (14)

  1. 一种DNA编码分子库，包括DNA标签，所述DNA标签包括第一引物区和第二引物区，所述第一引物区一端连接有化合物，其特征在于，所述DNA编码分子库还包括短链DNA和夹板DNA，所述短链DNA的一端与所述第一引物区结合，所述短链DNA的另一端与所述夹板DNA通过碱基配对结合，且所述夹板DNA与所述DNA标签通过碱基配对结合；所述短链DNA在靠近所述化合物的一端连接有光交联基团。
  2. 如权利要求1所述的DNA编码分子库，其特征在于，所述光交联基团包括苯基叠氮、二苯甲酮、丙基吖啶中的至少一种；和/或
    所述光交联基团连接在靠近所述化合物一端的所述短链DNA的第1-3个碱基内上。
  3. 如权利要求1所述的DNA编码分子库，其特征在于，所述夹板DNA与所述DNA标签通过7-10个碱基互补配对。
  4. 如权利要求1所述的DNA编码分子库，其特征在于，所述夹板DNA与所述短链DNA通过7-10个碱基互补配对。
  5. 如权利要求1所述的DNA编码分子库，其特征在于，所述短链DNA具有20-25个碱基。
  6. 一种DNA编码分子库的化合物筛选方法，其特征在于，包括如下步骤：
    将权利要求1-5任一项所述的DNA编码分子库中加入DNA连接酶进行连接处理，然后与蛋白质靶点孵育后光照处理；
    将所述光照处理后的产物进行电泳分离，收集与所述蛋白质靶点结合的DNA标签；
    将与所述蛋白质靶点结合的DNA标签进行DNA测序，以筛选出对应的化合物。
  7. 如权利要求6所述的DNA编码分子库的化合物筛选方法，其特征在于，所述蛋白质靶点包括纯化蛋白质和/或非纯化蛋白质。
  8. 如权利要求6所述的DNA编码分子库的化合物筛选方法，其特征在于，所述蛋白质靶点包括修饰蛋白质和/或非修饰蛋白质。
  9. 如权利要求6所述的DNA编码分子库的化合物筛选方法，其特征在于，所述蛋白质靶点包括固载蛋白质和/或非固载蛋白质。
  10. 如权利要求6所述的DNA编码分子库的化合物筛选方法，其特征在于，所述光照处理的条件为：波长365nm，时间30s。
  11. 如权利要求6所述的DNA编码分子库的化合物筛选方法，其特征在于，所述电泳分离为12%SDS-PAGE凝胶电泳分离。
  12. 如权利要求6-11任一项所述的DNA编码分子库的化合物筛选方法，其特征在于，所述电泳分离后还包括切胶、萃取和沉淀处理步骤。
  13. 如权利要求12所述的DNA编码分子库的化合物筛选方法，其特征在于，所述切胶后还包括用1X PBS浸泡处理步骤；和/或
    所述沉淀为乙醇沉淀。
  14. 如权利要求6-11任一项所述的DNA编码分子库的化合物筛选方法，其特征在于，与所述蛋白质靶点结合的DNA标签进行DNA测序前还包括PCR扩增步骤。
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