A single-molecule method for detecting the formation of DNA adducts catalyzed by drug-metabolizing enzymes in vitro

Abstract

The present invention belongs to the technical field of pharmacological and toxicological analysis of small molecule compounds, and specifically discloses a method for single-molecule manipulation detection of in vitro drug metabolizing enzyme-catalyzed DNA adduct formation, comprising the steps of: a) fixing a double-stranded DNA molecule between a fixed surface and a movable surface; b) mechanical stretching to measure the length of the double-stranded DNA molecule; c) applying a reaction solution containing a small molecule compound and a drug metabolizing enzyme component, and reacting; d) washing away the small molecule compound and the drug metabolizing enzyme component; e) mechanical stretching to detect the change in the length of the double-stranded DNA, thereby judging whether the small molecule compound forms an embedded adduct with DNA based on the change in length, or judging the amount and / or reaction kinetics of the embedded adduct formed by the small molecule compound and DNA. The present invention uses a single-molecule mechanical manipulation method to detect the amount and reaction kinetics of the embedded adduct formed by the drug metabolizing enzyme-catalyzed small molecule compound and DNA.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmacological and toxicological analysis of small molecule compounds, and more specifically, relates to a method for single-molecule manipulation detection of the formation of DNA adducts catalyzed by in vitro drug metabolizing enzymes, and is a method for using single-molecule manipulation technology to detect the number and reaction kinetics of embedded DNA adducts formed by small molecule compounds under the catalysis of in vitro drug metabolizing enzymes. Background Art

[0002] DNA adducts are covalent bonds formed between endogenous or exogenous compounds and DNA molecules. DNA adducts are an important and common type of DNA damage and one of the important causes of tumorigenesis. The formation of DNA adducts can further trigger a series of events such as DNA chain breaks, base substitutions and base deletions, thereby causing DNA mutations and the occurrence of cancer. The mutagenic, teratogenic and carcinogenic effects of chemical carcinogens and genotoxic compounds in the environment are related to the ability to form DNA adducts. Important chemotherapeutic drugs used in clinical practice, such as compounds derived from natural products such as mitomycin, cause DNA damage by forming adducts with DNA, and ultimately induce programmed cell death. Therefore, the detection of DNA adducts can not only help understand the occurrence of tumors, but is also crucial for the treatment of tumors.

[0003] Various exogenous carcinogens and natural products such as aflatoxin B1 and other compounds, under the catalysis of drug metabolizing enzymes in the liver (such as cytochrome P450 enzymes), covalently bind to DNA to form DNA adducts. These DNA adducts are also called bulky DNA adducts due to their large molecular weight and volume. Some bulky DNA adducts contain planar aromatic ring structures that can be embedded between double strands of DNA, thereby affecting processes such as DNA transcription and replication. Compared with non-embedded DNA adducts, embedded DNA adducts are more difficult to be repaired by the DNA damage repair system, and the probability of DNA mutation or DNA damage is greatly increased, thereby leading to the occurrence of cancer. Therefore, the detection of embedded DNA adducts is particularly important, which can help understand the pharmacological and toxicological mechanisms of environmental carcinogens and anti-tumor drugs.

[0004] There are several methods for detecting DNA adducts, including 32 P-post-labeling, immunoassay, chromatography, and mass spectrometry can detect the number and kinetics of DNA adducts formed after nucleic acids react with small molecules under certain catalytic conditions. However, these traditional biochemical analysis methods require purification of DNA samples and discontinuous steps to digest or label DNA adducts, making it difficult to monitor the process of DNA adduct formation in real time. 32The P-post-labeling method for detecting DNA adducts is to detect single nucleotides, and it is impossible to distinguish whether the formed DNA adducts are embedded adducts or non-embedded adducts, etc., with different covalent binding modes. In addition, some of the generated adducts, such as doxorubicin-DNA adducts, are unstable. According to a report by Pei et al., doxorubicin-DNA adducts are lost in large quantities during the purification of DNA after the addition reaction (Analytical Chemistry 2020, 92 (6), 4504-4511). In addition, the labeling efficiency of the ligand or the affinity of the antibody in the immunoassay also depends on the structure of the ligand. Different antibodies need to be found to analyze different DNA adducts, which is not suitable for large-scale sample analysis. Therefore, it is very important to find a rapid, label-free and purification-free method to systematically compare the ability of compounds to form embedded DNA adducts.

[0005] On the other hand, single-molecule manipulation technology has been proven to be a powerful technology for studying the binding of small molecules and proteins to DNA. It can provide information such as the binding mode, binding kinetics, binding affinity and sequence specificity of small molecules and DNA. Due to its high sensitivity, single-molecule manipulation technology requires very little sample volume, which is very suitable for direct detection of trace compounds extracted from natural products. Almaqwash et al. reported the single-molecule detection of the formation of adducts between phenanthrene platinum and DNA using optical tweezers (J.Am.Chem.Soc.2019,141,4,1537–1545). However, unlike platinum compounds, many chemical carcinogens and anti-tumor drugs derived from natural products require metabolic enzyme activation to covalently bind to nucleic acids. For example, environmental carcinogens such as aflatoxin B1 and aristolochic acid require the catalysis of liver drug metabolizing enzymes to form DNA adducts. At present, there is no method for using single-molecule manipulation to detect the formation of DNA adducts catalyzed by drug metabolizing enzymes in vitro.

[0006] Exogenous compounds (such as environmental pollutants, chemicals, and drugs) are metabolized by drug-metabolizing enzymes in the body. Drug-metabolizing enzymes, also known as xenobiotic-metabolizing enzymes, are expressed in a variety of tissues (such as the liver, lungs, kidneys, intestines, and plasma). Among them, the microsomal enzyme activity of the liver is the highest, and it is the main site of metabolism of endogenous or exogenous compounds. The catalytic systems commonly used in in vitro experiments include recombinant drug-metabolizing enzymes (cytochrome P450 enzymes, etc.), liver subcellular components (cytoplasm, liver S9 enzyme components, microsomal enzymes), enzyme systems in hepatocytes and plasma, etc. The application of these in vitro catalytic systems to achieve high-sensitivity detection of DNA adducts at the single-molecule level remains a challenging topic. Summary of the invention

[0007] In view of the above defects or improvement needs of the prior art, the purpose of the present invention is to provide a method for single-molecule manipulation detection of in vitro drug metabolism enzyme catalysis to form DNA adducts, which can be used to determine whether small molecule compounds form embedded DNA adducts under the catalysis of in vitro drug metabolism enzymes by measuring the length change of double-stranded DNA molecules caused by the formation of embedded adducts under the catalysis of in vitro drug metabolism enzymes through single-molecule mechanical stretching experiments, and can be used to further determine the binding ability (such as the number of adducts generated) and reaction kinetics (such as how the reaction changes over time) of embedded DNA adducts. The present invention reports for the first time the use of drug metabolism enzyme components to catalyze the formation of DNA adducts in single-molecule manipulation experiments. By introducing drug metabolism enzymes in single-molecule experiments, it can be used for quantitative detection of the ability of known and unknown small molecule compounds such as aromatic ring compounds to form embedded adducts with DNA; it can also be used for the analysis of potential genotoxic components in active ingredients containing composite active ingredients and natural products; it can also be used for the analysis of the catalytic ability of in vitro drug metabolism systems (liver microsomes, liver S9 enzyme components, CYP450 enzymes).

[0008] To achieve the above object, the present invention provides a method for detecting the intercalation and covalent binding of small molecule compounds to double-stranded DNA by drug metabolizing enzymes using single-molecule manipulation, characterized in that the method comprises the following steps:

[0009] a) fixing a double-stranded DNA molecule between a first surface and a second surface, wherein the first surface remains in a fixed position and the second surface is a movable surface;

[0010] b) Measuring the length of the double-stranded DNA molecule by mechanically stretching the double-stranded DNA molecule;

[0011] c) applying a reaction solution comprising a small molecule compound and a drug metabolizing enzyme component to the double-stranded DNA molecule and performing a reaction;

[0012] d) after the reaction reaches a preset reaction time, washing away small molecule compounds and drug metabolizing enzyme components;

[0013] e) using the same mechanical conditions as in step b) to mechanically stretch the double-stranded DNA molecules and detect the change in length of the double-stranded DNA, thereby determining whether the small molecule compound forms an intercalation adduct with the DNA based on the change in length, or determining the amount and / or reaction kinetics of the intercalation adduct formed between the small molecule compound and the DNA.

[0014] As a further preferred embodiment of the present invention, in step a), the fixation is to directly or indirectly attach at least one base of one strand of the double-stranded DNA to the first surface, and directly or indirectly attach at least one base of the other strand of the double-stranded DNA to the second surface;

[0015] Alternatively, the fixation is to attach at least one base of one strand of the double-stranded DNA directly or indirectly to the first surface, and at least one base at a different position of the strand to the second surface directly or indirectly.

[0016] As a further preferred embodiment of the present invention, in the step a), the first surface is preferably a glass slide surface, and the second surface is preferably a magnetic sphere surface.

[0017] As a further preference of the present invention, in the step c), the drug metabolizing enzyme component is preferably a liver drug metabolizing enzyme component or a drug metabolizing enzyme component in the lung, kidney, intestine, heart, placenta, skin, or blood; more preferably, the liver drug metabolizing enzyme component includes at least one of a liver subcellular component, a recombinant liver drug metabolizing enzyme, and a hepatocyte; the liver subcellular component includes at least one of a liver microsome, a liver S9 enzyme component, a cytoplasm, and a drug metabolizing enzyme purified from hepatocytes, and the recombinant drug metabolizing enzyme is preferably a cytochrome P450 enzyme.

[0018] As a further preferred embodiment of the present invention, in step c), the drug metabolizing enzyme component is preferably a liver S9 enzyme component; the reaction solution containing the liver S9 enzyme component, in addition to glucose-6-phosphate disodium salt and glucose-6-phosphate dehydrogenase, also contains at least one of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and nicotinamide adenine dinucleotide phosphate (NADP).

[0019] As a further preferred embodiment of the present invention, in the step b), the external force applied by the mechanical stretching is 0.1 piconewton to 60 piconewtons; preferably, the mechanical stretching is applied by a single-molecule magnetic tweezers device.

[0020] As a further preferred embodiment of the present invention, in the step b), the external force applied by the mechanical stretching is at least 5 different external forces satisfying the range of 0.1 piconewton to 30 piconewton;

[0021] Correspondingly, the step e) will obtain the length changes of the double-stranded DNA corresponding to at least five external force conditions between 0.1 and 30 piconewtons; based on these length changes of the double-stranded DNA, the number of intercalated DNA adducts generated by the covalent binding of the small molecule compound to the double-stranded DNA can be fitted using the following formula 1:

[0022]

[0023] Where F is the force, x(F) is the length of the double-stranded DNA under the force F, and N bp is the number of base pairs of the double-stranded DNA molecule; x dsDNAis the contour length of double-stranded DNA, corresponding to 0.34 nm / base pair; γ is the compound insertion fraction per base pair; A is the persistence length of double-stranded DNA, equal to 50 nm; S is the elastic modulus of double-stranded DNA, equal to 1250 piconewtons; k B is the Boltzmann constant, T is the absolute temperature of the detection;

[0024] The γ value obtained by fitting is equal to the number of embedded adducts inserted in each base pair, and correspondingly, N bp ×γ is the total number of intercalation adducts formed between small molecule compounds and double-stranded DNA.

[0025] As a further preferred embodiment of the present invention, in the step b), the external force applied by the mechanical stretching is an external force satisfying 0.1 piconewton to 60 piconewtons, which can be used to determine whether the small molecule compound forms an intercalation adduct with double-stranded DNA.

[0026] As a further preferred embodiment of the present invention, in the step b), the external force applied by the mechanical stretching is an external force satisfying 10 piconewtons to 50 piconewtons;

[0027] In the step e), based on the change in the length of the double-stranded DNA, the number of intercalated DNA adducts generated by the covalent binding of the small molecule compound to the DNA is calculated using the following formula 2, and the specific quantitative amount is the number N of intercalated adducts inserted into each base pair:

[0028]

[0029] Where, ΔL 10-50pN Represents the length change of double-stranded DNA under an external force of 10 piconewtons to 50 piconewtons; N bp represents the number of base pairs of the double-stranded DNA molecule; Δx represents the increase in the contour length of the double-stranded DNA caused by each compound intercalation and binding, Δx = 0.34 nm;

[0030] Correspondingly, N bp ×N is the total number of intercalated DNA adducts formed by small molecule compounds on a single double-stranded DNA molecule.

[0031] As a further preferred embodiment of the present invention, in the step b), the external force applied by the mechanical stretching is an external force satisfying 0.1 piconewton to 60 piconewton;

[0032] The step d) includes a plurality of different pre-set reaction times;

[0033] In the step e), the detection of the length change of the double-stranded DNA is specifically to detect the length change value of the double-stranded DNA under the detection external force at different reaction times, so as to determine the reaction rate constant k of the small molecule compound to form an intercalation DNA adduct based on the change of the length change value over time;

[0034] Preferably, when the external force in step b) is an external force of 10 piconewtons to 50 piconewtons, in step e), in addition to determining the reaction rate constant k of the small molecule compound to form an intercalation DNA adduct, the number of intercalation DNA adducts generated by the covalent binding of the small molecule compound to DNA can also be calculated using the following formula 2 to obtain the change in the number of intercalation DNA adducts generated by the covalent binding of the small molecule compound to DNA over time, specifically quantitatively determining the change in the number of intercalation adducts N in each base pair over time:

[0035]

[0036] In the formula, ΔL 10-50pN Represents the length change of double-stranded DNA under an external force of 10 piconewtons to 50 piconewtons corresponding to a certain reaction time; N bp represents the number of base pairs of the double-stranded DNA molecule; Δx represents the increase in the contour length of the double-stranded DNA caused by each compound intercalation and binding, Δx = 0.34 nm;

[0037] Correspondingly, N bp ×N is the total number of intercalation adducts formed between the small molecule compounds and DNA during the reaction time.

[0038] Through the above technical scheme conceived by the present invention, compared with the prior art, the present invention reports for the first time the use of drug metabolizing enzyme components to catalyze the formation of DNA adducts in single-molecule manipulation experiments. By introducing drug metabolizing enzymes in single-molecule experiments, the number and reaction kinetics of the formation of embedded adducts between small molecule compounds and DNA are detected, thereby expanding the application of precision measurement methods based on single-molecule manipulation in the pharmacological and toxicological analysis of environmental carcinogens, genotoxic compounds, and anti-tumor drugs. For example, this method can be used for quantitative detection of the ability of known and unknown small molecule compounds such as aromatic ring compounds to form embedded adducts with DNA; it can also be used for the analysis of potential genotoxic components in active ingredients containing composite active ingredients and natural products; it can also be used for the analysis of the catalytic ability of in vitro drug metabolism systems (liver microsomes, liver S9 enzyme components, CYP450 enzymes).

[0039] The present invention can detect any compound that covalently binds to double-stranded DNA and causes a change in the length of both ends of the double-stranded DNA. Taking the detection of intercalated DNA adducts as an example, according to the report of Biebricher et al. (Nature communications, 2015, 6 (1), 1-12): each intercalation of a compound molecule will cause the contour length of DNA to increase by 0.34 nanometers. The method of the present invention can be used to determine whether a covalent binding double-stranded DNA reaction will occur and generate intercalated DNA adducts, and can also be further used to determine the number of intercalated DNA adducts and reaction kinetics. For example, the force-stretch curve of double-stranded DNA can be measured, such as using a jump force test to measure the length of double-stranded DNA under different forces from 0.1 piconewton to 60 piconewton. The method of the present invention is particularly useful for determining whether a small molecule compound containing a planar aromatic ring forms an intercalated DNA adduct with a double-stranded DNA molecule under the catalysis of an in vitro liver metabolic enzyme system.

[0040] After the formation of the embedded DNA adduct, the method of the present invention can wash away the non-covalently bound compounds and free small molecule compounds by solution exchange in the microfluidic reaction pool, and detect the number of embedded adducts on a single DNA by measuring the elongation of double-stranded DNA at the single molecule level. The method of the present invention does not require specific labeling of the ligand or the antibody bound to the ligand, and can be widely used in the detection of embedded DNA adducts of different structures.

[0041] The present invention has universal applicability for detecting exogenous covalently bound DNA compounds activated by the liver or other tissues expressing drug metabolizing enzymes, for example, the ability of known and unknown small molecule compounds to covalently bind to DNA can be analyzed. The scope of use of the present invention for pharmacological and toxicological research includes, for example, analysis of the ability of exogenous environmental carcinogens such as aflatoxin B1 and anti-tumor drugs to form embedded adducts with DNA; analysis of potential genotoxic components that covalently bind to DNA in composite active ingredients and natural products; and comparison and analysis of the catalytic ability of in vitro liver catalytic systems.

[0042] Based on the method of the present invention, the drug metabolizing enzyme liver S9 enzyme component can be used in particular. The following examples show that under the catalysis of the liver S9 enzyme component, the formation of aflatoxin B1 DNA adducts leads to an increase in DNA length, confirming the formation of aflatoxin B1-DNA adducts, an embedded DNA adduct, which is a carcinogenic and mutagenic compound, further proving the carcinogenicity of aflatoxin B1. In addition, the present invention can improve the catalytic effect of the liver S9 enzyme by further adding a NADPH regeneration system (glucose-6-phosphate disodium salt and glucose-6-phosphate dehydrogenase) to the liver S9 enzyme component.

[0043] In addition to being able to determine whether a small molecule compound forms an embedded adduct with double-stranded DNA under the catalysis of a drug metabolizing enzyme (in this case, the external force applied by mechanical stretching may preferably be 0.1 piconewton to 60 piconewtons), the present invention can also analyze the change in DNA length caused by the adduct under an external force of 0.1 piconewton to 30 piconewtons based on the worm chain model (corresponding to formula 1; at this time, at least 5 different external force conditions are required); and, the length change of DNA under any fixed external force between 10 piconewtons and 50 piconewtons can be used to directly calculate the number of embedded DNA adducts (formula 2) and the reaction kinetics of DNA adduct formation (formula 3), thereby simplifying the measurement process and reducing the detection time (this is because within the range of 10 piconewtons to 50 piconewtons, the change in DNA length caused by drug embedded adducts is basically unchanged).

[0044] In summary, the present invention measures the formation of DNA adducts by small molecule compounds under the catalysis of drug metabolizing enzymes in vitro, without the need for DNA purification or labeling, and is more conducive to real-time detection of reaction kinetics. In addition, expensive (fluorescent or radioactive or some other group) labeled nucleotides are not used, and can be widely used in the analysis of covalent binding of genotoxic environmental carcinogens or nucleic acid-targeted anti-tumor drugs to nucleic acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a schematic diagram of the reaction and operation flow diagram of single-molecule magnetic tweezers measuring the formation of intercalated DNA adducts catalyzed by drug metabolizing enzymes in an embodiment of the present invention. Figure 1 a in the figure corresponds to the reaction schematic diagram. Small molecule compounds containing aromatic planar rings interact with DNA and non-covalently embed into DNA. After a period of drug metabolizing enzyme-catalyzed reaction, the activated small molecule compounds covalently bind to DNA to form embedded DNA adducts. Figure 1 b in the figure corresponds to the schematic diagram of the operation process, first anchoring the magnetic ball to the double-stranded DNA on the surface of the glass slide, and measuring the height of the magnetic ball; then, adding compounds and drug metabolizing enzyme components to catalyze the reaction for a period of time to form DNA adducts; then, washing away the free and non-covalently bound small molecule compounds through a liquid replacement experiment; by measuring and comparing the height of the magnetic ball before and after the reaction, the change in height can be used to calculate the number of DNA adducts.

[0046] Figure 2 Schematic diagram of the reaction process of the microfluidic reaction pool and the connection between DNA and magnetic balls on the glass slide in the embodiment. Figure 2 a in the figure corresponds to the schematic diagram of the microfluidic reaction pool in the single-molecule stretching experiment used in the embodiment. The glass slide at the bottom and the cover glass at the top can be divided into two independent 20-50 μl chambers of the microfluidic reaction pool formed by the glass slide and the cover glass by double-sided tape and silicone. The arrow indicates the direction of liquid flow. Figure 2 b in the figure corresponds to a schematic diagram of the reaction process. After the amino groups on the surface of the glass slide react with the chemical cross-linking reagent Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester), the excess Sulfo-SMCC is washed away and then reacted with the double-stranded DNA with a -SH modification at one end. The -Biotin at the other end of the double-stranded DNA is connected by combining with the streptavidin on the surface of the magnetic ball.

[0047] Figure 3 It is a schematic diagram of the working principle. Figure 3 The a in the figure corresponds to the liver metabolic enzyme that catalyzes the covalent reaction of aflatoxin B1 with DNA. Figure 3 The b in it corresponds to the NADPH regeneration principle. Specifically, glucose-6-phosphate dehydrogenase uses glucose-6-phosphate as a substrate, converts NADP into NADPH, and generates 6-phosphogluconolactone.

[0048] Figure 4 This is a comparison of the results of the single-molecule stretching experiment of aflatoxin B1 and double-stranded DNA with and without the catalysis of liver metabolic enzymes. Figure 4 The a in the figure corresponds to the catalysis of liver metabolic enzymes. Specifically, under the catalysis of liver S9 enzyme components, 200 micromoles of aflatoxin B1 reacted with DNA for a period of time. After the free and non-covalently bound aflatoxin B1 were eluted by the liquid exchange experiment, the length of the double-stranded DNA (inverted triangle legend) was significantly increased compared with the length of naked DNA (circular legend), indicating the formation of covalently bound aflatoxin B1-DNA adducts. Figure 4 b in the figure corresponds to the absence of drug metabolizing enzyme catalysis. Specifically, in the absence of catalysis by liver S9 enzyme components, 200 micromoles of aflatoxin B1 non-covalently embedded in double-stranded DNA led to an increase in DNA length (diamond legend). After solution exchange and elution, the double-stranded DNA length (inverted triangle legend) recovered to the length of naked DNA (circle legend), indicating that non-covalently bound aflatoxin B1 can dissociate from DNA and be eluted in the solution exchange experiment.

[0049] Figure 5 is an example of the fitting calculation result of the embodiment. Figure 5 a in the figure corresponds to the change in the contour length of the double-stranded nucleic acid measured by fitting the worm chain model. Specifically, the solid black line in the figure is the force-extension curve of the double-stranded DNA calculated according to the worm chain model; the square points are the force-extension curve of the double-stranded DNA after 200 micromolar aflatoxin B1 was activated in the liver S9 enzyme component at 37 degrees Celsius and added to the microfluidic reaction pool containing DNA, reacted at room temperature for 1 hour and eluted by changing the liquid; Formula 1 can be used to fit the number of embedded DNA adducts in each base pair. Figure 5b in the figure corresponds to the quantitative graph of the elongation results of double-stranded DNA under different forces. Specifically, after aflatoxin B1 reacts with DNA for 1 hour under the catalysis of liver S9 enzyme components, the quantitative graph of the elongation results of double-stranded DNA under different forces after eluting non-covalently bound and free compounds is shown; from the results, it can be seen that the increase in DNA length between 10 piconewtons and 50 piconewtons is basically unchanged, so the change in DNA length under any external force in this range can be substituted into formula 2 to obtain the number of embedded DNA adducts per base pair.

[0050] Figure 6 The time-dependent change of the elongation of double-stranded DNA after elution caused by the formation of aflatoxin B1-DNA adduct; the reaction rate constant k of aflatoxin B1-DNA adduct formation can be obtained by fitting the curve using formula 3. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0052] In general, the present invention is to fix double-stranded DNA between a fixed surface and a movable surface, use single-molecule manipulation technology to measure the length of double-stranded DNA, then add small molecule compounds to be tested and components of the in vitro liver metabolic enzyme system, and after a certain reaction time, wash away free compounds and non-covalently bound compounds by means of changing the liquid, etc. By measuring the length change between the two ends of the double-stranded DNA molecule before and after the reaction, the binding amount of the adduct can be accurately quantified based on the worm-like chain model and the change in the contour length of the DNA caused by the binding of a single small molecule compound.

[0053] like Figure 1 As shown, the method of the present invention may include the following steps:

[0054] a. Immobilize double-stranded DNA molecules between the glass slide surface and the magnetic ball; the method of the present invention is not limited to magnetic tweezers, and any device that allows external force to stretch double-stranded DNA and simultaneously measure the change in the stretched length of the DNA can be used to implement the method of the present invention. For example, double-stranded DNA molecules can be immobilized between two microspheres in optical tweezers, between the sample surface and the microsphere in acoustic tweezers, or between the sample surface and the needle tip in an atomic force microscope.

[0055] b. Use single-molecule manipulation experiments to measure the length of double-stranded DNA under force;

[0056] c. providing small molecule compounds and drug metabolizing enzyme systems to the system;

[0057] d. Fluid exchange experiments to elute free and non-covalently bound compounds;

[0058] e. Measure the change in length of double-stranded DNA under force relative to the length of the original DNA after washing away free and non-covalently bound small molecules. Accurately quantify the amount of embedded adducts based on the length change.

[0059] Wherein, the double-stranded DNA molecule used in step a can be a random sequence or a double-stranded DNA of a specific nucleic acid sequence. It can be a PCR product, a plasmid fragment, or an extracted genomic DNA fragment. When PCR is used to generate double-stranded DNA, biotin and thiol can be introduced into the 5' end of the double-stranded DNA by using oligonucleotide primers modified with biotin and thiol (Nucleic Acids Research. 2014, 42 (13), 8789-8795). When genomic DNA is used, restriction endonucleases can be used to digest the genomic DNA, and ligases can be used to connect the modified oligonucleotides to the ends of the nucleic acids. Preferably, the present invention uses sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (Sulfo-SMCC) to connect the thiol groups of the nucleic acid and the amino groups modified on the glass surface (such as Figure 2 shown).

[0060] In step b, a single molecule manipulation experiment is used. The implementation of the method of the present invention can be implemented by a magnetic tweezers device. Of course, the implementation of the method of the present invention is not limited to the above-mentioned equipment, and any device that allows the measurement of the force-extension curve of a double-stranded nucleic acid can be used to implement the method of the present invention. For example, optical tweezers, atomic force microscopy, or acoustic tweezers can be used. Of course, compared with optical tweezers and atomic force microscopy, magnetic tweezers are easier to parallelize the measurement of multiple molecules for high-throughput measurement.

[0061] The present invention uses a single-molecule manipulation experiment to measure the force-extension curve of double-stranded DNA, and the force-extension curve of double-stranded DNA can be fitted by a worm-like chain model (Nature methods 4.6 (2007): 517-522).

[0062]

[0063] Where F is the force, x(F) is the length of the DNA under the force F, and N bp is the number of base pairs of the sample DNA used in the experiment, x dsDNA is the contour length of DNA, γ is the compound insertion fraction per base pair, A is the persistence length of DNA, S is the elastic modulus, k B is the Boltzmann constant, and T is the absolute temperature. According to the measurement report by Wang et al., the contour length x of double-stranded DNA dsDNA = 0.34 nm / base pair, persistence length A pure DNA =50 nanometers, elastic modulus S = 1250 piconewtons (Biophysical journal, 1997, 72 (3), 1335-1346). According to Biebricher et al. (Nature communications, 2015, 6 (1), 1-12), each insertion of a small compound molecule will cause the contour length of the DNA base pair to increase, which is approximately equal to the contour length of a DNA base pair, so the γ value is equal to the number of embedded adducts inserted in each base.

[0064] Using Formula 1, based on the force-stretching curve measured by magnetic tweezers, the change in nucleic acid contour length can be obtained by fitting the worm chain model, and the number of adducts embedded in each base can be accurately analyzed. When analyzing Formula 1, the test results under at least 5 different external forces can be used for fitting.

[0065] According to formula 1, the elongation of double-stranded DNA under 10-50 piconewtons accounts for 95-98% of the change in DNA contour length. The following examples of the present invention prove that the embedding and addition of small molecule compounds such as aflatoxin B1 do not change the persistence length of DNA (this phenomenon can also be observed in other small molecule-DNA adducts such as doxorubicin-DNA adducts, and is universal). Therefore, the amount of DNA adducts generated under any external force between 10-50 piconewtons can also be used to quantify the number of adducts. According to structural studies and reports by Biebricher et al. (Nature communications, 2015, 6 (1), 1-12), each embedded compound molecule will cause the DNA base pair contour length to increase by 0.34 nanometers. The number of embedded adducts N inserted in each base is calculated based on the elongation of DNA under 10 piconewtons:

[0066]

[0067] In the formula, ΔL 10-50pN Represents the elongation of DNA under a force between 10 and 50 piconewtons. Here, 10 piconewtons is preferred. N bprepresents the number of base pairs of the sample DNA used in the experiment, and Δx represents the increase in DNA contour length after intercalation and binding of each compound (i.e., 0.34 nm).

[0068] Taking the liver S9 enzyme component as an example, in actual operation, the method for detecting the formation of intercalated DNA adducts catalyzed by the liver drug metabolizing enzyme component using the single molecule manipulation technology based on the present invention may include the following specific steps:

[0069] 1) constructing sample double-stranded DNA molecules and fixing the double-stranded DNA molecules in microfluidic reaction pools and magnetic balls;

[0070] 2) Apply external force to the double-stranded DNA molecule and measure the length of the double-stranded DNA;

[0071] 3) Providing liver S9 enzyme components to catalyze the reaction between small molecule compounds and double-stranded DNA;

[0072] 4) Washing away the compounds and liver metabolites after the reaction is complete;

[0073] 5) Apply external force to the double-stranded DNA molecule and measure the change in the length of the double-stranded DNA after the reaction.

[0074] In order to distinguish between covalent binding and non-covalent binding of compounds after drug metabolizing enzyme catalysis, the present invention verifies that in a microfluidic reaction pool, a solution exchange method is used to wash away non-covalently bound compounds and free small molecule compounds, thereby achieving accurate measurement of DNA length changes caused by covalently bound adducts. In addition, by using the negative feedback system (feedback) of the single-molecule magnetic tweezers device to reduce the drift of the instrument, the present invention achieves accurate measurement of DNA length changes of at least several nanometers (~5 nanometers) after a hundred times of channel volume exchange in the microfluidic channel, thereby achieving a minimum detection limit of several adducts per thousand bases.

[0075] In the following examples, after preparing DNA with thiol and biotin labeled at both ends by PCR, the two ends of the double-stranded DNA are fixed to the glass slide and the surface of the magnetic ball by coupling reaction in a microfluidic reaction pool. Figure 1As shown, the length of naked DNA without small molecules is measured using a magnetic tweezers single-molecule manipulation system. Specifically, the height of the permanent magnet is modulated to control the magnetic field strength, and the height of the magnetic ball under different pulling forces is measured. The embedding of the compound causes an increase in the length of the double-stranded DNA. After the catalysis of the enzyme catalyst, the binding of the compound and the double-stranded DNA to form an adduct is irreversible and cannot be washed away by elution with a buffer solution, causing an increase in the length of the double-stranded DNA. The force-stretching curve of the double-stranded DNA is analyzed using a worm model to obtain the contour length of the double-stranded DNA. Thus, the increase in the length of the double-stranded DNA caused by the covalent binding of the compound to the double-stranded DNA is measured to determine the amount of embedded DNA adducts formed.

[0076] The following are specific embodiments:

[0077] Example 1 Preparation of double-stranded DNA for single molecule detection

[0078] 1.1 Double-stranded DNA sample preparation

[0079] Double-stranded DNA with thiol and biotin labeled at the 5' end was prepared by PCR reaction. Using λ-DNA as template, primers labeled with 5' biotin and 5' thiol (Shanghai Shenggong Biotechnology Co., Ltd.) were used to PCR amplify the double-stranded DNA with a product length of 6618 base pairs. Ex Taq enzyme (catalog number: RR001A, Beijing Baoriyi Biotechnology Co., Ltd.) with a final concentration of 5 activity units / μl, 20 mM 10×Ex Taq Buffer (Mg 2+ plus) (catalog number: 9152A, Beijing Bao Ri Yi Biotechnology Co., Ltd.), 2.5 mmol of dNTP Mixture (catalog number: 4030Q, Beijing Bao Ri Yi Biotechnology Co., Ltd.), 2 μg of λ-DNA (catalog number: SD0011, Shanghai Thermo Fisher Scientific Co., Ltd.), 1 μmole of 5'biotin primer (Shanghai Sangon Biotechnology Co., Ltd.), 1 μmole of 5'thiol primer (Shanghai Sangon Biotechnology Co., Ltd.), and then add sterile water to make up to 200 μl and mix well.

[0080] Table 1: Primer sequences

[0081] Primer name sequence 5' Biotin 5'-Biotin-ATTACAAAGTTACCTGTCAAACGGT-3' 5'thiol 5'-thiol-CATATCGAGGAACGCCTTACGT-3'

[0082] 1.2 Purification and recovery of double-stranded DNA

[0083] The target nucleic acid fragment was purified by agarose gel electrophoresis and gel cutting under a blue light irradiator. The target DNA purified by agarose gel was recovered using an agarose gel DNA recovery kit (Beijing Tiangen Biochemical Technology Co., Ltd.).

[0084] Example 2 Immobilization of double-stranded DNA in microfluidic reaction pool

[0085] 2.1 Assembly of microfluidic reaction cell

[0086] Take 24×54 mm and 22×22 mm glass slides (Rebus, Shanghai) and place them on the stand respectively and add 20% Decon90 detergent (Dikang, USA). After ultrasonic treatment for 40 minutes, use ultrapure water to wash away the detergent residue. After drying, place the 22×22 mm glass slide in a clean culture dish for use. The 24×54 mm glass slide is placed in a 10 molar potassium hydroxide solution for overnight treatment, taken out and rinsed with deionized water, and then placed in a container containing ultrapure water for 40 minutes of ultrasonic treatment, and then washed with ultrapure water. After drying, the 24×54 mm glass slide is placed in a methanol solvent containing 1% 3-aminopropyltriethoxysilane ((3-aminopropyl)triethoxysilane, CAS No.: 919-30-2, Cool Chemical Technology (Beijing) Co., Ltd.), soaked in the dark for 25 minutes to modify the amino group, then rinsed with methanol three times, washed with ultrapure water 5-6 times, and dried for use. The dried 24×54 mm glass slide and 22×22 mm glass slide are glued together with double-sided tape or silicone to construct a microfluidic channel. The resulting microfluidic reaction pool, such as Figure 2 As shown in a in .

[0087] 2.2 Fixed reference microspheres

[0088] In the magnetic tweezers experiment, polystyrene microspheres with a diameter of 3 μm fixed on the glass surface were used as reference spheres to calibrate the height of the focal plane. 30 μl of polystyrene microspheres (Cat. No. 17134-15, PolyScience, USA) diluted 100 times with phosphate buffer were added to the microfluidic reaction pool as reference spheres. After waiting for 15 minutes, the free microspheres were washed away with 1× phosphate buffer.

[0089] 2.3 Immobilization of double-stranded DNA on glass surface

[0090] Immobilize double-stranded DNA on the glass surface of the microfluidic reaction pool. Add 100 μL of Sulfo-SMCC solution (sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, Thermo Fisher Scientific, USA, Cat. No. 22322) with a concentration of 1 mg / mL in a ratio of DMSO to 1× phosphate buffer to the microfluidic reaction pool. After reacting for 25 minutes, place the microfluidic reaction pool vertically and elute the remaining SMCC with 500 μL of 1× phosphate buffer. Dilute the prepared double-stranded DNA with an appropriate amount of 1× phosphate buffer to a concentration of 5 ng / μL, pipette 30 μL into the microfluidic reaction pool, react for 45 minutes, and then add 200 μL of blocking solution (mixed with 0.1 g bovine serum albumin, 5 μL β-mercaptoethanol, and 10 mL 1× phosphate buffer) prepared in advance for blocking (process Figure 2 (as shown in b in the figure).

[0091] 2.4 Immobilization of streptavidin-coated superparamagnetic spheres on double-stranded nucleic acids

[0092] Immobilized streptavidin-coated superparamagnetic beads (Dynabeads TM M-280 Catalog No.: 35136, Shanghai Thermo Fisher Scientific Co., Ltd.) on double-stranded nucleic acids. After the magnetic beads were diluted with 1× phosphate buffer at a ratio of 1:200, 20 μl of magnetic beads were added to the microfluidic reaction pool and reacted for 10 minutes, and then the unattached magnetic beads were eluted with 200 μl of 1× phosphate buffer.

[0093] Example 3 Force-stretch curve of double-stranded DNA measured by single-molecule magnetic tweezers

[0094] The single-molecule magnetic tweezers device in this embodiment is produced by BioPSI, Singapore. The magnetic tweezers device controls the height of the permanent magnet through a motor to change the strength of the magnetic field and the magnitude of the pulling force applied to the magnetic ball. The height of the magnetic ball is obtained by Fourier transforming the diffraction ring image of the magnetic ball and the diffraction ring image of the microspheres in different focal planes stored during the calibration measurement. The camera sampling frequency of the magnetic tweezers in this solution is 200 Hz.

[0095] There are many options for mechanical manipulation of nucleic acids using magnetic tweezers, such as constant force, force-jump and force-climbing methods. The preferred experimental method of this embodiment is the force-jump mode. According to the experimental requirements, different forces of different sizes at different intervals from 0.1 to 75 piconewtons are set to measure the length of double-stranded DNA under different forces, thereby obtaining an accurate force-stretching curve. Preferably, this embodiment selects a jump force experiment to measure at intervals of 1 piconewton between 0.1 piconewton and 10 piconewtons, stays at each force for 5 seconds, and measures the height of the magnetic ball after stabilization. The height of the magnetic ball is measured at intervals of 5 or 10 piconewtons between 10 piconewtons and 75 piconewtons. The measurement of the force-magnetic ball height can also be measured by a linearly increasing force-climbing experiment between 0 piconewtons and 75 piconewtons.

[0096] The force-extension curve of naked double-stranded DNA itself is calculated based on the worm chain model (Formula 1), and the contour length x of double-stranded DNA is dsDNA = 0.34 nm / base pair, persistence length A pure DNA =50 nm, elastic modulus S = 1250 piconewtons. The force-extension curve after adding the compound is calculated based on the difference (change in DNA length) of the force-magnetic ball height comparison of naked DNA measured on the same DNA, and the accurate contour length change value and the number of adducts embedded and bound on each base are fitted using formula 1.

[0097] Example 4 Rat liver S9 enzyme component catalyzes aflatoxin B1-DNA adduct

[0098] 4.1 Components of the Rat Liver S9 Enzyme Catalyzed Reaction System

[0099] For the formation of aflatoxin B1-DNA adducts catalyzed by the rat liver S9 enzyme component, a 300 μl reaction system was prepared in 1× phosphate (pH 5.0) containing 10 mM MgCl2. 7.4) Buffer was added with a final concentration of 200 μM aflatoxin B1 (AFB1, cas number: 1162-65-8, Shanghai McLean Biochemical Technology Co., Ltd.), 2 mmol of reduced nicotinamide adenine dinucleotide phosphate (NADPH, cas number: 2646-71-1, Shanghai Aladdin Reagent Co., Ltd.), 0.5 mmol of nicotinamide adenine dinucleotide phosphate (NADP, cas number: 24292-60-2, Shanghai Aladdin Reagent Co., Ltd.), 5 mmol of glucose 6-phosphate disodium hydrate (G-6-P-Na2, cas number: 3671-99-6, Shanghai Aladdin Reagent Co., Ltd.), 1 unit / ml of glucose 6-phosphate dehydrogenase (catalog number: G5885, Sigma-Aldrich) and 1 mg / ml of male rat liver induced S9 enzyme component (catalog number: S10013, Wuhan Plat Biopharmaceutical Technology Co., Ltd.).

[0100] 4.2 Single-molecule magnetic tweezers measurement of DNA length changes caused by aflatoxin B1-DNA adducts

[0101] The steps of measuring the formation of aflatoxin B1-DNA adducts in this protocol are as follows: Figure 4 As shown, the force-stretching curves ( Figure 4 Then, a mixed solution of aflatoxin B1 and S9 enzyme components was prepared, activated in a 37°C water bath for 30 minutes, and then added to a microfluidic reaction pool to react with double-stranded DNA at room temperature (23-25°C) for a fixed time. The force-stretching curve of the double-stranded DNA after adding the reaction mixture was measured ( Figure 4 The reaction conditions and time are set according to the experimental requirements. After the reaction is completed, 2-3 ml of 1× phosphate buffer (100-150 times the volume of the flow chamber) is used to wash away the free compounds that are not covalently bound to DNA in the microfluidic reaction pool. The force-stretching curve of the double-stranded DNA after the elution experiment is completed is measured ( Figure 4 The inverted triangle in figure a is used to determine the amount of adducts formed by the reaction of the compound with DNA by counting the increase in DNA length. Figure 4The b in the figure shows the elution result after the reaction between aflatoxin B1 solution and DNA without adding enzymes or other catalysts. Since aflatoxin B1 and DNA are in a non-covalent embedding binding mode, the non-covalently bound compounds and free compounds will be washed away after the elution experiment with the buffer solution, and finally the DNA height will return to its original length, and the force-stretching curve coincides with that of naked DNA.

[0102] like Figure 4 As shown, the results have demonstrated that when the liver microsomal S9 enzyme component is present, aflatoxin B1 can form an intercalated DNA adduct with DNA. After washing away the free and non-covalently bound compounds, the increase in the length of double-stranded DNA caused by covalently bound aflatoxin B1 can be detected in the examples of the present invention.

[0103] Example 5 Determination of the quantity of embedded aflatoxin B1-DNA adducts

[0104] When using single-molecule magnetic tweezers to perform force-extension experiments on DNA, the force-extension curve of double-stranded DNA at less than 30 piconewtons is fitted by the worm-like chain model, such as Figure 5 As shown, it describes the relationship between the elongation of DNA and the force F, which can be used to calculate the number of embedded DNA adducts:

[0105]

[0106] Where F is the force, x(F) is the length of the DNA under the force F, A is the persistence length of the DNA, S is the elastic modulus, and k B is the Boltzmann constant, T is the absolute temperature, N bp is the number of bases in the sample DNA used in the experiment, x dsDNA is the contour length of DNA, and γ is the number of adducts in each base pair. According to Wang et al. dsDNA = 0.34 nm / base pair, A pure DNA =50 nanometers, S = 1250 piconewtons (Biophysical journal, 72(3), 1335-1346). The DNA used in this example has a N bp =6618 base pairs.

[0107] The force-extension curve was analyzed using the worm chain model to quantify the number of adducts embedded in DNA (adducts / base). The force-extension curve of naked DNA was obtained by fitting the worm chain model reported in the literature. Figure 5The smooth curve shown in a in FIG. The DNA force-extension curve is obtained by calculating the length change of the double-stranded DNA relative to the naked DNA after adding aflatoxin B1 catalyzed by the S9 enzyme component and reacting for 1 hour and changing the liquid. Figure 5 The square mark result shown in a in the figure is then fitted by the worm chain model to obtain the persistence length of the aflatoxin B1-DNA adduct, A AFB1-DNA =46±7 nm. This value is similar to that of naked double-stranded DNA, indicating that the formation of aflatoxin B1-DNA adducts does not change the persistence length of DNA, and the increase in the length of double-stranded DNA is mainly caused by the change in contour length caused by the embedding of small molecules. The γ after the formation of aflatoxin B1-DNA adducts can be obtained by fitting the worm chain model, and the amount of embedded aflatoxin B1-DNA formed after the catalytic reaction of the S9 enzyme component can be calculated.

[0108] Example 6 Another method for calculating the amount of intercalated aflatoxin B1-DNA adducts

[0109] The number of embedded aflatoxin B1-DNA adducts can also be calculated based on the change in DNA length under external force. According to the worm chain model (Formula 1), the elongation of DNA under 10-50 piconewtons accounts for 95-98% of the change in DNA contour length. Therefore, the number of adducts can also be quantified by the elongation of DNA under a fixed external force of 10-50 piconewtons after the generation of aflatoxin B1-DNA adducts. Previous studies have shown that each embedded compound molecule will cause the height of the DNA base pair to increase by 0.34 nanometers. The number of embedded DNA adducts N (adduct / base) is calculated based on the elongation of DNA under 10-50 piconewtons, taking 10 piconewtons as an example:

[0110]

[0111] Where, ΔL 10pN Represents the change in the elongation of DNA under an external force of 10 piconewtons, N bp Represents the number of bases in the sample DNA used in the experiment, N bp = 6618 base pairs, Δx = 0.34 nanometers. In this embodiment, the elongation of DNA under different forces after the S9 enzyme component catalyzes the reaction of aflatoxin B1 with DNA for 1 hour to form an intercalation adduct and the eluted DNA is measured. Figure 5 b. In this embodiment, the elongation after elution at 10 piconewtons is 208±39 nanometers, and the number of aflatoxin B1-DNA generated is calculated by formula 2 to be 92±17 per 10 3The base pairs are consistent with the number of adducts calculated by the γ calculation of the worm chain model. In this embodiment, the length change of double-stranded DNA under 10 piconewtons is preferably used for calculation, and any fixed external force between 10-50 piconewtons can also be used for measurement and calculation.

[0112] Example 7 Determination of the formation kinetics of intercalated aflatoxin B1-DNA adducts

[0113] The aflatoxin B1 reaction solution catalyzed by the S9 enzyme component (for specific preparation, see Example 4) was added to the microfluidic reaction pool, and reacted with DNA for different times, followed by an elution experiment. The curve obtained by the force-stretching experiment was compared with the force-elongation curve of naked DNA to quantify the length change of DNA under 10 piconewtons, and then substituted into Formula 3 to fit the change of DNA elongation after elution with reaction time, thereby obtaining the reaction rate constant k for the formation of aflatoxin B1-DNA adduct. The fitting result is shown in Figure 6 As shown:

[0114] ΔL=ΔL max ×(1-e (-kt) ) (Formula 3)

[0115] Where ΔL represents the length change of the DNA after elution and the naked double-stranded DNA after different reaction times, ΔL max Represents the maximum change in the length of double-stranded DNA after the reaction reaches saturation. The preferred external force range for length change measurement is a fixed external force between 10 piconewtons and 50 piconewtons. A fixed external force between 0.1 piconewtons and 60 piconewtons can also be used for reaction kinetics measurement.

[0116] The above embodiments are only examples. For example, in addition to using the liver S9 enzyme component to catalyze the reaction of the compound and double-stranded DNA to form an adduct, the present invention is also applicable to recombinant drug metabolizing enzymes (P450 enzymes) and liver subcellular components (cytoplasm, S9 enzyme components and microsomal enzymes) and hepatocytes, or in vitro drug metabolizing enzyme systems in other tissues (such as plasma). In addition, when using the present invention to determine whether a small molecule compound forms an embedded adduct with a nucleic acid under the catalysis of a drug metabolizing enzyme, the external force applied by mechanical stretching can preferably be 0.1 piconewton to 60 piconewtons to avoid excessive stretching phase transition of double-stranded DNA, and excessive external force and multiple operations can easily cause nucleic acid chain breakage.

[0117] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting the in vitro drug metabolizing enzyme-catalyzed intercalation and covalent binding of small molecule compounds to double-stranded DNA using single-molecule manipulation, characterized in that: The following steps are involved: a) fixing a double-stranded DNA molecule between a first surface and a second surface, wherein the first surface remains in a fixed position and the second surface is a movable surface; b) Measuring the length of the double-stranded DNA molecule by mechanically stretching the double-stranded DNA molecule; c) applying a reaction solution comprising a small molecule compound and a drug metabolizing enzyme component to the double-stranded DNA molecule and performing a reaction; the small molecule compound is aflatoxin B1, and the drug metabolizing enzyme component comprises at least one of a liver S9 enzyme component or a cytochrome P450 enzyme; d) after the reaction reaches a preset reaction time, washing away small molecule compounds and drug metabolizing enzyme components; e) using the same mechanical conditions as in step b) for mechanical stretching, mechanically stretching the double-stranded DNA molecules, detecting the length change of the double-stranded DNA, thereby judging whether the small molecule compound forms an intercalation adduct with the DNA based on the length change; judging the number and / or reaction kinetics of the intercalation adduct formed between the small molecule compound and the DNA based on whether the intercalation adduct of aflatoxin B1 does not change the persistence length of the DNA; the length change is the length change between the two ends of the double-stranded DNA molecule before and after the reaction; Among them, ① when judging the number of embedded adducts formed by the small molecule compound and DNA: In the step b), the external force applied by the mechanical stretching is an external force satisfying 10 piconewtons to 50 piconewtons; In the step e), based on the change in the length of the double-stranded DNA, the number of intercalated DNA adducts generated by the covalent binding of the small molecule compound to the DNA is calculated using the following formula 2, and the specific quantitative amount is the number N of intercalated adducts inserted into each base pair: In the formula, ΔL 10-50pN Represents the length change of double-stranded DNA under an external force of 10 piconewtons to 50 piconewtons; N bp represents the number of base pairs of the double-stranded DNA molecule; Δx represents the increase in the contour length of the double-stranded DNA caused by each compound intercalation and binding, Δx = 0.34 nm; Correspondingly, N bp ×N is the total number of intercalated DNA adducts formed by small molecule compounds on a single double-stranded DNA molecule; ②When judging the reaction kinetics of the small molecule compound forming an embedded adduct with DNA, In the step b), the external force applied by the mechanical stretching is an external force satisfying 10 piconewtons to 50 piconewtons; The step d) includes a plurality of different pre-set reaction times; In the step e), the length change of the double-stranded DNA is detected, specifically, the length change value of the double-stranded DNA under the detection external force at different reaction times is detected, and the change in the number of intercalated DNA adducts generated by the covalent binding of small molecule compounds to DNA over time is calculated using the following formula 2, which is specifically quantified as the change in the number N of intercalated adducts in each base pair over time: In the formula, ΔL 10-50pN Represents the length change of double-stranded DNA under an external force of 10 piconewtons to 50 piconewtons corresponding to a certain reaction time; N bp represents the number of base pairs of the double-stranded DNA molecule; Δx represents the increase in the contour length of the double-stranded DNA caused by each compound intercalation and binding, Δx = 0.34 nm; Correspondingly, N bp ×N is the total number of intercalation adducts formed between the small molecule compounds and DNA during the reaction time.

2. The method according to claim 1, characterized in that: In the step a), the fixation is to attach at least one base of one strand of the double-stranded DNA directly or indirectly to the first surface, and at least one base of the other strand of the double-stranded DNA directly or indirectly to the second surface; Alternatively, the fixation is to attach at least one base of one strand of the double-stranded DNA directly or indirectly to the first surface, and at least one base at a different position of the strand to the second surface directly or indirectly.

3. The method according to claim 1, characterized in that: In the step a), the first surface is a glass slide surface, and the second surface is a magnetic sphere surface.

4. The method according to claim 1, characterized in that: In the step c), the drug metabolizing enzyme component is a liver S9 enzyme component; the reaction solution containing the liver S9 enzyme component, in addition to glucose-6-phosphate disodium salt and glucose-6-phosphate dehydrogenase, also contains at least one of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and nicotinamide adenine dinucleotide phosphate (NADP).

5. The method according to claim 1, characterized in that: In the step b), the mechanical stretching is applied by a single-molecule magnetic tweezers device.

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