WO2019157692A1 - Dna 编码分子库及无靶点限制的化合物筛选方法 - Google Patents

Dna 编码分子库及无靶点限制的化合物筛选方法 Download PDF

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WO2019157692A1
WO2019157692A1 PCT/CN2018/076802 CN2018076802W WO2019157692A1 WO 2019157692 A1 WO2019157692 A1 WO 2019157692A1 CN 2018076802 W CN2018076802 W CN 2018076802W WO 2019157692 A1 WO2019157692 A1 WO 2019157692A1
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dna
compound
molecular library
protein
screening
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PCT/CN2018/076802
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English (en)
French (fr)
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周海鹏
托斯特·迪恩
赵劲
黄湧
韩珂珩
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/CN2018/076802 priority Critical patent/WO2019157692A1/zh
Publication of WO2019157692A1 publication Critical patent/WO2019157692A1/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

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  • the invention belongs to the technical field of biochemistry, and particularly relates to a DNA coding molecular library and a screening method for a compound without target restriction.
  • 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 target-free screening method for a compound, aiming at solving the existing DNA encoding molecular library for drug screening, only using purification and solidification.
  • the protein target is loaded, thereby limiting the technical problems of the range of applications of the DNA-encoding molecular library.
  • 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 A short-stranded DNA having 15-20 bases, the short-stranded DNA is bound to the first primer region, and 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 polymer After culturing the DNA encoding molecular library with the protein target, the DNA polymer is added for DNA amplification;
  • the DNA amplified 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.
  • a unique short-chain DNA (defined as PC-DNA in the present specification) is introduced, since all DNA tags have the same PCR at both ends in the DNA-encoding molecular library.
  • the primer region so that only one PC-DNA is required, it is possible to bind a PCR primer region to which one end of the compound is attached to all DNA tags to form a double-stranded structure.
  • 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 in Example 2 having 47 different DNA molecules tagged library compound screening is a schematic view of the present invention, in addition to the large number of molecules of the library background sequence comprising additionally two sequences tag (with a GLCBS, with There is CBS) as a positive control; among them, 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 a short-stranded DNA having 15-20 bases, the short-stranded DNA is combined with the first primer region, and the short-stranded DNA is linked to a light-crossing at one end of the compound Joint group.
  • 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 (PC-DNA) is also introduced, since all the DNA tags have the same PCR primer region at both ends in the molecular library. Therefore, only one type of PC-DNA is required to bind to the PCR primer region (ie, the first primer region, which is bound by base complementary pairing) at one end of the compound on all the DNA tags to form a double-stranded structure.
  • 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, so that the DNA coding molecule can be conveniently obtained from the DNA.
  • the library is screened for compounds that bind to protein targets.
  • 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 bound to 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.
  • 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 photocrossing
  • the linking group is linked 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 embodiment of the present invention further provides a screening method for a compound of a DNA encoding molecular library, and the flow principle thereof is as shown in FIG. 1 , and includes the following steps:
  • S02 electrophoretically separating the product amplified by the above DNA, and collecting a DNA tag bound to the protein target;
  • the DNA encoding molecular library unique to the embodiment of the present invention is incubated with the protein target, a part of the compound can bind to the target, so that the PC-DNA carries
  • the photocrosslinking group is located near the protein target. Under light conditions, the photocrosslinking group can covalently capture the protein target. If the compound does not bind to the protein target, the crosslink between the protein target and the PC-DNA cannot be occur.
  • PC-DNA By adding DNA polymerase, all PC-DNA can be extended to a double-stranded pair with the bound DNA tag under the action of DNA polymerase, and the sequence information (coding region DNA sequence) contained in the DNA tag of the molecular library is also All of them are replicated onto PC-DNA such that the PC-DNA covalently cross-linked to the protein target carries the corresponding DNA tag information.
  • Compounds that are not cross-linked to protein targets have a smaller molecular weight and can be separated from the compound cross-linked to the protein target under electrophoresis conditions, thus achieving separation from the "target-DNA” conjugate.
  • the chemical structure of the selected compound can be read by DNA sequencing of the tag information on the isolated "target-DNA” conjugate.
  • the method is applicable to a DNA-encoding molecular library with a small molecule compound attached to any end, which is not limited by the coding direction, and can be screened by adding the short-chain DNA using the existing DNA-encoding molecular library, and the label redesign of the molecular library is not required. Or rebuild.
  • 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. After incubating a library of DNA-encoding molecules with PC-DNA with a non-loaded, unmodified protein target, a portion of the compound in the DNA-encoding library can bind to the protein target, allowing light to be carried on the PC-DNA.
  • the linker group is near the protein target, and under light conditions, the photocrosslinking group can crosslink with the protein target; if the small molecule compound does not bind to the protein target, the protein target and PC-DNA Crosslinking 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 polymerase 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; Molecular library compounds that are not cross-linked to protein targets, due to their smaller molecular weight, are at the bottom of the gel, thus achieving complete separation from the "target-DNA" conjugate.
  • the gel-separated tape is obtained by cutting, extracting, and precipitating to obtain a purified "target-DNA” conjugate. More preferably: the separated "target-DNA” strip is cut from the gel, using 1X The PBS (phosphate buffer) was soaked overnight for extraction and then ethanol precipitated to obtain purified "target-DNA”.
  • PBS phosphate buffer
  • the "target-DNA” conjugate is stable and not destroyed by strong denaturation conditions such as SDS-PAGE gel electrophoresis because of the covalent linkage between the protein and the PC-DNA and the corresponding replicated DNA tag. Structure; gel electrophoresis separation method can better remove compounds that cannot bind to protein targets, reduce background, and make 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.
  • Desthiobiotin is a known ligand that binds selectively to avidin protein and has high binding capacity.
  • another DNA strand does not carry any small molecule compound, and the corresponding coding region is a mixed sequence of "DDD" (defining Russian-Japanese background DNA), where D represents three bases of A, C, and G. Any of the bases.
  • the DNA encoding molecular library (the molecular library is added with a photocrosslinking group)
  • the PC-DNA of the group is combined with the target protein avidin incubation, light treatment (light conditions: 365 nm, 30 s), DNA polymerase extension, gel electrophoresis separation, gelation, extraction concentration, ethanol precipitation, etc.
  • the "avidin-DNA” sample was obtained, and after the sample was subjected to PCR amplification, Sanger sequencing was performed, and the sequencing result is shown in Fig. 2b.
  • a library of DNA-encoding molecules including a library of 4 7 (ie, 16384) different DNA tag sequences.
  • a sequence with GLCBS and a sequence with CBS was added for the positive control.
  • GLCBS is a high binding small molecule of protein target CA-II
  • CBS is a medium binding small molecule.
  • the high-binding GLCBS in the positive control was enriched nearly 500-fold, and the medium-binding CBS was enriched nearly 200-fold, while other compounds in the molecular library, due to lack and CA-II
  • the combined chemical structure is basically not enriched.
  • the above data validated the feasibility of our proposed screening strategy for introducing PC-DNA into the molecular library and performing gel electrophoresis separation for non-fixed, unmodified protein target screening.

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Abstract

一种DNA编码分子库，包括DNA标签，DNA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编码分子库还包括一种具有15-20个碱基的短链DNA，短链DNA与第一引物区相结合，且短链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。该方法利用短链DNA实现配体诱导的光交联，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化合物，可应用于任何蛋白质靶点。

Description

DNA编码分子库及无靶点限制的化合物筛选方法 技术领域
本发明属于生物化学技术领域，具体涉及一种DNA编码分子库及无靶点限制的化合物筛选方法。
 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编码分子库及无靶点限制的化合物筛选方法，旨在解决现有DNA编码分子库进行药物筛选时，只能使用纯化、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编码分子库还包括一种具有15-20个碱基的短链DNA，所述短链DNA与所述第一引物区相结合，且所述短链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编码分子库与蛋白质靶点孵育光照处理后，加入DNA聚合物进行DNA扩增；
将所述DNA扩增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，引入了一种特有的短链DNA（本说明书中定义为PC-DNA），由于在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标签上连接有化合物的一端的PCR引物区，形成双链结构。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中具有4 7个不同DNA标签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，包含另外两个标签序列（一个带有GLCBS、一个带有CBS）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编码分子库还包括一种具有15-20个碱基的短链DNA，所述短链DNA与所述第一引物区相结合，且所述短链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上述DNA编码分子库中，还引入了一种特有的短链DNA（PC-DNA），由于在分子库中，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标签上连接有化合物的一端的PCR引物区（即第一引物区，通过碱基互补配对结合），形成双链结构。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，如此可方便地从DNA编码分子库中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选出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）。
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，其流程原理如图1所示，包括如下步骤：
S01：将本发明实施例的上述DNA编码分子库与蛋白质靶点孵育光照处理后，加入DNA聚合物进行DNA扩增；
S02：将上述DNA扩增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，将本发明实施例特有的DNA编码分子库与蛋白质靶点孵育之后，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，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。加入DNA聚合酶，可以在DNA聚合酶的作用下，所有PC-DNA都被延伸为与结合的DNA标签完全配对的双链，同时分子库DNA标签上含有的序列信息（编码区DNA序列）也就都转被复制到PC-DNA上，这样与蛋白质靶点共价交联的PC-DNA带有对应的DNA标签信息。没有和蛋白质靶点交联的化合物由于具有较小的分子量，在电泳条件下，就可以和与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”偶合物上的标签信息进行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。
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聚合酶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”偶合物的彻底分离。
更进一步优选地，凝胶分离后的胶带，通过切胶、萃取、沉淀，获得纯化的“靶点-DNA”偶合物。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以及对应复制的DNA标签之间是共价连接，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，验证我们提出的引入PC-DNA的DNA编码分子库，并通过DNA聚合酶延伸，进行化合物筛选的策略，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），将其连接到一个DNA链（即DNA标签）上，这个DNA链中有一个TTT的碱基序列对之进行编码（定义为目标DNA）；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（定义俄日背景DNA），其中D代表A，C，G三种碱基中的任意一种。
将上述两种DNA链分别以1:10，1:100的比例进行混合之后，按照上文说明书中所提出的筛选过程：首先将DNA编码分子库（该分子库中加入连接有光交联基团的PC-DNA）与靶点蛋白avidin孵育相结合、光照处理（光照条件为：365 nm，30s）、DNA聚合酶延伸、凝胶电泳分离、切胶、萃取浓缩、乙醇沉淀等一系列步骤，获得“avidin-DNA”样品，再将该样品经过PCR扩增之后，进行Sanger测序，测序结果如图2b所示。
从图2b可知：在筛选之前，1:10和1:100的混合物中，在编码区仅仅能够看到DDD的混合序列，这反映了体系中过量的背景DNA。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，编码区的序列变为了TTT序列，即证明了目标DNA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：
一个DNA编码分子库：包括有4 7（即16384）个不同DNA标签序列的分子库，除了大量的背景序列之外，我们还加入了一个带有GLCBS的序列以及一个带有CBS的序列，用作于阳性对照，用该DNA编码分子库进行化合物筛选的整个过程如图3所示。
GLCBS和CBS的结构式如图3a中所示。在本实施例中，GLCBS为蛋白质靶点CA-II的高结合力小分子，CBS为中等结合力小分子。将含有上述所有序列（16384 + 两个阳性对照）以及含有PC-DNA的分子库进行合成之后，对非固载无修饰的蛋白质靶点CA-II进行筛选（筛选过程跟上述实施例1相同），对筛选后的分子库成员进行PCR扩增和高通量DNA测序来解码，最终数据以散点图的方式显示在图3b之中。
从图3b中可以看到，阳性对照中高结合力的GLCBS被富集了近500倍，中等结合力的CBS被富集了近200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。以上数据验证了我们所提出来的向分子库中引入PC-DNA，并进行凝胶电泳分离的筛选策略针对于非固载、无修饰蛋白质靶点筛选的可行性。
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 (12)

  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编码分子库还包括一种具有15-20个碱基的短链DNA，所述短链DNA与所述第一引物区相结合，且所述短链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。
  3. 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个碱基上。
  4. 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1-3任一项所述的DNA编码分子库与蛋白质靶点孵育光照处理后，加入DNA聚合物进行DNA扩增；
    将所述DNA扩增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。
  5. 如权利要求4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。
  6. 如权利要求4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。
  7. 如权利要求4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。
  8. 如权利要求4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。
  9. 如权利要求4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。
  10. 如权利要求4-9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。
  11. 如权利要求10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。
  12. 如权利要求4-9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，将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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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022135445A1 (zh) * 2020-12-23 2022-06-30 重庆大学 一种dna单双链可控转化的dna编码化合物库合成方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013036810A1 (en) * 2011-09-07 2013-03-14 X-Chem, Inc. Methods for tagging dna-encoded libraries
CN107130299A (zh) * 2016-09-30 2017-09-05 深圳劲宇生物科技有限公司 一种dna编码分子库及化合物筛选方法
CN107130300A (zh) * 2016-10-08 2017-09-05 深圳劲宇生物科技有限公司 一种dna编码分子库的合成方法及dna模板

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013036810A1 (en) * 2011-09-07 2013-03-14 X-Chem, Inc. Methods for tagging dna-encoded libraries
CN107130299A (zh) * 2016-09-30 2017-09-05 深圳劲宇生物科技有限公司 一种dna编码分子库及化合物筛选方法
CN107130300A (zh) * 2016-10-08 2017-09-05 深圳劲宇生物科技有限公司 一种dna编码分子库的合成方法及dna模板

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CAO, C. ET AL.: "A DNA-Templated Synthesis of Encoded Small Molecules by DNA Self- assembly", CHEM. COMMUN., vol. 50, 31 December 2014 (2014-12-31), pages 10997 - 10999, XP055473848, DOI: 10.1039/C4CC03380A *
LI, G. ET AL.: "Design, Preparation, and Selection of DNA-Encoded Dynamic Libraries", CHEM. SCI., vol. 6, 31 December 2015 (2015-12-31), pages 7097 - 7104, XP055473781, DOI: 10.1039/C5SC02467F *
LI, G. ET AL.: "Novel Encoding Methods for DNA-Templated Chemical Libraries", CURRENT OPINION IN CHEMICAL BIOLOGY, vol. 26, 28 January 2015 (2015-01-28), pages 25 - 33, XP055473833, DOI: 10.1016/j.cbpa.2015.01.004 *
LI, Y.Z. ET AL.: "Multistep DNA-Templated Synthesis Using a Universal Template", JACS, vol. 135, 14 November 2013 (2013-11-14), pages 17727 - 17730, XP055473763, DOI: 10.1021/ja409936r *
SHI, B.B. ET AL.: "Selecting a DNA-Encoded Chemical Library against Non-immobilized Proteins Using a ''Ligate-Cross-Link-Purify'' Strategy", BIOCONJUGATE CHEM., vol. 28, 25 July 2017 (2017-07-25), pages 2293 - 2301, XP055632869, DOI: 10.1021/acs.bioconjchem.7b00343 *
ZHAO, P. ET AL.: "Selection of DNA-Encoded Small Molecule Libraries Against Unmodified and Non-Immobilized Protein Targets", ANGEW. CHEM. INT. ED., vol. 53, 7 July 2014 (2014-07-07), pages 10056 - 10059, XP055473842, DOI: 10.1002/anie.201404830 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022135445A1 (zh) * 2020-12-23 2022-06-30 重庆大学 一种dna单双链可控转化的dna编码化合物库合成方法

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