一种dna编码化合物库药物分子垂钓方法

DNA coding compound library drug molecule fishing method

文档序号：3692 发布日期：2021-09-17 浏览：58次中文

1. A method for fishing a drug molecule in a DNA coding compound library, which is characterized by comprising the following steps:

step S1, coupling the first section of compound with the first section of DNA fragment, mixing and dispersing, connecting the coupled compound with the second section of molecular building block and the second section of DNA fragment, carrying out isothermal amplification on the two sections of DNA fragments, and mixing uniformly to obtain a compound library coded by DNA;

step S2, preparing metal nano-particle probe to couple with target protein or target protein binding receptor protein, adding into the prepared DNA coding compound library, incubating, performing molecular fishing, and obtaining targeted drug information through PCR amplification and DNA sequencing;

step S3, preparing a metal nanoparticle probe and a magnetic bead probe respectively, coupling the metal nanoparticle probe and the magnetic bead probe with Raman signal molecules, coupling the metal nanoparticle probe or the magnetic bead probe with a target protein or a target protein binding receptor protein respectively, and adding a screened compound;

and step S4, detecting the Raman signal, and judging whether the screened compound has the effect of inhibiting the combination of the target protein and the target protein receptor.

2. The drug molecule fishing method according to claim 1, wherein the metal nanoparticle probe is a gold nanoparticle probe or a silver nanoparticle probe.

3. The drug molecule fishing method according to claim 1, wherein the raman signal molecule is 4-MBA or 4-ABP.

4. The method for fishing with drug molecules according to claim 1, wherein the binding receptor is a receptor protein having a binding activity to a target protein or a nucleic acid.

5. The method for molecular fishing of claim 1, wherein the binding receptor is KRAS-PDE δ protein.

6. The method for molecular fishing of claim 1, wherein the determination in step S4 is specifically as follows:

if the SERS signal is obviously weakened from strong to weak or from weak to strong, the compound to be screened is a targeted drug aiming at a specific target;

and if the SERS signal is not obviously changed, the compound to be screened is not a targeted drug for a specific target.

7. The method for fishing with drug molecules according to claim 1, further comprising providing a positive control, wherein the positive control is an inhibitor known to have an activity of inhibiting the binding of the target protein to the target protein-binding receptor.

8. The method for fishing with drug molecules according to claim 7, wherein the inhibitor is a small molecule compound, a protein or a nucleic acid.

9. The method of claim 1, further comprising recovering the targeted drug.

10. Use of the method of molecular fishing of drugs according to any of claims 1 to 9 for obtaining targeted drugs by molecular fishing in a mixture.

Background

Molecular Fishing (molecular Fishing) is a High-Throughput biological analysis method for realizing discovery of lead compounds by affinity selection of ligands from complex biological samples based on interaction of drug targets and active molecules, has the characteristics of strong specificity, High efficiency, low requirement on sample pretreatment and the like, and is an important technical progress on the basis of the traditional High Throughput Screening (HTS). Generally consists of 4 parts, target immobilization (enzymes, cells, etc.), affinity fishing, ligand elution and LC-MS analysis. The molecular fishing technique includes an offline-mode molecular fishing technique and an online-mode molecular fishing technique. In the offline-mode molecular fishing, affinity fishing and ligand structure analysis are performed independently, and the molecular fishing has relatively low requirements on equipment and technology, so that the method is widely applied to ligand screening experiments in laboratories, including affinity ultrafiltration, magnetic nanoparticle fishing and hollow fiber separation. In the molecular fishing technology in the online mode, affinity separation and ligand structure analysis are performed simultaneously, and compared with the offline mode, the molecular fishing technology has the characteristics of high automation degree, high simplicity and high sensitivity. Meanwhile, the on-line dynamic monitoring can be realized by the synchronous operation of affinity separation and ligand structure analysis, the interaction kinetic parameters of the components and the target are obtained, and the research and the improvement of experimental mechanisms including the bioaffinity chromatography, the capillary electrophoresis method and the BIA technology are facilitated.

Among them, a Biomolecule Interaction Analysis (BIA) technique based on Surface Plasmon Resonance (SPR) is a novel biosensing technique. The biosensing instrument developed by the technology is named Biacore and mainly comprises an SPR optical detection system, a microfluidic chuck and 3 core parts of a sensor chip. When in analysis, one biomolecule is coupled on the surface of the sensing chip, another biomolecule solution interacting with the biomolecule is injected into the SPR optical detection system, flows through the surface of the sensing chip and is combined with the biomolecule coupled on the sensing chip, the mass of the substance on the surface of the chip is increased, the refractive index is changed, and the interaction change between the biomolecules can be monitored in real time through an SPR response value. The Biacore directly measures the change of the optical reflectivity of the Biacore through the physical optical phenomenon of surface plasmon resonance without using a fluorescent label and an isotope label, tracks the interaction between biomolecules in real time, and has extremely high sensitivity, resolution and specificity. However, the Biacore technology has high requirements on samples and huge sample consumption, and has obvious limitations.

In the field of modern new drug development, establishing a large-scale candidate drug molecular library for high-throughput screening aiming at biological targets is one of indispensable means for obtaining lead compounds in new drug development, and large pharmaceutical companies all have large-scale molecular libraries and screening platforms thereof in the world at present. However, high-throughput screening based on single molecules requires high cost, expensive experimental equipment, complex management and operation, large time span, and limited and single synthesized compound, which greatly limits the discovery efficiency of lead compounds. In recent years, with the development of high throughput technology, DNA Encoding Chemical Library (DEL) technology has gradually become an emerging screening method in the field of drug development. The principle of DEL screening is to link a specific DNA sequence with a small molecule compound at the molecular level, and the DNA sequence is used as a marker sequence of the small molecule, so that the small molecule in each reaction corresponds to a unique DNA fragment. The structural information of the required small molecule compound is selected by carrying out PCR amplification and sequencing on the unique DNA barcodes connected with the target compound. The method can realize the completion of billions-level high-flux screening in a very small system, greatly improves the discovery efficiency of lead compounds, and is one of important means for the research and development of new drugs. However, conventional DELs only act on a single protein target and do not act at the level of protein interaction. With the research on cancer targeted inhibition, drug screening aiming at a single target point obviously cannot meet the requirement of a protein interaction mechanism.

Surface-Enhanced Raman scattering (SERS) is a signal enhancement technique based on physical and chemical enhancement, and is widely used in the biomedical field, such as detection of nucleic acids, proteins, cells, tissues, etc. It is based on that when metal is attached to rough molecular surface or positioned in gap or tip of metal material, the electromagnetic field generated by local surface plasma excitation is amplified, so that the Raman signal of the object to be measured adsorbed on the metal surface is enhanced 10³-10⁶And high sensitivity detection is performed on low concentration analytes. SERS techniqueThe method can provide inherent molecular fingerprint information of an ultrahigh-sensitivity measured object in a liquid environment, has strong specificity and high sensitivity, can accurately and sensitively detect the characteristic peak of the protein, and has great significance in high-throughput screening of drugs because the characteristic peak changes obviously when the small molecular compound influences the protein interaction. At present, no report exists that DEL and SERS are combined for molecular fishing and are applied to small molecule drug screening.

Disclosure of Invention

In order to solve the technical problems, the invention discloses a molecular fishing method of a small molecular compound, which combines molecular fishing with DEL and carries out high-sensitivity and high-specificity high-throughput screening on small molecules influencing the interaction of target proteins by surface enhanced Raman scattering.

The invention provides a high-throughput screening method of a targeted drug, which comprises the following steps:

and step S4, detecting the Raman signal, and judging whether the screened compound has the effect of inhibiting the combination of the target protein and the target protein receptor.

In certain embodiments, the metal nanoparticle probes are gold nanoparticle probes or silver nanoparticle probes.

In certain embodiments, the Raman signal molecule is 4-MBA or 4-ABP.

In certain embodiments, the binding receptor is a receptor protein having binding activity capable of binding to a target protein or nucleic acid.

In certain embodiments, the binding receptor is a KRAS-PDE delta protein.

In some embodiments, the determination in step S5 is specifically:

if the SERS signal is obviously weakened from strong to weak or from weak to strong, the compound to be screened is a targeted drug aiming at a specific target;

and if the SERS signal is not obviously changed, the compound to be screened is not a targeted drug for a specific target.

In certain embodiments, the method further comprises providing a positive control that is an inhibitor known to have activity of inhibiting binding of the target protein to a target protein binding receptor.

In certain embodiments, the inhibitor may be a small molecule compound, a protein, or a nucleic acid.

In certain embodiments, the method further comprises recovery of the targeted drug.

The invention also provides application of the high-throughput screening method in molecular fishing in a mixture to obtain a targeted drug.

Compared with the prior art, the invention has the following effects:

1) the invention combines Raman signal detection and a DNA coding compound library for the first time to be applied to the technical field of inhibitor screening based on molecular fishing, and the inhibitor screening method acts on the level of protein interaction to form a simple and sensitive inhibitor screening method.

2) The method has the advantages of low cost, small measurement interference, strong signal, high sensitivity, capability of detecting low-abundance protein, capability of realizing high-flux molecular fishing, simple and convenient operation, no need of cleaning in the whole detection process, reusability of a sample bank after detection and capability of providing a new strategy for a screening approach of the inhibitor.

3) Compared with the existing Biacore technology in the field of molecular fishing, the invention only needs one ten thousandth of the dosage, obviously saves the sample cost and achieves the aim of screening the drugs in a tiny system.

4) The DEL is combined for molecular fishing, the traditional molecular fishing only can act on a single protein level, and the obtained result has high false positive; the molecular fishing-based drug screening can act on the protein interaction level, so that the molecular fishing-based drug screening can be widely applied to the research of disease-targeted drugs and has obvious advantages.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.

FIG. 1 is a schematic diagram of the process of creating a library of DNA encoding compounds of example 1.

FIG. 2 is a schematic diagram of the molecular fishing and SERS detection processes in examples 2, 3 and 4.

FIG. 3 is a CCK8 kit used in example 5 to test that Deltarasin has significant killing effect on Hep3B tumor cells in vitro.

FIG. 4 shows that D1350124 has a significant killing effect on Hep3B tumor cells in example 5 by using a CCK8 kit.

Detailed Description

The invention will be better understood from the following examples. However, it is easily understood by those skilled in the art that the description of the embodiment is only for illustrating and explaining the present invention and is not for limiting the present invention described in detail in the claims. Unless otherwise specified, reagents, methods and equipment used in the present invention are conventional methods, and test materials used therein are available from commercial companies, unless otherwise specified.

EXAMPLE 1 preparation of libraries of DNA encoding compounds

1. Oligonucleotide-compound ligation reaction

One compound (1.25. mu. mol) was dissolved in DMSO and diluted to 230. mu.L, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) (12. mu.L 100mM in DMSO) was addedAnd N-hydroxysuccinimide (NHS) (10. mu.L 330mM in DMSO/H)₂O₂1) after 20 minutes of activation at 30 ℃ Triethylamine (TEA)/HCl (50. mu.L 500mM pH 10.0) and the corresponding oligonucleotide (30. mu.L 500. mu.M) were added and stirred overnight at 30 ℃. After the reaction was terminated by adding Tris-HCl (20. mu.L, 500mM, pH 8.0) and stirring at 30 ℃ for 1h, triethylamine acetic acid (TEAA) (500. mu.L, 100mM, pH 7.0) was added. After HPLC purification, drying under reduced pressure, redissolving 100 μ LH20, and performing concentration quality inspection and LC-ESI-MS quality inspection by using an ultraviolet spectrophotometer. A total of 40 different oligonucleotide-compounds were ligated using the method described above. The structure of the oligonucleotide is 5' -H₂N-(CH₂)₁₂GGA GCT TGT GAA TTC TGG NNN NNN NNN GGA CGT GTG TGA ATT GTC-3', wherein NNN NNN NNN represents the nucleotide base linked to the compound, and 5 compounds and DNA fragments are exemplified, and the detailed information is shown in Table 1. The above-mentioned compounds were mixed and divided equally into 200 parts.

TABLE 1 first Compound species and first DNA fragment

2. Connecting the second chemical molecule building block

Each of the 200 molecular blocks contains a methylene carboxylic acid structure, and an exemplary list of 5 are shown in table 2.

TABLE 2 second Compound species and second DNA fragment

DEAE-agarose resin (100. mu.L) was transferred to a SpinX column, washed with water (0.8mL) and acetic acid (0.8mL,10mM, pH 5), the resin was resuspended in acetic acid (200. mu.L, 10mM, pH 5), an aliquot of oligonucleotide-compound (360pM) was added, and incubated for 15min to immobilize on the resin. The solution was transferred to a Glen Research column and washed with acetic acid (1mL,10mM) and methanol (MeOH 2 mL). Resin bound oligonucleotide-compound was added freshly prepared EDC-HCl (50mM, 500. mu.L) and acetic acid (5mM) and an aliquot of 50mM methylene carboxylic acid was dissolved in Dimethylformamide (DMF)/MeOH 3:2 at a flow rate of 25. mu. Lmin-1 for 20 min. The methylene carboxylic acids were activated six times with EDC-HCl in total, i.e. reacted at a flow rate of 25. mu. Lmin-1 for a total of 120 min. The column was washed with MeOH (3mL) and acetic acid (1mL,10mM), and the DEAE agarose resin solution was resuspended in acetic acid (400. mu.L, 10mM), transferred to a SpinX column and centrifuged at 6,000rpm for 1 min. The DEAE agarose resin solution was resuspended in sodium acetate buffer (400. mu.L, 1M, pH 8.7), incubated for 2min, centrifuged at 13,200rpm slightly and the yield of oligonucleotide-compound in the eluate was 5%. DEAE agarose resin solution was incubated with sodium acetate/acetic acid buffer (200. mu.L, 3M, pH 4.7) for 2min, centrifuged at 13,200rpm for 1min, and the yield of oligonucleotide-compound in the eluate was 50%. EtOH (1.8mL) was added to the eluate and incubated overnight at-20 ℃. After the sample was centrifuged at 4 ℃ for 20min (13,200rpm), the remaining DNA was washed with 85% EtOH and centrifuged at 4 ℃ for 20min (13,200rpm), the supernatant was removed, and the sample was dried with a rotary vacuum concentrator, dissolved in 50. mu.L of water and assayed for concentration with UV light.

3. Klenow polymerase encodes the second oligonucleotide fragment

200 parts of oligonucleotide in total, and the structure is as follows: 5 '-GTA GTC GGA TCC GAC CAC NNNNNNNNNNNN GAC AAT TCA CAC ACG TCC-3', wherein NNNNNNNN represents an oligonucleotide linked to a corresponding compound. The secondary pool of modified oligonucleotide-compounds obtained in step 1 (16pM) and the corresponding second oligonucleotide fragment (24pM) were dissolved in Klenow polymerase buffer to a final volume of 44. mu.L, wherein the second oligonucleotide fragment is shown in Table 2. The reaction mixture was incubated at 50 ℃ for 10min and cooled to room temperature to allow local complementary ligation of the two fragments. dNTP (5. mu.L, 33. mu.M) was added, and Klenow polymerase (5U) was reacted at 25 ℃ for 30 min. The reaction was purified on an ion exchange column and eluted with QE buffer (50. mu.L). 120 μ L of each sub-pool were pooled to generate a pool of DNA encoding compounds DEL8000 at a final concentration of 360nM, stored at-20 ℃.

Example 2 molecular fishing

Synthesis of silver nano-ion probes (AgNPs): getOne 250mL three-neck flask is soaked in an acid jar overnight, taken out the next day, washed clean and then washed with water for injection for three times, so as to ensure that the wall is pure and has no impurities. Drying in oven, and cooling to room temperature. During which 0.018g of silver nitrate powder was accurately weighed and carefully poured into a three-necked flask. 100mL of ultrapure water is added, the mixture is shaken up, a condenser tube is connected, the temperature of an electric heating jacket is set to be 100 ℃, the solution is stirred and heated to be slightly boiled, and 2mL of 1% sodium citrate solution is added quickly. Observing the color change of the solution: and (3) slightly cooling to 90 ℃, continuing magnetic stirring, and maintaining for 40min to obtain the silver nanoparticle dispersion system. Measuring the absorbance curve of the silver nanoparticles by using an ultraviolet spectrophotometer: the silver nanoparticles are diluted by 5 times and then detected, the maximum absorption wavelength range (420-480nm) is observed to be correct, and other miscellaneous peaks are not existed, so that the synthetic particle size can be preliminarily judged to be uniform. Using the formula: a ═ kbc (a ═ Amax, k ═ 3 × 10¹¹M^-1cm^-1And b ═ 1cm) the concentration of silver nanoparticles was calculated. Storing at 4 deg.C for use.

The AgNPs were concentrated to above 0.29nM, freshly prepared Tween 20 was added, reacted for 30min, 5.0. mu.L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) (2.5mM) and 5.0. mu. L N-hydroxysuccinimide (NHS) (2.5mM) were added, reacted for 1h, centrifuged to remove excess activator, and resuspended. Adding 5 mu L of Domain Z (0.05mg/mL) for reaction for 2h, washing after centrifugation, adding 3 mu L of PDE delta (0.05mg/mL) for coupling overnight, washing once after centrifugation, adding DEL8000 for coupling overnight, removing supernatant after centrifugation, resuspending and precipitating with 1% acetic acid (500 mu L), rapidly oscillating at 37 ℃ until obvious silver mirror reaction is separated out, taking the supernatant for centrifugation, and taking the supernatant as a sample to be detected.

EXAMPLE 3 Compound Structure determination

And determining the information of the DNA fragment by PCR amplification and DNA sequencing according to the oligonucleotide fragment connected with the compound, thereby obtaining the structural information of the compound.

mu.L of the supernatant was used as a template, and 10. mu.M of primers (5'-GCC TCC CTC GCG CCA TCA GGG AGC TTG TGA ATT CTG G-3' (SEQ ID NO.11) and 5'-GCC TTG CCA GCC CGC TCA GGT AGT CGG ATC CGA CCA C-3' (SEQ ID NO.12) in terms of primer structure) 4. multidot.2 mM dNTP, 1.25U of Taq enzyme and PCR buffer were added. The PCR steps are as follows: DNA was denatured at 94 ℃ for 2min, 40s 4 cycles at 94 ℃, 40s at 54 ℃, 40s at 72 ℃, 40s at 94 ℃, 30s at 64 ℃, 30s at 72 ℃ and 7 min at 72 ℃. After the reaction, the corresponding samples were mixed and purified by ion exchange column, and extracted with 50. mu.L of QE buffer.

And determining the information of the DNA fragment according to the PCR amplification and DNA sequencing results, thereby obtaining the corresponding compound D1350124 and the structural information of the compound. According to the technical process, fishing of the compound library can be realized, and the structural information of the compound can be obtained.

Example 4 surface enhanced Raman Spectroscopy detection

1. Preparation of gold (silver) nanoparticles

1.1 preparation of gold nanoparticles

Gold nanoparticles (AuNPs): a250 mL three-neck flask is taken and washed by aqua regia (1 part of concentrated nitric acid is added with 3 parts of concentrated hydrochloric acid), taken out the next day and washed clean, and then washed by water for injection for three times, so as to ensure that the wall is pure and free of impurities. Drying in oven, and cooling to room temperature. The flask was charged with 2mL of 1% HAuCl₄Adding water to 200mL, connecting with a condenser tube, heating to 150 ℃ by an electric heating jacket, stirring, heating and refluxing to slightly boil. Adding 2mL of 1% sodium citrate, controlling the temperature, observing the color change from light yellow to black (110-120 ℃), then to red (90 ℃), cooling to 90 ℃, and keeping the temperature for 40min to obtain the gold nanoparticle dispersion system.

1.2 preparation of silver nanoparticles

Silver nanoparticles (AgNPs): and (3) soaking a 250mL three-neck flask in an acid jar overnight, taking out the flask the next day, washing the flask clean, and washing the flask three times with injection water to ensure that the wall is pure and has no impurities. Drying in oven, and cooling to room temperature. During which 0.018g of silver nitrate powder was accurately weighed and carefully poured into a three-necked flask. 100mL of ultrapure water is added, the mixture is shaken up, a condenser tube is connected, the temperature of an electric heating jacket is set to be 100 ℃, the solution is stirred and heated to be slightly boiled, and 2mL of 1% sodium citrate solution is added quickly. Observing the color change of the solution: and (3) slightly cooling to 90 ℃, continuing magnetic stirring, and maintaining for 40min to obtain the silver nanoparticle dispersion system. Measuring the absorbance curve of the silver nanoparticles by using an ultraviolet spectrophotometer: the silver nanoparticles are diluted by 5 times and then detected, the maximum absorption wavelength range (420-480nm) is observed to be correct, and other miscellaneous peaks are not existed, so that the synthetic particle size can be preliminarily judged to be uniform. Using the formula: the concentration of silver nanoparticles was calculated at a ═ kbc (a ═ Amax, k ═ 3 × 1011M-1cm-1, b ═ 1 cm). Storing at 4 deg.C for use.

2. Preparation of magnetic nanoparticles

The commercialized carboxyl magnetic beads produced by Baimeige are prepared at the concentration of 0.5mg/mL, washed by a magnetic bead preservation solution and magnetically separated for three times, resuspended in the preservation solution to prevent the aggregation of the magnetic beads, and preserved at 4 ℃ for later use.

3. Synthesis of KRAS-PDE delta protein inhibitor two Raman probes (Raman signal molecule coupled on silver nanoparticle) related to screening

In order to reduce the non-site specific binding and improve the targeting efficiency of the Protein when the Protein is coupled, an Fc tag is added at the C end of the PDE delta Protein, and an Fc segment which can effectively and specifically recognize the PDE delta is used by using a Protein A structural domain B.

1mL of AgNPs (0.25nM) was mixed with 20. mu.L of Tween 20 for 20min to prevent the AgNPs from coagulating. Then, 10. mu.L of 2.5mM N-hydroxysuccinimide (NHS) and 10. mu.L of 2.5mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) were added and activated by mixing for 1 hour, centrifuged (3500g,6min), the supernatant aspirated, and the AgNPs were resuspended in an equal volume of water for injection. 4 μ L of Domain B (0.05mg/mL) was added rapidly for coupling for 1.5 h, centrifuged (3500g,6min), and the supernatant aspirated and the AgNPs resuspended in an equal volume of water for injection. The assembly onto the AgNPs surface was performed with the Raman signal molecule 4-Mercaptobenzoic acid (4-Mercaptobenzoic acid,4-MBA), 2. mu.L of PDE delta (0.05mg/mL) and 25. mu.L of 4-MBA (1.0mM) were added for coupling for 1.5 hours, centrifuged (3500g,6min), the supernatant aspirated and then resuspended with an equal volume of water for injection to give AgNPs4-MBA @ Domain B @ PDE delta probe.

Add 1. mu.L ethanolamine (15mM) and 1. mu. LBSA (0.5mg/mL1) to block for 20min, centrifuge (3500g,6min), aspirate the supernatant and resuspend the AgNPs with an equal volume of water for injection.

To accomplish the coupling of KRAS4B to magnetic beads, a biotinylated C-terminal K-Ras4B polypeptide was synthesized and coupled to streptavidin-modified magnetic beads (300nm,0.5 mg/mL).

Deltarasin (a small molecule KRAS-PDE delta inhibitor) was used as a positive drug. Two kinds of probes (silver probe: magnetic probe 500. mu.L: 150. mu.L) were mixed in the presence of Deltarasin (2.0. mu.L, 5. mu.g/mL) and in the absence of Deltarasin, and the two sets of mixtures were each subjected to magnetic separation, washed 3 times with water for injection and then resuspended with an equal volume of water for injection.

After magnetic enrichment, SERS detection is carried out under 633nm exciting light, obvious Raman signal change can be seen, and the Raman signal is obviously reduced after the positive drug is added. The higher the concentration of positive drug, the lower the signal peak.

Two kinds of probes (silver probe: magnetic probe: 500. mu.L: 150. mu.L) were mixed with the addition of D1350124 compound (2.0. mu.L, 5. mu.g/mL), and the mixture was magnetically separated, washed 3 times with water for injection and then resuspended in an equal volume of water for injection. After magnetic enrichment, SERS detection is carried out under 633nm exciting light, obvious Raman signal change can be seen, and after the compound D1350124 is added, the Raman signal is obviously reduced. The higher the concentration of D1350124, the lower the signal peak.

Example 5 cell killing assay compound No. D1350124

The CCK8 kit is used for detecting that Deltarasin and D1350124 both have obvious in vitro killing effect on Hep3B tumor cells. The results are shown in fig. 3 and 4, and after 48 hours of incubation of Deltarasin and D1350124, the number of Hep3B cells changed, and the killing effect of the cells in vitro has metering dependence, and the killing effect is obviously enhanced with the increase of the drug concentration.

The feasibility of the technical approach for drug molecule fishing by combining DNA-encoding compound libraries based on surface enhanced raman spectroscopy was demonstrated both at the molecular and cellular level.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims.

SEQUENCE LISTING

<110> Jilin university

<120> a DNA coding compound library drug molecule fishing method

<130> 2021

<160> 12

<170> PatentIn version 3.3

<210> 1

<211> 9

<212> DNA