CN114764089A - Method for identifying operable cut DNA coding seedling head compound - Google Patents
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Abstract
The invention relates to an identification method of operable cut DNA coding head compounds, which comprises the steps of obtaining DNA head compounds through screening analysis according to a DNA coding compound library, carrying out the re-synthesis of potential head compounds on DNA with an operable cut group according to a synthesis strategy of the DNA coding compound library, releasing the head compounds under specific conditions, carrying out affinity incubation with a target spot, and carrying out molecular identification on eluted substances by utilizing mass spectrometry, thereby accurately identifying the head compounds. The invention can realize the accurate and rapid discovery of DNA coding head compounds, and avoid the influence of DNA by operating a cutting group.
Description
Technical Field
The invention belongs to the field of drug screening, and particularly relates to a method for identifying operably cut DNA coding head compounds based on liquid chromatography-mass spectrometry.
Background
DNA Encoded Library (DEL) is gradually developing as an important driving force for drug discovery as an important means for discovery of novel drug lead compounds. DEL has advantages in that it can efficiently complete the construction and screening of molecular libraries in the billion or even billion levels in a short time, and exhibits time and cost advantages in new drug discovery (WO2005058479, WO2018166532, CN 103882532).
Conventionally, the DNA coding seedling-end compound is determined by calculating and screening an enrichment value based on sequencing data by bioinformatics, and then referring to synthesis history and corresponding combination information of corresponding molecular building blocks, so that the high-reliability target binding DNA coding seedling-end compound is determined. However, the synthesis of DELs by combinatorial chemistry yields large numbers of molecules in a short time, and the residues of intermediates and by-product formation in the process cannot be completely purified away after each step of synthesis, so that the end product of the DEL contains all of the molecules generated by the synthetic route. These molecules have the same probability of binding to the target protein during incubation with the target protein. Therefore, the DNA ends of the intermediate products and by-products in the DEL synthesis can still obtain complete DNA strand information through ligation, resulting in that the final product, intermediate product and by-product of a DEL molecule in the synthetic route are labeled with the same DNA sequence information, and the structure of a compound bound to a target protein cannot be precisely located after screening, sequencing and data analysis are finished.
Disclosure of Invention
The invention provides an operable cut-off DNA coding seedling head compound identification method, which can accurately identify small molecular seedling head compounds combined with target protein and avoid the influence of the existence of DNA on the reliability and authenticity of a final detection result.
The invention provides a method for identifying a head-of-shoot compound encoded by operable cleaved DNA, comprising the steps of:
(1) synthesis of DNA-encoded head-of-shoot compounds with cleavable groups: screening data signals aiming at a target protein DNA coding compound library, sequencing the data signals according to the enrichment degree from high to low, determining the molecular sequence information and the synthesis path of potential DNA coding seedling compounds, and synthesizing all potential DNA coding seedling compounds on DNA with an operable cutting group by using the synthesis path;
(2) release of shoot head compound: cutting the DNA coding sprout compound with the cleavable group, and filtering out the DNA part through a filter to obtain a sprout compound without DNA;
(3) preparation of sample set: preparing a pre-screening sample of the DNA-free shoot head compound by using a screening buffer solution; the experimental group incubates the sample before screening, the target protein and the magnetic beads for fixing the protein together; incubating the sample before screening with the magnetic beads for fixing the protein by the reference group; respectively eluting the experimental group and the reference group by using an elution buffer solution, and respectively collecting eluates as an experimental group elution sample and a reference group elution sample;
(4) structural identification by mass spectrometry: determining the molecular weight of a test sample by adopting a high performance liquid chromatography-mass spectrometry combined system, wherein the test sample comprises a sample before screening, an experimental group elution sample and a reference group elution sample; all product molecular weights found in the sample before screening are respectively extracted and compared in the mass spectra of the experimental group elution sample and the reference group elution sample, and if the ratio of the signal-to-noise ratio of the extracted ion intensity of a certain molecular weight in the experimental group elution sample divided by the signal-to-noise ratio of the extracted ion intensity of the molecular weight in the reference group elution sample is more than 1, the molecule is judged to be a shoot compound combined with the target protein; wherein the signal-to-noise ratio is the extracted ion intensity divided by the background ion intensity.
Further, the structure of the DNA coding the seedling-end compound on the DNA with the operable cutting group is as follows:
DNA-L-A-M
wherein,
the DNA in the structural formula comprises a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers, and the nucleotide chain is connected with L through one or more chemical bonds or groups;
l is a connecting chain or nothing;
a is an operable cleavage group;
m is a shoot apex compound moiety.
Still further, the operable cleavable group is:
more specifically, the DNA on the DNA with the operable cleavage group encodes a shoot head compound having the structure:
further specifically, the cleavage operation of the step (2) is cleavage of an operable cleavage group by light irradiation. Preferably, the illumination wavelength is 306-400 nm.
Further, the specific steps of synthesizing the potential DNA-encoded shoot apex compound on the DNA with the operable cleavage group in step (1) include: carrying out chemical reaction assembly on reaction building blocks of all dimensions and DNA in sequence according to a synthesis route, a post-treatment operation and a purification mode which are followed by the shoot head compound during library synthesis; after the reaction of each dimension is finished, carrying out alcohol precipitation operation, and then putting all reaction products into the circulation reaction of the next dimension.
Furthermore, the alcohol precipitation operation is to add 10% of sodium chloride solution with the volume ratio of 5.0mol/L and 3 times of absolute ethyl alcohol in sequence, then to freeze for 40 minutes below minus 20 ℃, and then to carry out centrifugation and supernatant removal operation.
More specifically, the DNA-encoded miaow compound synthesized in step (1) is subjected to an alcohol precipitation operation, then filtered through a filter and lyophilized.
Further, step (1) further comprises: after obtaining the DNA coding the head compound, the yield condition of the target product synthesized on the DNA and the condition of the by-product are analyzed by liquid chromatography-mass spectrometry. And determining the molecular weight and purity information of all intermediate products, byproducts and target products contained in the reaction route according to a mass spectrometric detection verification report for synthesizing all potential DNA-coded miaow compounds on the DNA with the operable cutting group.
Further, step (2) further comprises: after the cleavage operation, the conversion rate in the cleavage step was obtained by liquid chromatography-mass spectrometry analysis.
Further, the concentration of the shoot apex compound containing no DNA in step (2) is calculated from the concentration and purity of the DNA-encoding shoot apex compound before cleavage and the cleavage conversion rate. That is, the concentration of the shoot apex compound is obtained by multiplying the concentration of DNA before cleavage by the purity of the shoot apex compound having an operable cleavage cluster in the sample before cleavage and by the cleavage conversion rate.
Furthermore, the amount of the target protein in the experimental group in the step (3) is 70-140 pmol, and the amount of the DNA-free shoot head compound in the sample before screening is 1000 pmol.
Further, the step (3) specifically comprises: dissolving target protein and a sample before screening in a screening buffer solution in the experimental group, wherein the final concentration of the target protein is 0.7-1.4 mu M; and rotationally incubating at room temperature for 1 hour, adding the protein into the target protein affinity coupled immunomagnetic beads for protein fixation, and fixing at room temperature for 30 minutes.
Further, the step (3) specifically includes: in the reference group, a sample before screening is dissolved by using a screening buffer solution, the final concentration of a DNA-free seedling-end compound is 10 mu M, then target protein affinity coupled immunomagnetic beads are added, and the mixture is fixed for 30 minutes at room temperature.
Further, 65. mu.L of each elution buffer was used in step (3).
Further, the elution buffer in step (3) has the following components: 1)50mM HEPES pH 7.5,300mM NaCl; or 2)40mM Tris,300mM NaCl, pH 8.0; 3) methanol.
Further, the elution operation in the step (3) is: after removal of the supernatant, incubation with elution buffer was performed for 1min, the supernatant was removed, and elution buffer was added to dissociate at 95 ℃ for 10 min.
Further, the high performance liquid chromatography-mass spectrometry system in the step (4) specifically includes: setting the sample injection volume of the liquid chromatogram to be 10 mu L, the detection time to be 6 minutes, and setting the mass spectrum to be in a full scanning mode; and carrying out sample injection setting according to the sequence of the sample before screening, the experimental group elution sample and the reference group elution sample.
Further, the step (4) specifically includes: the structural assignment of a molecular weight molecule was determined by comparison of the major and minor analyses in the synthesis of DNA-encoded headpiece compounds with cleavable groups.
The method comprises the following steps:
(1) synthesis of DNA-encoded head-of-shoot compounds with cleavable groups: screening data signals aiming at a target protein DNA coding compound library, sequencing according to the enrichment degree from high to low, determining the molecular sequence information and the synthesis path of potential DNA coding seedling compounds, and synthesizing all potential DNA coding seedling compounds on DNA with an operable cutting group by using the synthesis path;
(2) release of shoot-head compound: cutting off the DNA coding seedling head compound with the cleavable group, and filtering out corresponding DNA through a filter to obtain a seedling head compound without DNA;
(3) preparation of sample set: a final concentration of 10. mu.M of the DNA-free heading compound and 70pmol of the target protein (no target protein added to the reference group) were placed in a total volume of 50. mu.L of a selection buffer (50mM Tris, 150mM NaCl, 1mM TCEP, 0.01% Tween20, pH 8.0) in a 1.5mL centrifuge tube, and the centrifuge tube was placed on a rotary mixer and incubated at 25 ℃ for 60min at 20 rpm. Then 20 mul of magnetic beads after 3 times and 150 mul of screening buffer solution are put into the solution after the micromolecules and the target protein are incubated, the centrifugal tube is placed on a rotary mixer and incubated for 30min at the temperature of 25 ℃, and the target protein and the complex of the target protein and the DNA-free seedling head compound combined by the target protein are separated from the solution by a magnetic frame through the affinity effect of the target protein label and the specific magnetic beads. The separated magnetic beads were resuspended in 50. mu.L of dissociation buffer, washed on a rotary mixer at 20rpm for 1min at 25 ℃ to wash away non-specifically bound small molecule complexes, and the magnetic beads were separated from the supernatant using a magnetic stand. Resuspend the washed magnetic beads in 50. mu.L of dissociation elution buffer (50mM Tris, 150mM NaCl, pH 8.0), heat to 95 ℃ in a metal bath for 10min for elution, denature the target protein to elute the DNA-free bead bound to the protein, separate the magnetic beads with a magnetic frame, and obtain the experimental and reference elution samples, respectively.
(4) Identification of the structure by mass spectrometry: measuring the molecular weight of a test product by adopting a high performance liquid chromatography-mass spectrometry combined system, wherein the test product comprises a sample before screening, an experimental group elution sample and a reference group elution sample; all product molecular weights found in the sample before screening are respectively extracted and compared in the mass spectra of the experimental group elution sample and the reference group elution sample, and if the ratio of the signal-to-noise ratio (signal-to-noise ratio ═ extraction ion strength/background ion strength) of the extraction ion strength of the molecular weight corresponding to a certain molecule in the experimental group elution sample divided by the signal-to-noise ratio of the extraction ion strength of the molecular weight in the reference group elution sample is more than 1, the small molecule is judged to be the vaccine head compound combined with the target protein.
Drawings
FIG. 1: a flow chart of the method of the invention;
FIG. 2: the high performance liquid chromatogram of the elution sample of the experimental group in the embodiment 1 of the invention;
FIG. 3: extracting the molecular weight of small molecular weight vaccine head compounds in the eluent of the experimental group from a high performance liquid mass spectrogram of the sample eluted by the experimental group in the embodiment 1 of the invention to obtain the extracted ion current intensity;
FIG. 4 is a schematic view of: in the embodiment 1 of the invention, the extracted ion current intensity is obtained by extracting the molecular weight of the small molecular weight head compound in the eluent of the experimental group from the high performance liquid chromatography mass spectrogram of the elution sample of the reference group;
FIG. 5 is a schematic view of: the mass spectrogram of the potential small-molecule plantlet head compound finally extracted in the embodiment 1 of the invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples, but it should not be construed that the scope of the present invention is limited to the examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Example 1 affinity screening of ROCK 2 protein to determine potential shoot-head Compounds
1) Stepwise synthesis of potential shoot head compounds on DNA with operable cleavage groups was performed following the synthesis strategy of a library of DNA-encoding compounds. And filtering the synthesized sample through a filter to obtain a purified sample. Dissolving the purified sample in H2Preparing 1.0 millimole per liter solution in O/DMSO (96:4), and then placing the solution under the illumination condition of 365 nanometers at the temperature of 0 ℃ and the illumination intensity of 800J for 40 minutes. The reaction solution was sampled for mass spectrometric monitoring to determine the conversion of the reaction (80%).
The reaction solution is filtered by a filter, and the collected filtrate is the solution containing the potential head-end compound.
2) Preparation of screening buffer: 50mM Tris, 150mM NaCl, 1mM TCEP, 0.01% Tween20, pH 8.0; preparation of elution buffer: 50mM Tris, 150mM NaCl, pH 8.0
3) In the experimental group, potential small molecule head compounds at a final concentration of 10. mu.M and 70pmol of target protein (no target protein added to the reference group) were dispensed in a total volume of 50. mu.L of screening buffer (50mM Tris, 150mM NaCl, 1mM TCEP, 0.01% Tween20, pH 8.0) in 1.5mL centrifuge tubes, and the tubes were placed on a rotary mixer and incubated at 25 ℃ for 60min at 20 rpm. Then 20 mu L of magnetic beads which are balanced by 150 mu L of screening buffer solution after 3 times are put into the solution after the incubation of the small molecules and the target protein, the centrifugal tube is placed on a rotary mixer and incubated for 30min at the temperature of 25 ℃ at 20rpm, and the complex of the target protein and the small molecule compound combined with the target protein is separated from the solution by a magnetic frame through the affinity action of the target protein label and the specific magnetic beads. The separated magnetic beads were resuspended in 50. mu.l of dissociation buffer (50mM Tris, 150mM NaCl, pH 8.0), washed on a rotary mixer at 20rpm for 1min at 25 ℃ to remove non-specifically bound small molecule complexes, and the magnetic beads and supernatant were separated using a magnetic stand. And (3) resuspending the washed magnetic beads with 50 μ l of dissociation elution buffer (50mM Tris, 150mM NaCl, pH 8.0), heating to 95 ℃ in a metal bath, eluting for 10min, denaturing the target protein to elute small molecules bound to the protein, separating the magnetic beads from the eluted small molecules by using a magnetic frame, and obtaining the supernatant as an experimental group elution sample (the reference group elution sample without the target protein).
4) Molecular weight determination was performed on test samples including pre-screening samples, experimental elution group samples, and reference elution group samples using high performance liquid mass spectrometry with Water UPLC-MS XeVo G2-XS Q-TOF (Waters usa). Analyzing liquid phase mass spectrum data of a sample before screening, extracting a target signal with molecular weight of 412.1580, extracting in an experimental group elution sample and a reference group elution sample by using the molecular weight to respectively obtain two extraction ion intensity graphs of figure 3 and figure 4, dividing a signal-to-noise ratio of the extraction ion intensity of the experimental group elution sample (figure 3) by a signal-to-noise ratio of the extraction ion intensity of the reference group elution sample to obtain a ratio of 60, wherein the ratio is more than 1, and determining that the structure of a compound corresponding to the molecular weight is a small molecular compound combined with the target protein.
In summary, the above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (19)
1. A method for identifying a head-of-shoot compound encoded by operable cleaved DNA, comprising: the method comprises the following steps:
(1) synthesis of DNA-encoded head-of-shoot compounds with cleavable groups: screening data signals aiming at a target protein DNA coding compound library, sequencing the data signals according to the enrichment degree from high to low, determining the molecular sequence information and the synthesis path of potential DNA coding seedling compounds, and synthesizing all potential DNA coding seedling compounds on DNA with an operable cutting group by using the synthesis path;
(2) release of shoot-head compound: cutting the DNA coding sprout compound with the cleavable group, and filtering out the DNA part through a filter to obtain a sprout compound without DNA;
(3) preparation of sample set: preparing a pre-screening sample of the DNA-free shoot head compound by using a screening buffer solution; the experimental group incubates the sample before screening, the target protein and the magnetic beads for fixing the protein together; incubating the sample before screening with the magnetic beads for fixing the protein by the reference group; respectively eluting the experimental group and the reference group by using an elution buffer solution, and respectively collecting eluates as an experimental group elution sample and a reference group elution sample;
(4) identification of the structure by mass spectrometry: measuring the molecular weight of a test sample by adopting a high performance liquid chromatography-mass spectrometry combined system, wherein the test sample comprises a sample before screening, an experimental group elution sample and a reference group elution sample; all product molecular weights found in a sample before screening are respectively extracted and compared in the mass spectra of an experimental group elution sample and a reference group elution sample, if the ratio of the signal-to-noise ratio of the extracted ion intensity of a certain molecular weight in the experimental group elution sample to the signal-to-noise ratio of the extracted ion intensity of the molecular weight in the reference group elution sample is more than 1, the molecule is judged to be a shoot compound combined with the target protein; wherein the signal-to-noise ratio is the extracted ion intensity divided by the background ion intensity.
2. The method of claim 1, wherein: the structure of the DNA coding the head-shoot compound on the DNA with the operable cutting group is as follows:
DNA-L-A-M
wherein,
the DNA in the structural formula comprises a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers, and the nucleotide chain is connected with the L through one or more chemical bonds or groups;
l is a connecting chain or none;
a is an operable cleavage group;
m is a shoot apex compound moiety.
3. The method of claim 2, wherein: the operable cleavable group is:
4. the method of claim 3, wherein: the structure of the DNA coding the seedling-end compound on the DNA with the operable cutting group is as follows:
5. the method according to claims 1 to 4, wherein: the cleavage operation of the step (2) is to cleave the operable cleavage group by illumination; preferably, the illumination wavelength is 306-400 nm.
6. The method of claim 1, wherein: the specific steps for synthesizing the potential DNA-encoded head compounds on the DNA with the operable cleavage group in the step (1) comprise: carrying out chemical reaction assembly on reaction building blocks of all dimensions and DNA in sequence according to a synthesis route, a post-treatment operation and a purification mode which are followed by the shoot head compound during library synthesis; after the reaction of each dimension is finished, alcohol precipitation operation is carried out, and then all reaction products are put into the circulation reaction of the next dimension.
7. The method of claim 6, wherein: the alcohol precipitation operation is to add 5.0mol/L sodium chloride solution with 10 percent volume ratio and 3 times volume of absolute ethyl alcohol in sequence, then to freeze for 40 minutes below minus 20 ℃, and then to carry out centrifugation and supernatant removal operation.
8. The method of claim 7, wherein: and (2) carrying out alcohol precipitation on the DNA coding head-of-seedling compound after the synthesis in the step (1), filtering by a filter and freeze-drying.
9. The method of claim 1, wherein: the step (1) further comprises the following steps: after obtaining the DNA coding head-of-seedling compound, the yield condition of the target product synthesized on the DNA and the condition of the by-product are analyzed by the liquid chromatography-mass spectrometry.
10. The method of claim 1, wherein: the step (2) further comprises: after the cleavage operation, the conversion rate in the cleavage step was obtained by liquid chromatography-mass spectrometry analysis.
11. The method according to claims 9 to 10, wherein: the concentration of the DNA-free shoot apex compound in the step (2) is calculated by the concentration and purity of the DNA-encoding shoot apex compound before cutting and the cutting conversion rate.
12. The method of claim 1, wherein: in the experimental group in the step (3), the amount of the target protein is 70-140 pmol, and the amount of the DNA-free seedling head compound in the sample before screening is 1000 pmol.
13. The method of claim 1, wherein: the step (3) specifically comprises the following steps: dissolving target protein and a sample before screening by using a screening buffer solution in an experimental group, wherein the final concentration of the target protein is 0.7-1.4 mu M; and rotationally incubating at room temperature for 1 hour, adding the protein into the target protein affinity coupled immunomagnetic beads for protein fixation, and fixing at room temperature for 30 minutes.
14. The method of claim 1, wherein: the step (3) specifically comprises the following steps: in the reference group, a sample before screening is dissolved by using a screening buffer solution, the final concentration of a DNA-free seedling-end compound is 10 mu M, then target protein affinity coupled immunomagnetic beads are added, and the mixture is fixed for 30 minutes at room temperature.
15. The method of claim 1, wherein: 65. mu.L of each elution buffer described in step (3) was used.
16. The method of claim 1, wherein: the elution buffer solution in the step (3) comprises the following components: 1)50mM HEPES pH 7.5,300mM NaCl; or 2)40mM Tris,300mM NaCl, pH 8.0; 3) methanol.
17. The method of claim 1, wherein: the elution operation in the step (3) is: after removal of the supernatant, incubation with elution buffer was performed for 1min, the supernatant was removed, and elution buffer was added to dissociate at 95 ℃ for 10 min.
18. The method of claim 1, wherein: the high performance liquid chromatography-mass spectrometry system in the step (4) specifically comprises: setting the sample injection volume of the liquid chromatogram to be 10 mu L, the detection time to be 6 minutes, and setting the mass spectrum to be in a full scanning mode; and carrying out sample injection setting according to the sequence of the sample before screening, the experimental group elution sample and the reference group elution sample.
19. The method of claim 9, wherein: the step (4) specifically comprises the following steps: the structural assignment of a molecular weight molecule was determined by comparison of the major and minor analyses in the synthesis of DNA-encoded headpiece compounds with cleavable groups.
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