CN109023535B - Method for screening DNA coding compound by using antibody to non-recognition marker protein or cell lysate - Google Patents
Method for screening DNA coding compound by using antibody to non-recognition marker protein or cell lysate Download PDFInfo
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Abstract
The invention discloses a method for screening a DNA coding compound aiming at a protein without a recognition marker or a cell lysate by using an antibody, which comprises the following steps: (1) coating the antibody on the immunomagnetic globin G; (2) adding a solution containing target protein to the magnetic beads combined with the antibodies, and washing off substances not combined with the antibodies; (3) adding a DNA coding compound library for screening; (4) washing away unbound compounds; (5) extracting the bound compound by heating; (6) the structure of the compound was decoded by next-generation sequencing, and synthesis and subsequent functional validation were performed. The invention avoids the complicated process of purifying the protein, solves the problem that the purified protein does not contain post-translational modification, can effectively improve the screening efficiency and reduce the screening cost.
Description
Technical Field
The invention belongs to the field of drug screening, and particularly relates to a method for screening a DNA coding compound, in particular to a method for screening a DNA coding compound by using an antibody to a protein without an identification marker or a cell lysate.
Background
As a novel small molecule drug screening method, the DNA coding compound library can greatly improve the flux and speed of drug screening, reduce the cost of manpower and material resources and improve the screening efficiency. In the existing method, the purified target protein with an identification tag (tag) is usually fixed on a solid phase carrier for compound screening, so that the synthesis and purification processes of the target protein are required, the process is complicated, the time consumption is long, the cost is high, and the purified protein is difficult to have posttranslational modification under physiological conditions, so that the original protein structure and biological activity are difficult to maintain, and the success rate of obtaining effective compounds is reduced. Meanwhile, it is difficult to screen for a protein without tag or a protein with altered biological activity after purification by this method.
The invention of Chinese patent application CN201610871861.6 discloses a DNA coding molecule library and a compound screening method, which uses light energy to make a compound combined with protein and a combined target point thereof generate a cross-linking reaction so as to protect the combined compound. While unbound DNA-encoding compounds are degraded by exodnase. And finally, finding out a compound structure interacting with the target protein through sequencing and decoding. The method can be used for screening a library of compounds encoded by the DNA of the marker-free target protein. The limitations of this approach are: although this method can be used for label-free proteins, it is disadvantageous to obtain effective data because of the high amount of impurities in the cell or tissue lysate, which results in high background noise.
Therefore, there is a need to develop an alternative method for screening libraries of DNA-encoded compounds that is suitable for label-free or cell lysate (lysate) screening to overcome the above-mentioned drawbacks.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for screening a DNA coding compound of a protein or a cell lysate (lysate) without an identification marker (tag), so that a complicated process for purifying the protein is avoided, the problem that the purified protein does not contain posttranslational modification is solved, the screening efficiency can be effectively improved, and the screening cost is reduced. The invention utilizes the characteristic of high specificity of the antibody, can effectively reduce the interference of background noise and improve the success rate of screening.
In order to solve the problems in the prior art, the method utilizes the antibody to fix the target protein, and can be suitable for screening a DNA coding compound library without tag or cell lysate.
The technical scheme adopted by the invention is as follows:
a method of screening for compounds encoded by DNA directed against a protein or cell lysate free of identifying markers using an antibody comprising the steps of:
(1) coating the antibody on immunomagnetic globin G (Dynabeads protein G);
(2) adding a solution containing target protein to the magnetic beads (beads) to which the antibody has been bound, and washing away substances that have not bound to the antibody;
(3) adding a DNA coding compound library for screening;
(4) washing away unbound compounds;
(5) extracting the bound compound by heating;
(6) the structure of the compound was decoded by next-generation sequencing, and synthesis and subsequent functional validation were performed.
As a preferable technical scheme of the invention, the following steps are added before the step (1): fixing a certain amount of uniformly vortexed and oscillated immunomagnetic beads (Dynabeads) by using a magnetic frame, and removing the upper layer liquid; separating the magnetic beads from the magnetic rack; the magnetic beads were washed once with Antibody binding buffer. The antibody binding buffer comprises 10mM DPBS (Du's phosphate buffer) and 0.02% Tween20 (Tween-20).
As a preferred technical solution of the present invention, in the step (1), the antibody coating process specifically includes: diluting the Antibody with Antibody binding buffer; incubation was reversed at room temperature for 30 minutes; the mixture was placed on a magnetic stand and the supernatant was removed. The antibody binding buffer composition was 10mM DPBS, 0.02% Tween 20.
As a preferable technical scheme of the invention, the following steps are added before the step (2): seeding the cells on a culture plate to grow a uniform monolayer of cells; washing the cells twice with DPBS at 3-5 ℃; scraping the cells into the cell lysate with a scraper; cell sap was lysed for 30 min; centrifuging at 13000rpm for 10 minutes; the supernatant was collected and stored at-80 ℃.
As the preferred technical scheme of the invention, the cell lysis process needs to be operated on ice, and the centrifugation temperature is 4 ℃. The cell lysate is NP40 cell lysate, and a protease inhibitor is added before use. During lysis of the cell fluid, vortexed once every 10 minutes.
As a preferred technique of the present inventionThe scheme is characterized in that the following steps are added before the step (2): by BS3Ligation Buffer (BS)3Coupling buffer) washing antibody coupled paramagnetic proteins G (IgG-coupled Dynabeads Protein G) twice, placing the mixture on a magnetic frame for precipitation, and then removing supernatant; with 5mM BS3Resuspending immunomagnetic beads (Dynabeads) in the working solution; incubation was reversed at room temperature for 30 minutes; joining BS3Quenching Buffer (BS)3Quenching Buffer) to terminate the binding reaction; incubation was inverted at room temperature for 15 minutes; bound Dynabeads were washed three times with cell lysate, placed on a magnetic rack for precipitation, and the supernatant discarded. This step is an optional step and is not necessary.
As a preferred technical scheme of the invention, the BS3Ligation Buffer (BS)3Conjugation buffer) ingredient was 20mM Sodium Phosphate, 0.15M NaCl (Sodium chloride), pH 7-9, fresh BS was prepared before each experiment3And (4) connecting a buffer solution. The BS3Quenching Buffer (BS)3Quenching Buffer) was 1M Tris-HCl (Tris-HCl), pH 7.5. The BS3The preparation method of the working solution comprises the following steps: by BS3Ligation buffer preparation 100mM BS3The storage liquid of (3); diluted to 5mM BS with binding buffer before each experiment3250. mu.L of working solution was used for each sample.
As a preferred technical solution of the present invention, the step (2) specifically includes: washing the beads once with cell lysate; incubating the beads and the cell lysate in an inverted manner for 1 hour at room temperature, placing the cell lysate and the beads on a magnetic frame for precipitation, and then removing supernatant; washing immunomagnetic beads (Dynabeads) -antibody-antigen conjugates with DPBS three times, placing the washed antibodies on a magnetic frame for precipitation each time, then removing supernatant, and gently blowing and resuspending; the immunomagnetic beads (Dynabeads) -antibody-antigen conjugates were resuspended with DPBS and the resuspension transferred to a clean 1.5ml centrifuge tube.
As a preferred embodiment of the present invention, in step (3), the library of DNA-encoding compounds is diluted in the screening solution, and the library of DNA-encoding compounds is incubated with Dynabeads-antibody-antigen conjugates in reverse at 4 ℃ for 1 hour, and then deposited on a magnetic rack, and the supernatant is discarded. The components of the screening solution are 50mM Tris-HCl (Tris hydrochloride), 150mM NaCl (sodium chloride), 0.1% v/v Tween20 (Tween 20), 1mM DTT (dimercaptothreitol) or 10mM beta-ME (beta-mercaptoethanol), 1mg/mL Sheared fish sperm DNA (protamine DNA), 1mg/mL BSA (bovine serum albumin), and the pH is 7.5.
As a preferred technical scheme of the present invention, the cleaning process in the step (4) specifically comprises: and washing with the screening solution for five times, placing the screening solution on a magnetic frame for precipitation each time, then abandoning the supernatant, and gently blowing and resuspending the supernatant.
As a preferred embodiment of the present invention, the specific method for extracting the compound in step (5) is: the binding solution was added to the conjugate and heated at 80 ℃ for 10 minutes to elute the bound compound. After one to three rounds of the screening procedures of steps (3) to (5), the total amount of the extracted DNA-encoding compound is in the range of 1X107 to 1X 109.
As a preferable technical scheme of the invention, the following steps are added before the step (6): determining the enrichment degree and total DNA amount of the tool compound (tool compound) in the eluent by utilizing qPCR; 7-10 rounds of PCR amplification were performed to achieve total DNA amounts greater than 30ng for next generation sequencing.
Compared with the prior art, the invention has the beneficial effects that: the invention provides an effective method for screening a DNA coding compound library aiming at target proteins in tag-free or cell lysate, which utilizes an antibody to fix the target proteins, can be suitable for screening the DNA coding compound library without tag or cell lysate, avoids the complex process of purifying the proteins, solves the problem that the purified proteins do not contain post-translational modification, can screen the proteins in a physiological state and unknown binding proteins thereof, enlarges the application range of screening the DNA coding compound library, improves the screening efficiency and reduces the screening cost. The flux of drug screening is improved relative to traditional high-flux screening, and is increased from 1000 ten thousand compounds to 100 hundred million compounds, and larger structural space can be covered. Meanwhile, the screening efficiency can be improved and reduced to 3-6 months from 9-18 months. The screening cost, including the cost of manpower and material, is reduced. Compared with the Chinese patent application CN201610871861.6, the method disclosed by the patent can be used for unlabeled protein, but when the method is used for cell or tissue lysate, the background noise is too high due to a large amount of impurities in the cell or tissue lysate, and the method is not favorable for obtaining effective data. The invention utilizes the characteristic of high specificity of the antibody, can effectively reduce the interference of background noise and improve the success rate of screening.
Drawings
The invention is further illustrated with reference to the following figures and examples.
FIG. 1 is a schematic flow diagram of the process of the present invention.
FIG. 2 is a graph showing the results of qPCR in example 1 of the present invention.
FIG. 3 is a graph showing the results of data analysis of the sequencing results in example 2 of the present invention.
Detailed Description
The invention will now be described in further detail with reference to the figures and examples.
Example 1: screening for positive compounds on unlabeled IDO1 to confirm feasibility of the method
Firstly, background: indoleamine 2, 3-dioxygenase IDO1 is the tryptophan catabolism rate-limiting enzyme, and in this example the purchased label-free IDO1 was used as the target protein to validate the method.
Secondly, the implementation method comprises the following steps:
as shown in fig. 1, the method specifically includes the following steps:
firstly, preparing magnetic beads:
dynabeads magnetic beads were vortexed and shaken for 30 seconds, 20. mu.L of the beads were pipetted into a 1.5ml Eppendorf tube (microcentrifuge tube, EP tube for short), the EP tube was placed on a magnetic holder to adsorb the beads, and the supernatant was removed. The beads were washed twice with 100. mu.L Antibody binding buffer.
II, antibody binding:
mu.g of the antibody to IDO1 was diluted with 100. mu.l of the lantibiody binding buffer and incubated at room temperature for 30 minutes with inversion. The EP tube was then placed on a magnetic rack to adsorb the magnetic beads and the supernatant removed.
Thirdly, the bound Dynabeads were washed twice with 200. mu.L Antibody binding buffer, settled on a magnetic rack and the supernatant discarded.
And fourthly, washing the combined Dynabeads once by using the screening solution, placing the Dynabeads on a magnetic frame for precipitation, and then discarding the supernatant. The components of the screening solution are 50mM Tris-HCl (Tris hydrochloride), 150mM NaCl (sodium chloride), 0.1% v/v Tween20 (Tween 20), 1mM DTT (dimercaptothreitol) or 10mM beta-ME (beta-mercaptoethanol), 1mg/mL Sheared fish sperm DNA (protamine DNA), 1mg/mL BSA (bovine serum albumin), and the pH is 7.5.
Fifth, 10pmol of IDO1 was added to 100. mu.L of the selection solution to prepare 0.1. mu.M IDO1 solution
Sixthly, the magnetic beads and the IDO1 solution are incubated for 1 hour in an inverted way at room temperature, placed on a magnetic rack for precipitation, and then the supernatant is discarded
And seventhly, washing the Dynabeads-antibody-antigen conjugate by using 200 mu L of screening solution for three times, placing the Dynabeads-antibody-antigen conjugate on a magnetic frame for precipitation each time, then removing supernatant, and gently blowing and resuspending.
Eighthly, 2nmol of a DNA-encoding compound pool (DEL) containing about 50000 compounds and 4pmol of a positive compound (IDO-IN-4) ligated with DNA were diluted IN 100. mu.L of the selection solution.
Nine, add DEL solution to Dynabeads-antibody-antigen conjugate, incubate in reverse at room temperature for 1 hour, place on magnetic rack for precipitation and discard supernatant.
And ten times of washing by 200 mu L of screening solution, placing the screening solution on a magnetic frame for precipitation each time, then abandoning supernatant, and gently blowing and resuspending.
Eleventh, 100. mu.L of the screening solution was added to the conjugate, and the conjugate was heated at 80 ℃ for 10 minutes to elute the bound compound.
And twelfth, reserving the eluent.
Thirteen, after each round of screening, about 3 μ L of eluate was retained, diluted 10-fold and used for qPCR to determine the enrichment degree and total DNA amount of the positive compound IDO-IN-4.
Fourteen, through one to three rounds of screening process, the total amount of extracted DNA coding compound is 1X107To 1X109Within the range.
The products after each round of screening were used to determine the enrichment degree of positive compounds by qPCR. The qPCR results are schematically shown in figure 2. FIG. 2 shows the degree of enrichment of IDO-IN-4 relative to compounds IN the pool of other DNA-encoding compounds after two rounds of screening, 100:1 before screening, one round of screening increasing to 1000: 1, after two rounds of screening, the screening is promoted to 1600:1, and after three rounds of screening, the screening reaches 2400:1, and the standard of being more than 2000:1 is reached, so that the screening is considered to be successful so far.
Example 2: screening of pools of DNA-encoded compounds for LAG3 expressed in cells
Firstly, background: LAG3 is an immune checkpoint inhibitory receptor expressed on the surface of T cells. There are no known small molecule compounds that can bind, and no intracellular domain binding proteins are known. Human LAG3 is a transmembrane protein and is therefore difficult to express and purify in bacterial or insect cells while retaining its physiological structure and activity. This method allows LAG3 and its associated proteins to be isolated from cells and a library of DNA encoding compounds to be screened against this complete.
Secondly, the implementation method comprises the following steps:
as shown in fig. 1, the method specifically includes the following steps:
1. preparation of cell lysis samples:
human T cells expressing LAG3 were seeded into suspension cell culture flasks and allowed to grow to saturation. Centrifugation was carried out at 800rpm for 10 minutes, the supernatant was discarded, and the cells were washed twice with 10ml of DPBS at 3-5 degrees Celsius, centrifuged, and the DPBS was discarded. The cells were placed on ice, 500ul of cell lysate was added, and after standing for 10 minutes, the lysate was pipetted into a 1.5ml Eppendorf tube, lysed on ice at 4 ℃ for 30 minutes, and vortexed once every 10 minutes. Centrifuge at 13000rpm for 10 minutes at 4 ℃. The supernatant was collected and stored at-80 ℃.
2. Preparing magnetic beads:
dynabeads magnetic beads were vortexed for 30 seconds, 20. mu.L of the vortexed vortex. The beads were washed twice with 100. mu.L of antibody binding buffer.
3. Antibody binding:
mu.g of LAG3 antibody was diluted with 100. mu.L of antibody coupling buffer and incubated at room temperature for 30 minutes in reverse. The EP tube was then placed on a magnetic rack to adsorb the magnetic beads and the supernatant removed.
4. By BS3Ligation Buffer (BS)3Coupling buffer) 100mM BS was prepared3The storage liquid of (3); BS was used before each experiment3Ligation buffer diluted to 5mM BS3250. mu.L of working solution was used for each sample.
5. By BS3IgG-conjugated Dynabeads Protein G was washed twice with the ligation buffer, placed on a magnetic rack for precipitation, and the supernatant was discarded.
6. With 250. mu.L of 5mM BS3The working solution was resuspended in Dynabeads and incubated for 30 minutes at room temperature with inversion.
7. Add 12.5. mu.L BS3The binding reaction was stopped with quench Buffer (Quenching Buffer) and incubated at room temperature for 15 minutes with inversion.
8. Bound Dynabeads were washed three times with cell lysate, placed on a magnetic rack for precipitation, and the supernatant discarded.
9. The beads were washed once with cell lysate.
10. The beads were incubated with the cell lysate in reverse at room temperature for 1 hour, placed on a magnetic rack for sedimentation and the supernatant discarded (100. mu.L of sample was retained).
11. Dynabeads-Ab-Ag conjugate was washed three times with 200. mu.L DPBS, each time placed on a magnetic rack for precipitation and the supernatant discarded, and resuspended by gentle pipetting.
12. Dynabeads-Ab-Ag conjugates were resuspended with DPBS and the resuspension was transferred to a clean Eppendorf tube.
13.2nmol of the DNA coding compound library (DEL) diluted in 100. mu.L of the selection medium.
DEL was incubated with Dynabeads-Ab-Ag conjugate in reverse at room temperature for 1 hour, placed on a magnetic rack to precipitate and the supernatant discarded.
15. Washing with 200 μ L of screening solution five times, depositing on a magnetic rack each time, discarding supernatant, and gently blowing and resuspending.
16. To the conjugate, 100. mu.L of the screening solution was added, and the conjugate was eluted by heating at 80 ℃ for 10 minutes.
17. The eluent is retained.
18. After each round of screening, about 3 μ L of the eluate was retained, diluted 10-fold and used for qPCR to determine the total DNA amount.
19. After one to three rounds of screening, the total amount of the extracted DNA-encoding compound was 1X107To 1X109Within the range.
20. 7-10 rounds of PCR amplification were performed to achieve total DNA amounts greater than 30ng for next generation sequencing.
21. FIG. 3 is a schematic representation of the results of the second generation sequencing, each axis representing a DNA label, with three different DNA labels per compound. Each dot in the graph represents a compound screened out, and the size of the dot represents the degree of enrichment of the compound in the product. The most enriched compound structures were selected from fig. 3 and subjected to separate synthesis and subsequent functional validation.
The above is only a specific application example of the present invention, and the protection scope of the present invention is not limited in any way; other variations and modifications will be apparent to persons skilled in the art in light of the above description. All embodiments need not be described or illustrated herein. The technical solutions similar to the above embodiments formed by equivalent transformation or equivalent replacement fall within the scope of the claims of the present invention.
Claims (12)
1. A method for screening a compound encoded by DNA directed against a protein or cell lysate free of a recognition marker with an antibody, comprising the steps of:
(1) coating the antibody on the immunomagnetic globin G;
(2) adding a solution containing target protein to the magnetic beads combined with the antibodies, and washing off substances not combined with the antibodies;
(3) adding a DNA coding compound library for screening; diluting the library of DNA-encoding compounds in step (3) in a selection solution comprising 50mM Tris-HCl, 150mM NaCl, 0.1% v/v Tween20, 1mM DTT or 10mM β -ME, 1mg/mL protamine DNA, 1mg/mL BSA, pH 7.5;
(4) washing away unbound compounds;
(5) extracting the bound compound by heating;
(6) the structure of the compound was decoded by next-generation sequencing, and synthesis and subsequent functional validation were performed.
2. The method of claim 1, wherein the following steps are added before step (1): fixing a certain amount of uniformly mixed immunomagnetic beads by using a magnetic frame, and removing upper-layer liquid; separating the magnetic beads from the magnetic rack; washing the magnetic beads once with antibody binding buffer; the antibody binding buffer comprises 10mM DPBS and 0.02% Tween 20.
3. The method according to claim 1, wherein the antibody coating process in step (1) comprises in particular: diluting the antibody with an antibody binding buffer; incubation was reversed at room temperature for 30 minutes; placing on a magnetic frame, and removing supernatant; the antibody binding buffer composition was 10mM DPBS, 0.02% Tween 20.
4. The method of claim 1, wherein the following steps are added before step (2): seeding the cells on a culture plate to grow a uniform monolayer of cells; washing the cells twice with DPBS at 3-5 ℃; scraping the cells into the cell lysate with a scraper; cell sap was lysed for 30 min; centrifuging at 13000rpm for 10 minutes; the supernatant was collected and stored at-80 ℃.
5. The method of claim 4, wherein the cell lysis process is performed on ice at a centrifugation temperature of 4 degrees Celsius; the cell lysate is NP40 cell lysate, and a protease inhibitor is added before use; during lysis of the cell fluid, vortexed once every 10 minutes.
6. The method of claim 1, wherein the following steps are added before step (2): by BS3Washing the antibody-coupled magnetoferritin G twice with a connection buffer solution, placing the antibody-coupled magnetoferritin G on a magnetic frame for precipitation, and then discarding the supernatant; with 5mM BS3Working solution resuspension immunomagnetic beads(ii) a Incubation was reversed at room temperature for 30 minutes; joining BS3Quenching the buffer solution to stop the binding reaction; incubation was inverted at room temperature for 15 minutes; and washing the combined immunomagnetic beads three times by using cell lysis solution, placing the washed immunomagnetic beads on a magnetic frame for precipitation, and then discarding the supernatant.
7. The method of claim 6, wherein the BS is configured as a base station3The ligation buffer was 20mM sodium phosphate, 0.15M NaCl, pH 7-9, and fresh BS was prepared before each experiment3Connecting a buffer solution; the BS3The quenching buffer solution is 1M Tris-HCl, and the pH value is 7.5; the BS3The preparation method of the working solution comprises the following steps: by BS3Ligation buffer preparation 100mM BS3The storage liquid of (3); diluted to 5mM BS with binding buffer before each experiment3And (4) 250 muL of working solution is needed for each sample.
8. The method according to claim 1, characterized in that in step (2) comprises in particular: washing the magnetic beads once by using cell lysis solution; the magnetic beads and the cell lysate are incubated for 1 hour in an inverted mode at room temperature, placed on a magnetic frame for precipitation, and then the supernatant is discarded; cleaning the immunomagnetic beads-antibodies-antigen conjugates with DPBS three times, placing the conjugates on a magnetic frame for precipitation each time, then discarding the supernatant, and gently blowing and resuspending the conjugates; the immunomagnetic beads-antibody-antigen conjugate was resuspended with DPBS and the resuspension transferred to a clean 1.5ml centrifuge tube.
9. The method of claim 1, wherein in step (3) the library of DNA-encoding compounds is diluted in the screening solution, the library of DNA-encoding compounds is incubated with the immunomagnetic bead-antibody-antigen conjugate in reverse at 4 degrees celsius for 1 hour, placed on a magnetic rack for sedimentation, and the supernatant is discarded.
10. The method according to claim 1, wherein the cleaning process in step (4) is specifically: and washing with the screening solution for five times, placing the screening solution on a magnetic frame for precipitation each time, then abandoning the supernatant, and gently blowing and resuspending the supernatant.
11. As claimed in claimThe method according to claim 1, wherein the specific method for extracting the compound in the step (5) is: adding the screening solution into the conjugate, and heating at 80 ℃ for 10 minutes to elute the conjugated compound; after one to three rounds of screening processes of steps (3) - (5), the total amount of the extracted DNA coding compound is 1X107To 1X109Within the range.
12. The method of claim 1, wherein the following steps are added before step (6): determining the enrichment degree and total DNA amount of the tool compound in the eluent by utilizing qPCR; 7-10 rounds of PCR amplification were performed to achieve total DNA amounts greater than 30ng for next generation sequencing.
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