WO2018098854A1 - Dna编码动态分子库的合成与筛选方法 - Google Patents

Dna编码动态分子库的合成与筛选方法 Download PDF

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WO2018098854A1
WO2018098854A1 PCT/CN2016/110102 CN2016110102W WO2018098854A1 WO 2018098854 A1 WO2018098854 A1 WO 2018098854A1 CN 2016110102 W CN2016110102 W CN 2016110102W WO 2018098854 A1 WO2018098854 A1 WO 2018098854A1
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dna
structural unit
reaction
molecular library
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French (fr)
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冯海涛
陈宇锋
路杨
杨东晖
张晓�
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杭州阿诺生物医药科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/04—Methods of creating libraries, e.g. combinatorial synthesis using dynamic combinatorial chemistry techniques
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/08—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/08—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
    • C40B50/10—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support involving encoding steps
    • 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 relates to a molecular library of DNA coding, in particular to a method for synthesizing and screening a dynamic molecular library encoded by DNA.
  • 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, and lead compounds have been obtained 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.
  • a major feature of DNA-encoding molecular libraries compared to conventional high-throughput screening libraries is the ability to include tens of millions to billions of different compounds in a micro-scaled system, enabling high-throughput screening Simple and fast, the 1-2 year screening cycle is reduced to 1-2 weeks.
  • conventional high-throughput screening molecular libraries can only reach 6-7 million compounds, while DNA-encoding molecular libraries far exceed this magnitude.
  • the technical problem solved by the invention is that the DNA coding molecular libraries synthesized by the existing methods are all static molecular libraries, the proportion of the compounds in the molecular library is unchanged, and the proportion of the compounds capable of binding to the target protein is small; screening When the protein target needs to be immobilized and separated by physical elution, the operation is complicated and the cost is high.
  • the object of the present invention is to provide a dynamic DNA encoding molecular library in which the proportion of the compound in the molecular library is not fixed.
  • the dynamic molecular library is formed in a solution and can rapidly enrich the compound bound to the target protein without solidification. Load, simplify operations, reduce costs, and shorten the development cycle.
  • the present invention provides the following technical solutions:
  • the invention provides a method for synthesizing a DNA-encoded dynamic molecular library, the method comprising the steps of:
  • each structural unit contains at least two reactive groups, and the structural unit is divided into a group A structural unit and a group B structural unit;
  • the modifying group is capable of reacting with a reactive group in a structural unit.
  • the reaction of the structural unit with the DNA containing the modifying group comprises reacting an alkynyl group with an azide group to form a triazole function, reacting an aldehyde or a ketone with ammonia to form an imine, reacting two thiol groups to form a disulfide bond,
  • the amino group reacts with an acid chloride or a carboxylic acid to form an amide bond, the amino group reacts with an isocyanate to form a carbamate linkage, or the N-hydroxysuccinimide linkage connects one or both of the amino group of the modifying group and the reactive group amino group in the structural unit.
  • the reaction of the structural unit with the DNA containing the modifying group is such that the amino group of the modifying group reacts with the carboxyl group of the reactive group in the structural unit to form an amide bond.
  • the reaction of step (b) is selected from the group consisting of aldehyde or ketone reaction with an amine, two thiol reactions, reaction of an affinity reagent with an unsaturated olefin, reaction of hydrazine with an aldehyde or a ketone, and a Diels-Alder reaction.
  • the reaction of step (b) is the reaction of an aldehyde with an amine.
  • reaction conditions of step (b) are heating to 90-95 ° C and annealing to room temperature.
  • it is maintained at 90-95 ° C for 1-2 minutes and at room temperature for 5-10 minutes.
  • the amount of the reaction substrate of step (b) is calculated in stoichiometric ratio.
  • the present invention provides a screening method for selecting a compound from a DNA-encoded dynamic molecular library prepared by the aforementioned synthetic method, the method comprising the steps of:
  • the equilibrium locking method is determined according to the type of reaction of step (b), which comprises adding a reducing agent, an acid, an oxidizing agent, or adjusting the pH, or lowering one of the temperatures.
  • the balance locking method is to add a reducing agent.
  • the separation and purification method comprises gel electrophoresis.
  • PCR amplification primers are designed based on the DNA base sequence encoding the compound.
  • the present invention also provides a method for synthesizing the aforementioned DNA-encoded dynamic molecular library and a method for screening compounds from the synthesized molecular library for drug screening.
  • the effects and benefits of the present invention are: by reversible dynamic chemical reaction, each group is arranged and combined, and each compound in the molecular library is spontaneously formed in the same solution, and the target can be rapidly enriched and targeted.
  • a protein-binding compound, and the system does not need to be immobilized, the operation is simple, the cost is reduced, and the development cycle is shortened.
  • Figure 1 shows the synthesis of a DNA-encoded dynamic molecular library and a compound screening process that binds to a target
  • Figure 2 is a decoding process of the compound to be screened
  • Example 3 is a result of synthesis and screening of the molecular library described in Example 1;
  • Example 4 is a gel electrophoresis pattern during the molecular library screening process described in Example 1.
  • This patent describes a novel method for synthesizing a dynamic DNA-encoding molecular library, introducing a coding DNA into dynamic combinatorial chemistry, and realizing efficient synthesis of a dynamic molecular library by a dynamic reversible chemical reaction between structural units linked to the encoded DNA.
  • the dynamic molecular library of the present invention is another type of combinatorial chemical molecular library.
  • the compounds in the dynamic molecular library are not synthesized in advance, but a simple structural group is added to the system; these groups are reversible
  • the chemical reactions are arranged in combination to spontaneously form individual compounds in the molecular library in the same solution.
  • the formation and dissociation of compounds are carried out at all times, under the dynamic equilibrium of thermodynamic control. After the addition of the protein target, the protein target binds to the compound, promotes the movement of the dynamic equilibrium, changes the composition of the molecular library, and the compound having strong binding ability to the protein is enriched, and the content of the compound not bound to the protein is lowered.
  • Compounds that bind to protein targets can be identified by analyzing the equilibrium shift after the addition of the protein target.
  • the synthetic step of the DNA-encoded dynamic molecular library of the present invention comprises: the structural unit is linked to the DNA containing the modifying group, wherein each structural unit contains at least two reactive groups, and the structural unit is Divided into a group A structural unit and a group B structural unit; a reactive group on each structural unit in the group A structural unit undergoes a reversible chemical reaction with an active group on each structural unit in the group B structural unit, The reversible reaction type between each structural unit in the group A structural unit and each structural unit in the group B structural unit is the same, and the reaction conditions are heating and annealing.
  • a screening method for selecting a compound that binds to a target protein from the dynamic molecular library encoded by the DNA of the present invention comprises adding a target protein to enrich a compound that binds to a target protein; and determining a method of equilibrium locking according to a type of reversible chemical reaction
  • the equilibrium locking method is selected from the group consisting of adding a reducing agent, an acid, an oxidizing agent, or adjusting the pH value, or decreasing the temperature; separating and purifying the compound bound to the target protein by gel electrophoresis, the compound carrying single-stranded DNA; using PCR to expand
  • the DNA encoding the compound bound to the target protein is amplified, and the PCR product is purified and subjected to DNA sequencing; the compound molecules corresponding to the base sequence are searched according to the sequencing result, thereby determining the structure of the compound to be screened.
  • the molecular library is encoded by two sets of double-stranded DNA of groups A and B.
  • the structural unit is composed of m and n different structural units in each group. Prior to molecular library synthesis, the two sets of structural units are first directly encoded with m and n different DNA tags for structural units. On the structural groups of Group A and Group B, there are functional groups FA and FB capable of reversible chemical reaction, respectively.
  • the number of chemical reactions occurring between the structural groups of Group A and Group B is Group A structural unit and The arrangement and combination of the B group structural units, that is, capable of forming a molecular library containing m x n compounds, each compound in the molecular library being the base in the coding region of the two DNAs on the two structural units
  • the sequence is encoded.
  • the reversible chemical reaction conditions between the structural units of Group A and Group B are heating and annealing, and the reaction types include, but are not limited to, the reaction of an aldehyde or a ketone with an amine to form an imine, and a reaction of forming a disulfide bond between the thiol groups, and affinity.
  • the number of structural units of Group A and Group B may be 2, and a molecular library containing 2 ⁇ 2 compounds may be synthesized; or 10 may be used to synthesize a molecular library containing 10 ⁇ 10 compounds; A molecular library of 100 ⁇ 100 compounds; the number of structural units is determined according to actual needs, that is, the method of the present invention can synthesize dynamic molecular libraries of different sizes according to actual needs.
  • the isolated DNA can be directly subjected to PCR amplification, and high-throughput sequencing is performed to decode the coding regions of the A and B chains, according to the obtained
  • the DNA barcode finds the corresponding chemical group, and the specific chemical structure of the selected structural group in the A group and the B group can be obtained.
  • the molecular library is synthesized according to the method of the present invention by using a structural unit known to be capable of binding to a known protein target, and compound screening is performed.
  • the compounds obtained by screening are indeed expected compounds capable of binding to known target proteins, and the reliability of the dynamic molecular library synthesis method and screening method of the present invention is verified.
  • a dynamic molecular library containing 100 ⁇ 100 compounds was synthesized, in which the structural units of Group A and Group B each contained 100 compounds, and the type of reversible chemical reaction between Group A structural units and Group B structural units was consistent, and A1 and B1 are known structural units capable of binding to the model target protein carbonate dehydrogenase CA-II.
  • the active group capable of reversible chemical reaction in A1 is an aldehyde group, and the corresponding complementary active group in B1 is an amino group.
  • the A and B chains of the coding DNA are encoded with TAAC and TCCG, respectively, and the other structural units are encoded by other sequences.
  • the synthetic steps of the DNA-encoded dynamic molecular library include:
  • Both the A group structural unit and the B group structural unit exist in the form of carboxylic acid, which are all linked to the DNA through the amide bond.
  • the 384-well plate is operated in parallel, and the ligation reaction of the 384 structural unit with the DNA can be rapidly realized.
  • A1 or B1 is first dissolved in anhydrous dimethyl sulfoxide (DMSO) to prepare a solution having a concentration of 900 mM.
  • DMSO dimethyl sulfoxide
  • EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
  • NHS N-hydroxysuccinimide
  • Peak area Peak height Peak width area% 1 3.964 117.8 28.2 0.0628 0.304 2 4.187 94.4 15.9 0.0874 0.244 3 13.54 252.1 16.4 0.2219 0.651 4 14.045 6387.9 350 0.2549 16.497 5 14.573 31870.5 1754 0.264 82.304
  • the HPLC assay showed that the retention time of A1 after ligation with DNA was 17.935 min, and the retention time of B1 after ligation with DNA was 14.573 min.
  • the DNA encoding all structural units differs only in the sequence of the coding region, and A1 and B1 are exemplified, the base sequence of the A-strand DNA linked to A1 is as shown in SEQ ID NO. 1; and the B-strand DNA linked to B1 is The base sequence is shown in SEQ ID NO.
  • SEQ ID NO. 1 ATGGCAGGCTACGAA TAAC CTGGAGCCAATAAGC
  • SEQ ID NO. 2 ATGGCAGGCTACGAATCCG CTGGAGCCAATAAGC
  • the solution was heated to 90 ° C for 1 minute and slowly annealed to room temperature for 5 minutes; the heating was carried out for 5 minutes and the annealing process was carried out for 30 minutes. This heating/annealing process completes the reaction of the structural units of Group A and Group B, that is, the DNA-encoded dynamic molecular library is synthesized.
  • the molecular library contains The product of the reaction of A1 with B1, that is, the aldehyde group in A1 reacts with the amino group in B1 to form an imine to link the A1 and B1 structural units.
  • the embodiment further comprises screening structural units bound to the model target protein carbonic acid dehydrogenase CA-II from the dynamic molecular library containing 100 ⁇ 100 compounds, and the screening steps include:
  • the synthesized molecular library was further cooled to 4 ° C, and the protein target CA-II was added, and the final concentration of the protein was 2 ⁇ M.
  • the solution was held at 4 ° C for 6 hours to allow the system to fully achieve dynamic equilibrium.
  • the reducing agent NaBH 3 CN (final concentration 10 mM) was added, and the reaction was maintained at 4 ° C for 16 hours, and the imine formed by dynamic equilibrium was reduced to an amine group, thereby locking the equilibrium.
  • the double-fold 40% acetic acid solution was used to quench the lock-balanced reduction reaction, which completed the screening process of the target protein CA-II by the molecular library.
  • the selected compound can bind to the target protein CA-II, and at the same time There are A chain and B chain DNA.
  • the A chain-B chain conjugate (which is capable of binding to the target protein CA-II) and the independent A-chain are combined by 10% or 15% TBE-Urea PAGE denaturing gel electrophoresis.
  • the B chain is separated (see Figure 4). Due to the strong denaturing conditions of gel electrophoresis, the isolated DNA is already single-stranded.
  • the strip of the A chain-B chain conjugate was excised and extracted with 1 x PB (phosphate buffer). The linked A-chain-B chain conjugates were extracted and directly purified by precipitation with ethanol.
  • the precipitate-purified A-chain-B chain conjugate was redissolved in water, diluted to a concentration of 1 nM, and 10 ⁇ L was taken for PCR amplification.
  • the experimental procedure for PCR amplification was as follows: 5 ⁇ L of PCR primer (5 ⁇ M), 1 ⁇ L of 1 mM dNTP mixture, 10 ⁇ L of 5 ⁇ reaction buffer solution and 0.5 ⁇ L of high-fidelity DNA polymerase were added to the system to dilute the total volume of the PCR system to 50 ⁇ L. .
  • the cells were cycled for 29 times at 92 ° C for 30 seconds, 55 ° C for 1 minute, and 72 ° C for 30 seconds, and then purified by 3% agarose gel electrophoresis, and further purified by PCR purification kit to obtain a final PCR product. . After the PCR product was quantified, 10 ng/ ⁇ L of the PCR product solution was taken and directly sent to DNA sequencing.
  • the primers include a forward primer and a reverse primer, the sequences of which are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.
  • Reverse primer sequence SEQ ID NO. 4 GCTTATTGGC TCCAG
  • a "pre-screening" molecular library sample was also sent for sequencing as a control.
  • the control sample was subjected to the same screening process as the screening sample, but no protein target was added.
  • the control sample was used to avoid false positives in the screening results, ie to exclude compound enrichment caused by non-added target proteins.
  • the samples screened by the target protein were tested by DNA sequencer and found that the sequences of TAAC and TCCG were enriched in the two coding regions, which greatly exceeded other background sequences, indicating that the structural units bound to CA-II in the molecular library were Enrichment, ie, the coding regions of the A and B strands linked to the compound after screening for the target protein CA-II are TAAC and TCCG (see Figure 3), respectively corresponding to structural units A1 and B1, thereby determining the target protein.
  • the structure of the compound in which CA-II is bound is as shown in Chemical Formula 3, which is a well-known compound capable of binding to the target protein CA-II, indicating that the screening method of the present invention is very accurate.
  • the above data fully demonstrates the feasibility of the DNA encoding dynamic molecular library technique of the present invention.
  • it is important to screen out compounds that bind to target proteins.
  • the present invention provides a rapid and accurate method for synthesizing and screening dynamic molecular libraries, which is of great significance.

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Abstract

一种合成DNA编码的动态分子库的方法，合成步骤包括将A组和B组结构单元中的结构单元与含修饰基团的DNA连接，其中，每个结构单元含有至少两个活性基团；然后A组中的每个结构单元上的一个活性基团与B组中的每个结构单元上的一个活性基团发生可逆化学反应，生成DNA编码的动态分子库。从分子库中筛选化合物的步骤包括加入靶蛋白，与靶蛋白结合的化合物富集后，加入试剂锁定平衡。利用凝胶电泳分离纯化连接在一起的DNA，通过PCR扩增和DNA测序确定碱基序列，然后确定该碱基序列对应的结构单元，从而筛选出与靶蛋白结合的化合物。

Description

DNA编码动态分子库的合成与筛选方法 技术领域
本发明涉及DNA编码的分子库，尤其涉及DNA编码的动态分子库的合成与筛选方法。
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，成为高通量筛选发展和应用中的严重制约。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，已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。
与常规高通量筛选分子库相比，DNA编码分子库的一个主要特点为能够在微升级的体系中，包含上千万至几十亿级别的不同的化合物，从而使高通量筛选变得简单而快捷，由1-2年的筛选周期降为1-2周。此外，常规高通量筛选分子库仅能够达到6-7百万个化合物，而DNA编码分子库则远远地超过了这个量级。
然而，对DNA编码分子库来说，如何高效的合成分子库中的数量如此之多的化合物，并且准确地在每一个不同化合物上连接不同的DNA条形码，是该领域中的一个技术核心。现有技术中有多种编码化合物的方法，包括组合化学中传统的“split-pool-split”方法、DNA模板控制合成、DNA routing、DNA双链连接等多种方法。然而，这些方法所合成的均为静态分子库，即每个化合物在分子库中所占的比例不变，其中能够与靶点蛋白结合的化合物的占比小， 在对靶点进行筛选时，需要用物理洗脱的方法将与蛋白质结合和不与蛋白质结合的化合物分离。该方法所用的蛋白靶点需要修饰，并且需要固载，操作复杂，成本高。
发明内容
本发明解决的技术问题是：现有方法所合成的DNA编码分子库均为静态分子库，化合物在分子库中所占的比例不变，能够与靶点蛋白结合的化合物的占比小；筛选时，蛋白靶点需要固载，并采用物理洗脱的方法分离，操作复杂，成本高。
本发明的目的是：提供一种化合物在分子库中所占比例不固定的动态DNA编码分子库，该动态分子库是在溶液中形成，能快速富集与靶点蛋白结合的化合物，无需固载，简化操作，降低成本，缩短研发周期。
具体来说，针对现有技术的不足，本发明提供了如下技术方案：
一方面，本发明提供了一种DNA编码的动态分子库的合成方法，该方法包括以下步骤：
(a)结构单元与含有修饰基团的DNA连接，其中，每个结构单元含有至少两个活性基团，所述结构单元被分为A组结构单元和B组结构单元；
(b)A组结构单元中的每个结构单元上的一个活性基团与B组结构单元中的每个结构单元上的一个活性基团发生可逆化学反应。
优选的，所述修饰基团能够与结构单元中的活性基团反应。
优选的，所述结构单元与含有修饰基团的DNA连接的反应包括炔基与叠氮基反应生成三氮唑官能团、醛或酮与氨反应生成亚胺、两个巯基反应生成二硫键、氨基与酰氯或羧酸反应生成酰胺键、氨基与异氰酸酯反应生成carbamate连接，或者通过N-羟基琥珀酰亚胺连接修饰基团氨基与结构单元中的活性基团氨基中的一种或两种。
优选的，所述结构单元与含有修饰基团的DNA连接的反应为修饰基团氨基与结构单元中的活性基团羧基反应生成酰胺键。
优选的，步骤(b)的反应选自醛或酮与胺反应、两个巯基反应、亲和试剂与不饱和烯烃反应、肼与醛或酮反应以及Diels-Alder反应中的一种。
优选的，步骤(b)的反应为醛与胺反应。
优选的，步骤(b)的反应条件为加热至90-95℃，退火至室温。
优选的，在90-95℃下维持1-2分钟，室温下维持5-10分钟。
优选的，步骤(b)的反应底物用量按照化学计量比计算。
另一方面，本发明提供了从前述合成方法制备的DNA编码的动态分子库中选出一种化合物的筛选方法，该方法包括以下步骤：
(c)加入靶蛋白，与靶蛋白结合的结构单元富集；
(d)平衡锁定；
(e)分离和纯化；
(f)PCR扩增，DNA测序解码；
(g)确定筛选出来的化合物结构。
优选的，根据步骤(b)的反应类型确定平衡锁定方法，所述平衡锁定方法包括加入还原剂、酸、氧化剂或者调节pH值，或者降低温度中的一种。
优选的，所述平衡锁定方法为加入还原剂。
优选的，所述分离和纯化方法包括凝胶电泳法。
优选的，根据编码化合物的DNA碱基序列来设计PCR扩增引物。
又一方面，本发明还提供了前述DNA编码的动态分子库的合成方法和从合成所得的分子库中筛选化合物的方法在药物筛选中的应用。
与现有技术相比，本发明的效果和益处在于：通过可逆的动态化学反应，各个基团之间进行排列组合，在同一溶液中自发形成分子库中的各个化合物，能快速富集与靶点蛋白结合的化合物，且体系无需固载，操作简单，成本降低，研发周期缩短。
附图说明
图1为DNA编码动态分子库的合成及与靶点结合的化合物筛选过程；
图2为被筛选化合物的解码过程；
图3为实施例1所述分子库的合成与筛选结果；
图4为实施例1所述分子库筛选过程中的凝胶电泳图。
具体实施方式
本专利描述了一种新型的动态DNA编码分子库的合成方法，将编码DNA引入到动态组合化学中，通过连接有编码DNA的结构单元之间的动态可逆化学反应实现动态分子库的高效合成。
本发明所述动态分子库是组合化学分子库的另一种类型，动态分子库中的化合物并非事先合成的，而是在体系中加入简单的结构基团；这些基团之间，通过可逆的化学反应进行排列组合，从而在同一溶液中自发形成分子库中的各个化合物。在动态分子库中，化合物的生成和解离在时刻进行着，处于热力学控制的动态平衡之下。在加入蛋白质靶点之后，蛋白质靶点与化合物结合，促进动态平衡的移动，分子库的成分发生变化，对蛋白质具有强结合力的化合物被富集，而不与蛋白质结合的化合物的含量降低。通过对加入蛋白质靶点之前之后的平衡移动进行分析，即可识别出与蛋白质靶点结合的化合物。
具体而言，本发明所述的DNA编码的动态分子库的合成步骤包括：结构单元与含修饰基团的DNA连接，其中，每个结构单元含有至少两个活性基团，所述结构单元被分为A组结构单元和B组结构单元；A组结构单元中的每个结构单元上的一个活性基团与B组结构单元中的每个结构单元上的一个活性基团发生可逆化学反应，A组结构单元中的各个结构单元与B组结构单元中的各个结构单元间的可逆反应类型相同，反应条件为加热、退火。
从本发明所述的DNA编码的动态分子库中选出一种与靶蛋白结合的化合物的筛选方法包括加入靶蛋白，富集与靶蛋白结合的化合物；根据可逆化学反应类型确定平衡锁定的方法，平衡锁定方法选自加入还原剂、酸、氧化剂或者调节pH值，或者降低温度中的一种；利用凝胶电泳分离纯化与靶蛋白结合的化合物，该化合物带有单链DNA；利用PCR扩增与靶蛋白结合的化合物的编码DNA，纯化PCR产物后进行DNA测序；根据测序结果查找该碱基序列对应的化合物分子，从而确定被筛选出的化合物结构。利用化学合成方法重新合成筛选出来的能够与靶蛋白结合的化合物，该化合物不带有DNA，对化合物进行系列结构表征以及与靶蛋白结合力的实验研究，进一步验证本发明所述动态分子库在药物筛选中的应用。
在本发明的一优选实施例中，分子库由A组和B组两组双链DNA编码的 结构单元组成，每组分别有m和n个不同的结构单元。在分子库合成之前，首先将这两组结构单元分别以m和n个不同的DNA标签对结构单元进行直接编码。在A组和B组的结构基团上，分别带有能够发生可逆化学反应的官能团FA和FB，因此A组和B组的结构基团之间发生的化学反应个数为A组结构单元与B组结构单元的排列组合，即能够形成一个包含有m x n个化合物的分子库，该分子库中的每一个化合物即被两个结构单元上的两条DNA中的编码区域中的碱基序列所编码。其中，A组和B组结构单元之间的可逆化学反应条件为加热、退火，反应类型包括但不限于醛或酮和胺生成亚胺的反应，巯基之间形成二硫键的反应，亲和试剂与不饱和烯烃之间的加成反应，肼和醛或酮之间的缩合反应以及Diels-Alder反应。
所述A组和B组结构单元的数量可以均为2，合成含2x 2个化合物的分子库；也可以均为10，合成含10x 10个化合物的分子库；还可以均为100，合成含100x 100个化合物的分子库；结构单元的数量根据实际需要确定，即本发明所述方法可以根据实际需要来合成大小不同的动态分子库。
利用分子库筛选化合物时，仅需要将分子库与蛋白质靶点进行孵育。在有蛋白质靶点存在的情况下，化合物与靶点的结合将促进动态平衡的转移，促进与靶点结合力强的结构单元之间的反应，而不与靶点结合的结构单元之间的反应将减少，从而实现对分子库中化合物的富集。在能够与靶点结合的化合物富集之后，将平衡锁定。在平衡锁定之后，通过凝胶电泳对已经连接在一起的A组和B组DNA链进行分离纯化，纯化后，DNA双链被打开，因此所获得的化合物上现仅有单链DNA。由于A链和B链DNA分别带有各自的引物结合区域，因此可以直接对分离出的DNA进行PCR扩增，再进行高通量测序对A链和B链的编码区域进行解码，根据获得的DNA条码查找对应的化学基团，即可获得A组和B组中被筛选出来的结构基团的具体化学结构。
利用含有已知的能够与已知的蛋白靶点结合的结构单元按照本发明所述的方法合成分子库，并进行化合物筛选。筛选得到的化合物确实为预期的能够与已知靶点蛋白结合的化合物，验证了本发明所述动态分子库合成方法和筛选方法的可靠性。
本发明所述分子库的合成，仅需要将A组和B组经DNA编码的结构单元按 化学计量比例混合，A组和B组的结构单元直接进行排列组合的可逆化学反应，在同一溶液中生成m x n个不同的化合物(m和n分别指A组结构单元和B组结构单元中的结构单元的数量)，操作非常简单。采用双链DNA编码化合物可以避免A组和B组DNA之间的耦合，避免DNA自身对化合物之间可逆化学反应的影响。
以下通过具体实施例对本发明作进一步说明。
实验中所用试剂和仪器的规格、型号与厂家见表1和表2。
表1试剂的规格和厂家
试剂 规格 厂家
对醛基苯甲酸 AR 百灵威
对氨甲基苯甲酸 AR 百灵威
含氨基修饰的DNA AR 上海生工
碳酸脱氢酶CA-Ⅱ AR 百灵威
氰基硼氢化钠 AR 百灵威
DNA聚合酶 AR NEB
EDC AR 百灵威
NHS AR 百灵威
磷酸缓冲液 AR 百灵威
DMSO AR 百灵威
DNA聚合反应预混溶液 AR NEB
三乙胺 AR 百灵威
乙酸 AR 百灵威
三氟乙酸 AR 百灵威
Glycogen AR Sigma
醋酸钠 AR 百灵威
柠檬酸铵 AR Fluka
PBS缓冲液 AR Bio-rad
溴乙锭 AR Sigma
PCR纯化试剂盒 100T Bio-rad
表2仪器的型号和厂家
Figure PCTCN2016110102-appb-000001
实施例1
本实施例合成了一个含有100x 100个化合物的动态分子库，其中A组和B组结构单元各含100个化合物，A组结构单元与B组结构单元间的可逆化学反应类型一致，且其中的A1与B1为已知的能够与模型靶点蛋白碳酸脱氢酶CA-II结合的结构单元，A1中能够发生可逆化学反应的活性基团为醛基，B1中对应的互补活性基团为氨基。在编码DNA的A链和B链中分别用TAAC和TCCG编码，其它结构单元则以其它序列编码。具体来说，DNA编码的动态分子库的合成步骤包括：
(a)结构单元与DNA通过酰胺键连接
A组结构单元与B组结构单元均以羧酸的形式存在，均与DNA通过酰胺键连接，实验中通过384孔板进行平行操作，能够快速实现384组结构单元与DNA的连接反应。以结构单元A1和B1为例(A1和B1的结构见化学式1和化学式2)，首先将A1或B1溶于无水二甲基亚砜(DMSO)，制备浓度为900mM的溶液。同时将1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)分别溶于无水DMSO中，浓度也分别为900mM。将三个溶液以1:1:1的比例混合，在室温下振荡1小时，实现对羧酸的活化。
Figure PCTCN2016110102-appb-000002
取40μL活化的羧酸溶液，将其直接加入80μL 5’-胺基DNA的溶液(100μM，pH＝7.2；100mM磷酸缓冲液)之中。该混合液在37℃下反应1小时后，加入乙醇，沉淀分离DNA。分离出来的DNA包括与结构单元结合的DNA以及未反应的DNA，再通过HPLC纯化出产物“结构单元-DNA”，并进行质谱表征。A1和B1分别与5’-胺基DNA反应后的HPLC测定条件相同，如表3所示，测定结果分别见表4和表5。
表3 A1和B1分别与5’-胺基DNA反应后的HPLC测定条件
Figure PCTCN2016110102-appb-000003
表4 A1与5’-胺基DNA反应后的HPLC测定结果
编号 时间(min) 峰面积 峰高 峰宽 面积％
1 2.964 95.6 4.5 0.2685 0.485
2 3.445 79.9 3.3 0.3556 0.405
3 3.765 366.4 65.5 0.0853 1.858
4 14.372 675.1 30.4 0.3113 3.422
5 17.935 17512.7 344 0.7419 88.776
6 21.163 997 133.1 0.1154 5.054
表5 B1与5’-胺基DNA反应后的HPLC测定结果
编号 时间(min) 峰面积 峰高 峰宽 面积％
1 3.964 117.8 28.2 0.0628 0.304
2 4.187 94.4 15.9 0.0874 0.244
3 13.54 252.1 16.4 0.2219 0.651
4 14.045 6387.9 350 0.2549 16.497
5 14.573 31870.5 1754 0.264 82.304
HPLC测定结果表明A1与DNA连接后的保留时间为17.935min，B1与DNA连接后的保留时间为14.573min。
标记所有结构单元的DNA仅编码区的序列不同，以A1和B1为例，所述与A1相连的A链DNA的碱基序列如SEQ ID NO.1所示；与B1相连的B链DNA的碱基序列如SEQ ID NO.2所示。
SEQ ID NO.1:ATGGCAGGCTACGAA TAAC CTGGAGCCAATAAGC
SEQ ID NO.2:ATGGCAGGCTACGAATCCG CTGGAGCCAATAAGC
(b)A组和B组结构单元反应
将连有DNA的A组和B组结构单元按照1:1的摩尔比加入，pH＝6.5，浓度为100mM的磷酸缓冲液之中，得到的A组和B组结构单元浓度均为1.0μM。将该溶液加热到90℃并维持1分钟，再缓慢退火至室温，维持5分钟；加热过程共5分钟，退火过程共30分钟。此加热/退火过程即完成了A组和B组结构单元的反应，即合成得到了DNA编码的动态分子库。该分子库包含 A1与B1反应的产物，即通过A1中的醛基与B1中的氨基反应形成亚胺来连接A1和B1结构单元。
本实施例还包括从所述的含100x100个化合物的动态分子库中筛选出与模型靶点蛋白碳酸脱氢酶CA-II结合的结构单元，其筛选步骤包括：
(c)加入靶蛋白，富集与靶蛋白结合的结构单元
将合成得到的分子库进一步冷却至4℃，加入蛋白质靶点CA-II，蛋白质的最终浓度为2μM。溶液在4℃保持6小时，使体系充分达到动态平衡。
(d)平衡锁定
体系充分平衡后，加入还原剂NaBH3CN(最终浓度10mM)，保持4℃反应16小时，动态平衡生成的亚胺被还原为胺基，从而锁定平衡。
用2倍体积的40％醋酸溶液淬灭锁定平衡的还原反应，即完成了分子库对靶点蛋白CA-II的筛选过程，筛选得到的化合物能够与靶点蛋白CA-II结合，且同时带有A链和B链DNA。
(e)分离和纯化
平衡锁定之后，通过10％或15％的TBE-Urea PAGE变性凝胶电泳，将已经连在一起的A链-B链偶合物(能够与靶点蛋白CA-II结合)与独立的A链和B链进行分离(见图4)。由于凝胶电泳的强变性条件，所分离出的DNA已经为单链。在凝胶电泳结束后，将A链-B链偶合物的胶条切出，用1x PB(磷酸缓冲液)进行萃取。萃取出连在一起的A链-B链偶合物，直接用乙醇进行沉淀纯化。
(f)PCR扩增与DNA测序解码
将沉淀纯化后的A链-B链偶合物重新溶解于水中，稀释到浓度为1nM，取10μL进行PCR扩增。PCR扩增的实验步骤如下：向体系中加入5μL PCR引物(5μM)，1μL浓度为1mM的dNTP混合物，10μL 5x反应缓冲溶液以及0.5μL高保真DNA聚合酶，将PCR体系的总体积稀释到50μL。再按照92℃ 30秒，55℃ 1分钟，72℃ 30秒的反应条件循环扩增29次，然后通过3％琼脂糖凝胶电泳进行纯化分离，并进一步通过PCR纯化试剂盒提纯得到最终PCR产物。对PCR产物进行定量之后，取10ng/μL的PCR产物溶液,直接送DNA测序。
所述引物包括正向引物和反向引物，其序列分别如SEQ ID NO.3和SEQ ID NO.4所示。
正向引物序列SEQ ID NO.3：TACCG TCCGATGCTT
反向引物序列SEQ ID NO.4：GCTTATTGGC TCCAG
(h)确定所筛化合物的结构
用DNA测序仪测序的样品除上述的分子库对靶点的筛选样品之外，还将一个“筛选前”分子库样品也送样测序作为对照。该对照样品为与筛选样品经过同样的筛选过程，但是没有加入蛋白质靶点。通过对照样品来避免筛选结果中的假阳性现象，即排除非加入的靶点蛋白引起的化合物富集。
从图3可以看到，在筛选前整个分子库中，在两个编码区的DNA序列并没有显出TAAC和TCCG的序列，说明结构单元A1与B1的反应产物在分子库中并没有被富集。
经靶蛋白筛选的样品用DNA测序仪测试后发现，在两个编码区中TAAC和TCCG的序列被富集，大大超过了其它的背景序列，说明分子库中和CA-II结合的结构单元被富集，即加入靶蛋白CA-II后筛选出来的与化合物连接的A链和B链DNA编码区分别为TAAC和TCCG(见图3)，分别对应结构单元A1和B1，从而确定与靶蛋白CA-II结合的化合物结构如化学式3所示，该化合物为公知的能够与靶蛋白CA-II结合的化合物，说明本发明的筛选方法很准确。
以上数据充分证明了本发明所述DNA编码动态分子库技术的可行性。在新药研究领域，筛选出与靶点蛋白结合的化合物至关重要，本发明提供了快速、准确的动态分子库合成与筛选方法，具有重大意义。
以上通过实施例描述了本发明的具体实施方式，本领域技术人员应理解的是，上文实施例仅出于举例的目的，不应认为以此限定本发明之保护范围，本领域技术人员在不脱离本发明精神的前提下可以对其进行修改、变化或替换，但是，依照本发明所作的各种等同变化，仍属于本发明所涵盖的范围。
Figure PCTCN2016110102-appb-000004
Figure PCTCN2016110102-appb-000005

Claims (15)

  1. DNA编码的动态分子库的合成方法，其特征在于，该方法包括以下步骤：
    (a)结构单元与含有修饰基团的DNA连接，其中，每个结构单元含有至少两个活性基团，所述结构单元被分为A组结构单元和B组结构单元；
    (b)A组结构单元中的每个结构单元上的一个活性基团与B组结构单元中的每个结构单元上的一个活性基团发生可逆化学反应。
  2. 根据权利要求1所述的合成方法，其特征在于，所述修饰基团能够与结构单元中的活性基团反应。
  3. 根据权利要求1或2所述的合成方法，其特征在于，所述结构单元与含有修饰基团的DNA连接的反应包括炔基与叠氮基反应生成三氮唑官能团、醛或酮与氨反应生成亚胺、两个巯基反应生成二硫键、氨基与酰氯或羧酸反应生成酰胺键、氨基与异氰酸酯反应生成carbamate连接，或者通过N-羟基琥珀酰亚胺连接修饰基团氨基与结构单元中的活性基团氨基中的一种或两种。
  4. 根据权利要求1-3任一项所述的合成方法，其特征在于，所述结构单元与含有修饰基团的DNA连接的反应为修饰基团氨基与结构单元中的活性基团羧基反应生成酰胺键。
  5. 根据权利要求1-4任一项所述的合成方法，其特征在于，步骤(b)的可逆化学反应选自醛或酮与胺反应、两个巯基反应、亲和试剂与不饱和烯烃反应、肼与醛或酮反应和Diels-Alder反应中的一种。
  6. 根据权利要求5所述的合成方法，其特征在于，步骤(b)的反应为醛与胺反应。
  7. 根据权利要求1-6任一项所述的合成方法，其特征在于，步骤(b)的反应条件为加热至90-95℃，退火至室温。
  8. 根据权利要求7所述的合成方法，其特征在于，在90-95℃下维持1-2分钟，室温下维持5-10分钟。
  9. 根据权利要求1-8任一项所述的合成方法，其特征在于，步骤(b)的反应底物用量按照化学计量比计算。
  10. 从权利要求1-9任一项所述的合成方法制备的DNA编码的动态分子库中选出一种化合物的筛选方法，其特征在于，该方法包括以下步骤：
    (c)加入靶蛋白，与靶蛋白结合的结构单元富集；
    (d)平衡锁定；
    (e)分离和纯化；
    (f)PCR扩增，DNA测序解码；
    (g)确定筛选出来的化合物结构。
  11. 根据权利要求10所述的筛选方法，其特征在于，根据步骤(b)的反应类型确定平衡锁定方法，所述平衡锁定方法包括加入还原剂、酸、氧化剂、调节pH值，或者降低温度中的一种。
  12. 根据权利要求10或11所述的筛选方法，其特征在于，所述平衡锁定方法为加入还原剂。
  13. 根据权利要求10-12任一项所述的筛选方法，其特征在于，所述分离和纯化方法包括凝胶电泳法。
  14. 根据权利要求10-13任一项所述的筛选方法，其特征在于，根据编码化合物的DNA碱基序列来设计PCR扩增引物。
  15. 权利要求1-9任一项所述DNA编码的动态分子库的合成方法和权利要求10-14任一项所述筛选方法在药物筛选中的应用。
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