CN111733459A - DNA coding compound library and screening method thereof - Google Patents
DNA coding compound library and screening method thereof Download PDFInfo
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- CN111733459A CN111733459A CN202010650666.7A CN202010650666A CN111733459A CN 111733459 A CN111733459 A CN 111733459A CN 202010650666 A CN202010650666 A CN 202010650666A CN 111733459 A CN111733459 A CN 111733459A
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 52
- 238000012216 screening Methods 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 17
- 108020004414 DNA Proteins 0.000 claims abstract description 46
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 17
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 17
- 238000012163 sequencing technique Methods 0.000 claims abstract description 11
- 108010058683 Immobilized Proteins Proteins 0.000 claims abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 4
- 238000004458 analytical method Methods 0.000 claims abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims abstract description 4
- 239000011324 bead Substances 0.000 claims description 49
- 239000007788 liquid Substances 0.000 claims description 15
- 239000011534 wash buffer Substances 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 9
- 239000012131 assay buffer Substances 0.000 claims description 8
- 239000000872 buffer Substances 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000011529 RT qPCR Methods 0.000 claims description 5
- 238000011002 quantification Methods 0.000 claims description 5
- 239000008176 lyophilized powder Substances 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- 238000001962 electrophoresis Methods 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 claims description 2
- 241000972773 Aulopiformes Species 0.000 claims description 2
- 102100036227 Cysteine and histidine-rich protein 1 Human genes 0.000 claims description 2
- 101000947448 Homo sapiens Cysteine and histidine-rich protein 1 Proteins 0.000 claims description 2
- 230000003321 amplification Effects 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 claims description 2
- 239000003480 eluent Substances 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 claims description 2
- 235000019515 salmon Nutrition 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 239000002699 waste material Substances 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- 238000009509 drug development Methods 0.000 description 5
- 239000002547 new drug Substances 0.000 description 4
- 150000003384 small molecules Chemical class 0.000 description 4
- 238000001712 DNA sequencing Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000013537 high throughput screening Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 238000012917 library technology Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000011176 pooling Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
- C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B30/00—Methods of screening libraries
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Abstract
The invention provides a standardized DNA coding compound library system with convenient operation, which adds a DNA sequence to mark reaction in the process of synthesizing a compound, realizes unique coding of each expected compound product molecule, contains a matched standardized reagent and can realize that the screening of a target compound can be completed locally by a user. The invention also provides a standardized operation process and a method for screening target targets by using the DNA coding compound library, which comprises the steps of selective combination of immobilized protein and DNA coding compounds, PCR, sequencing, DNA decoding information analysis and the like, and is suitable for screening the seedling-end compounds of the hydrophilic target protein.
Description
Technical Field
The invention relates to the technical field of DNA coding molecule libraries, in particular to a DNA coding molecule library and a screening method of compounds of the DNA coding molecule library.
Background
In the research and development of modern drugs, high-throughput and large-scale screening is an indispensable means in the research and development of new drugs by constructing a large candidate drug molecule library aiming at the drug target of diseases. Major pharmaceutical companies in the world today have large libraries of molecules and large screening platforms for new drug development. However, the traditional molecular library and screening platform have high cost, high technical threshold and complex management and operation, and become serious restrictions in the development and application of high-throughput screening.
In recent years, the technology of DNA encoding molecule libraries has been developed gradually, and this has become an emerging screening method in drug development. In the DNA coding molecule library, each compound is connected with a specific DNA chain to form a specific bar code, so that the specific coding of the compound is realized. The DNA coding molecule library can realize high-throughput screening of tens of millions or hundreds of millions in a very small system. The screening result can be decoded and analyzed by PCR amplification and DNA sequencing, and lead compounds are obtained for further drug development. In recent years, DNA coding molecule libraries have been widely recognized and applied in the field of new drug development, and become an important support technology in new drug development.
Drug screening is performed by using a DNA encoding molecular library, and most of the used targets are purified proteins. After modification, the protein target is immobilized on a solid phase such as magnetic beads, and then incubated with a library of molecules. Small molecules that cannot bind to the target protein are eluted and separated from small molecules that bind to the protein target. And eluting the combined small molecules under the protein denaturation condition, performing PCR amplification and DNA sequencing so as to read the coding sequence and obtain the chemical structure of the small molecules combined with the target.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a standardized and optimized convenient DNA coding molecular library system and a standardized method for screening compounds. By using the DNA coding molecular library system and the screening method, users can screen target compounds in local small laboratories, namely, the DNA coding molecular library technology can screen the target compounds better and more efficiently and screen and analyze the target compounds.
The invention provides the following technical scheme:
the present invention provides a library of DNA-encoded compounds, wherein each desired compound product molecule is uniquely encoded, characterized in that: the reaction is labeled by adding a DNA sequence during the synthesis of the compound to uniquely encode each desired product molecule of the compound.
Further, the DNA of the present invention encodes a library of compounds comprising about 1 to 5 hundred million molecules of compound per library-of-tubes reagent.
Further, the DNA coding compound library of the invention is prepared into freeze-dried powder.
Further, the DNA of the present invention encodes a library of compounds suitable for use in the screening of the head-of-shoot compounds for hydrophilic target proteins.
In another aspect, the invention provides a kit comprising a library of DNA encoding compounds according to any one of the preceding claims.
Furthermore, the kit of the invention also comprises magnetic beads, 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt (CHAPS), salmon sperm DNA, amplification primers and other reagents.
The kit can be implemented locally by a user, such as a small-sized or general-condition laboratory, and can conveniently realize the screening of the target compound. Preferably, the screening for the head of the hydrophilic target protein is performed.
In another aspect, the present invention provides a method of screening a library of standardised optimised DNA encoding molecules characterised in that it is carried out using a library of DNA encoding compounds according to any one of the above described invention or using a kit according to any one of the above described methods.
Further, the method for screening a library of DNA-encoding molecules of the present invention comprises the steps of:
(1): magnetic bead pretreatment: removing ethanol from the magnetic beads, Washing the magnetic beads by using PBS buffer solution, and resuspending the magnetic beads by Washing buffer/Assaybuffer;
(2): protein immobilization: adding the protein with the His tag into a magnetic bead tube, and mixing at low temperature to finish immobilization;
(3): the library of DNA-encoding compounds selectively binds: dissolving DNA coding compound library freeze-dried powder in a Selectionbuffer or an assoy buffer, adding a proper amount of sssDNA if the assoy buffer is selected, adding a magnetic bead waste liquid containing immobilized protein, adding the DNA coding compound solution into magnetic beads containing the immobilized protein, and mixing at low temperature;
(4): washing, separating magnetic beads, discarding a liquid part, resuspending the magnetic beads by Washing buffer, uniformly mixing, separating the magnetic beads and discarding the liquid, and repeatedly Washing for 2-3 times;
(5): and (3) elution: washing buffer resuspends the magnetic beads, heating the magnetic beads in a water bath at about 95 ℃ for about 20 minutes, centrifuging the magnetic beads for a short time, and separating the magnetic beads;
(6): adding the eluent into magnetic beads containing immobilized protein, repeating the steps (3) - (5), and repeating the screening for 2-3 rounds;
(7): the collected liquid is used for qPCR quantification and PCR library building;
(8): DNA electrophoresis detection, DNA recovery and sequencing;
(9): DNA decoding and information analysis;
the Washing buffer/Assay buffer comprises about 50mM PBS, about 0.5mM CHARPs, about 10mM midazoles; the Selection buffer comprises about 50mM PBS, about 0.5mM CHRPs, about 10mM imidazole, about 0.1mg/ml sssDNA.
Further, in the screening method of the present invention, the low temperature mixing in step (3) specifically comprises: mix on 3D rotator in a refrigerator at 4 ℃ for about 60 minutes.
Detailed Description
In order to make the purpose, technical solution and technical effect of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments obtained by a person of ordinary skill in the art without any inventive step in connection with the embodiments of the present invention shall fall within the scope of protection of the present invention.
Example 1: DNA coding compound freeze-dried powder
The reaction is labeled by adding a DNA sequence during the synthesis of the compound to achieve a unique code for each desired compound product molecule. The library reagents contain about 1-5 million molecules of compound per tube and provide three package sizes. The lyophilized powder of the DNA coding compound of the invention is obtained by preparing lyophilized powder by using the conventional technology in the field, and is suitable for screening the head of seedling compound of the hydrophilic target protein.
Example 2: target screening
1. Magnetic bead pretreatment
Adding 1mg magnetic beads into a 1.5ml EP tube, removing ethanol by a magnetic frame, adding 1ml PBS buffer, mixing uniformly, adsorbing by the magnetic frame, discarding the solution, and repeatedly washing for three times. 600ul Washing buffer/Assay buffer resuspend the beads in quadruplicates, 300ul (for protein immobilization) and 100ul × 3 (bead control), respectively.
2. Immobilized protein
About 80ug (or calculated according to the ratio of beads to protein) of protein with His tag is added into 300ul magnetic bead tube, and mixed for 30 minutes in 3D rotary instrument in refrigerator at 4 deg.C, to complete immobilization (immobilization condition can be adjusted according to actual conditions). The magnetic rack separates the magnetic beads and the liquid QC. The magnetic beads were resuspended in 500ul Washing buffer/Assay buffer, mixed, magnetically mounted, washed three times repeatedly for QC, finally resuspended in 300ul Washing buffer/Assay buffer, divided into 100ul × 3 groups, and placed on ice for use.
T-DELs selective binding
1 count
The DNA Encoded Chemical Libraries lyophilized powder was dissolved in 100ul of Selectionbuffer or assay buffer (plus 0.1mg/ml sssDNA). 100ul of immobilized protein-containing magnetic beads and 100ul of magnetic bead controls were loaded on a magnetic stand, the solution was discarded, 100ul of DNA Encoded Chemical Libraries solution was added, and mixed in a 3D rotator in a refrigerator at 4 ℃ for 60 minutes (conditions can be adjusted according to actual conditions).
4. Washing machine
The beads were separated by means of a magnetic stand and the liquid fraction was discarded. Resuspending the magnetic beads in 500ul Washing buffer, mixing, adsorbing by a magnetic frame, discarding the solution, and repeatedly Washing three times.
5. Elution is carried out
100ul Washing buffer resuspended beads and heated in a water bath at 95 ℃ for 20 minutes. And centrifuging for 5 seconds by using a palm centrifuge, adsorbing the magnetic beads by using a magnetic rack, and respectively collecting the liquid parts QC of the sample and the control group.
sssDNA was added to each of the two sets of fluids to a final concentration of 0.1 mg/ml).
6. Repeating two rounds of screening
The second round repeats: respectively taking another tube of the previously prepared 100ul immobilized protein and 100ul magnetic beads for comparison, adsorbing the magnetic beads by a magnetic frame, discarding the liquid part, correspondingly adding 100ul of the two groups of liquid collected in the step 5, and repeating the steps 3-5.
The third round repeats: the two liquid groups collected in the second round were repeated as in the second round. The final wash was performed with 500PBS and 50ul PBS as elution buffer, and the resuspension was performed as in step 5. The collected liquid was used for qPCR quantification and PCR pooling.
7.qPCR
The eluates from each round of screening were collected for qPCR quantification and grouped as detailed in the table below:
the qPCR program was proposed: 3min at 98 ℃; (98 ℃ for 20s, 68 ℃ for 20s) for 30 cycles; 10min at 72 ℃; storing at 12 deg.C.
8. Building warehouse
Library primers were synthesized according to the sequencing end sequences provided by the selected sequencing supplier.
Primer sequence conserved sequence at two ends of DNA code + sequencing end internal sequencing sequence
PCR procedure: same as above
DNA electrophoresis detection
Taking 5ul PCR product and DNA ladder sample, electrophoretic separation
Staining nucleic acid, and taking pictures
10. Glue recovery
According to the kit, the sequencing gel is recovered, and the sequencing gel can be recovered to an EP tube (Ultra Pure)
DNA sequencing
Based on the compound quantification results or PCR results obtained after three rounds of screening, multiplication by 300 times the sequencing depth is the amount of data suggested for sequencing, typically 1-10G.
12. Sample preservation
Freezing at-80 deg.C
DNA decoding and information analysis.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (9)
1. A library of DNA-encoded compounds, wherein each desired compound product molecule is uniquely encoded, characterized in that: the reaction is labeled by adding a DNA sequence during the synthesis of the compound to uniquely encode each desired product molecule of the compound.
2. The library of DNA encoding compounds of claim 1, wherein: the library reagent contains about 1 to 5 hundred million molecules of the compound per tube.
3. The library of DNA encoding compounds of claim 1, prepared as a lyophilized powder.
4. The library of DNA-encoding compounds of claim 1, which is suitable for use in the screening of hydrophilic target proteins for head-of-line compounds.
5. A kit comprising a library of DNA encoding compounds according to any one of claims 1 to 4.
6. The kit of claim 5, further comprising magnetic beads, 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonate inner salt (CHAPS), salmon sperm DNA, amplification primers and other reagents.
7. A method of screening a library of DNA-encoding molecules using a library of DNA-encoding compounds according to any one of claims 1 to 4 or using a kit according to claim 5 or 6.
8. The screening method according to claim 7, comprising the steps of:
(1): magnetic bead pretreatment: removing ethanol from the magnetic beads, Washing the magnetic beads by using PBS buffer solution, and resuspending the magnetic beads by using Washing buffer/Assay buffer;
(2): protein immobilization: adding the protein with the His tag into a magnetic bead tube, and mixing at low temperature to finish immobilization;
(3): the library of DNA-encoding compounds selectively binds: dissolving DNA coding compound library freeze-dried powder in a Selection buffer or an assoy buffer, adding a proper amount of sssDNA if the assoy buffer is selected, adding a magnetic bead waste liquid containing immobilized protein, adding the DNA coding compound solution into the magnetic bead containing the immobilized protein, and mixing at a low temperature;
(4): washing, separating magnetic beads, discarding a liquid part, resuspending the magnetic beads by Washing buffer, uniformly mixing, separating the magnetic beads and discarding the liquid, and repeatedly Washing for 2-3 times;
(5): and (3) elution: washing buffer resuspends the magnetic beads, heating the magnetic beads in a water bath at about 95 ℃ for about 20 minutes, centrifuging the magnetic beads for a short time, and separating the magnetic beads;
(6): adding the eluent into magnetic beads containing immobilized protein, repeating the steps (3) - (5), and repeating the screening for 2-3 rounds;
(7): the collected liquid is used for qPCR quantification and PCR library building;
(8): DNA electrophoresis detection, DNA recovery and sequencing;
(9): DNA decoding and information analysis;
the Washing buffer/Assay buffer comprises about 50mM PBS, about 0.5mM CHARPs, about 10mM midazoles; the Selection buffer comprises about 50mM PBS, about 0.5mM CHRPs, about 10mM Mimidazole, about 0.1mg/ml sssDNA.
9. The screening method according to claim 8, wherein: the low-temperature mixing in the step (3) is specifically as follows: mix on 3D rotator in a refrigerator at 4 ℃ for about 60 minutes.
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| CN202010650666.7A CN111733459A (en) | 2020-07-08 | 2020-07-08 | DNA coding compound library and screening method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN113403690A (en) * | 2021-06-21 | 2021-09-17 | 吉林大学 | DNA coding compound library drug molecule fishing method |
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