Research method for interaction between new pollutant and DNA

Abstract

The invention discloses a research method for interaction between a new pollutant and DNA (deoxyribonucleic acid). The method comprises the following steps: optimizing molecular structures of the new pollutant and deoxynucleoside by using a DFT (discrete fourier transform) method in quantum chemistry calculation software; utilizing Multiwfn software to obtain contribution of each atom to a molecular orbital and atomic charge distribution; searching a potential site of a new pollutant and a nucleophilic site of deoxynucleoside; molecular docking software is used for carrying out docking calculation on the new pollutant and the double-stranded DNA fragment, and an optimal binding position is searched; a UV-Vis ultraviolet spectrophotometer is used for carrying out ultraviolet spectrum titration on the interaction of the new pollutant and the double-stranded DNA; the method comprises the following steps: carrying out gradient elution by adopting an ultra-high performance liquid chromatography-mass spectrometry method, detecting deoxynucleoside by adopting a multiple reaction detection method, calculating a reaction rate, and identifying a deoxyribose unit which is easily subjected to an addition reaction with new pollutants; and analyzing the detection result. According to the invention, the computing resource cost is reduced during detection.

Description

Technical Field

[0001] The present invention relates to the technical field of research on the toxic effects of new pollutants, and in particular to a method for studying the interaction between new pollutants and DNA. Background Art

[0002] Emerging pollutants refer to a class of toxic and hazardous chemical substances that exhibit strong biological toxicity, high environmental persistence, and easy bioaccumulation after entering the environment, thus posing significant risks to the ecological environment and human health. There are many types of emerging pollutants, including pharmaceuticals and personal care products, drinking water disinfection by-products (such as halogenated benzoquinones), endocrine disruptors, persistent organic pollutants, microplastics, etc. Compared with traditional pollutants that have been widely recognized and strictly controlled, the particularity of emerging pollutants is that their environmental and health risks have recently received attention, and the existing environmental monitoring and management system has not yet been able to fully and effectively cover and control them, so they are called "new" pollutants. Emerging pollutants pose new challenges in the field of environmental protection. They enter the water, soil and atmospheric environment through various pathways such as industrial emissions, agricultural activities, and domestic sewage. They are characterized by wide distribution, low concentration levels (usually in the ng / L to μg / L level), environmental persistence, bioaccumulation and potential biological toxicity.

[0003] DNA, the core genetic material of living organisms, carries the genetic information necessary for nearly all life activities. The structural integrity and sequence accuracy of DNA molecules are crucial for normal cellular function, the stable transmission of genetic information, and individual health. When exogenous chemicals (such as new pollutants in the environment) enter an organism, they may interact with DNA through various pathways. These interactions can be divided into two categories: non-covalent and covalent binding. Non-covalent binding primarily involves electrostatic attraction, hydrophobic interactions, van der Waals forces, and binding to DNA through intercalation and groove binding. While non-covalent interactions do not directly alter the primary structure of DNA, they may affect DNA conformation, replication, and transcription. Covalent binding occurs when pollutants or their metabolic activation products form stable chemical bonds with nucleophilic sites on DNA bases (such as the N7 and O6 positions of guanine and the N1, N3, and N7 positions of adenine), forming DNA adducts. The formation of DNA adducts is a key initiating step in the induction of mutations and tumors by chemical carcinogens. They can interfere with normal DNA replication and repair, leading to gene mutations, chromosomal aberrations, and even cell apoptosis or carcinogenesis.

[0004] Therefore, in-depth research on the interaction mechanism between new pollutants and DNA, elucidating their binding mode, identifying key reaction sites, and evaluating their ability and efficiency to form DNA adducts are of vital scientific significance and practical application value for revealing the genotoxicity, carcinogenic potential and health risks of new pollutants.

[0005] At present, the methods for studying the interaction between chemicals and DNA mainly include: (1) computational simulation methods, such as molecular docking and quantum chemical calculations, which can predict the possible sites of interaction, binding energy and molecular conformation from a theoretical perspective, but their accuracy depends on the selection of force field parameters and computational models, and often requires high-performance computing resources; (2) spectroscopic methods, such as ultraviolet-visible absorption spectroscopy (UV-Vis), fluorescence spectroscopy, circular dichroism spectroscopy, etc., are often used to study non-covalent interactions. By monitoring the changes in spectral parameters, the binding mode, binding constant and other information can be inferred, but it is difficult to directly provide evidence of covalent binding and structural information; (3) chromatography-mass spectrometry technology, especially liquid chromatography-tandem mass spectrometry (LC-MS / MS), is currently one of the most effective and sensitive means to detect and confirm DNA adducts. It can perform qualitative and quantitative analysis of trace adducts in complex biological samples, but the instruments and equipment are expensive, the sample pretreatment is relatively complicated, and the professional skills of the analyst are required to be high; (4) other biological methods, such as gel electrophoresis, comet assay, γ-H2AX determination, etc., can be used to assess the degree of DNA or chromosome damage.

[0006] However, existing research methods often have some limitations. For example, a single computational simulation or spectroscopic method is difficult to fully reflect the full picture of the interaction between new pollutants and DNA, especially it is difficult to take into account both the preliminary screening of non-covalent interactions and the precise identification of covalent adducts. Methods that rely on large instruments such as complex biological experimental platforms face problems such as high equipment costs, cumbersome operations, and long experimental cycles, which are not conducive to rapid and economical high-throughput screening and preliminary risk assessment of a large number of new pollutants. In addition, in actual environmental exposure, organisms are often exposed to multiple low-dose pollutants at the same time, and their interactions with DNA may be more complex. There is an urgent need to develop research methods that are both accurate, economical, and comprehensive to effectively assess the potential genotoxicity of new pollutants. Summary of the Invention

[0007] The purpose of the present invention is to propose a method for studying the interaction between new pollutants and DNA to address the shortcomings of the existing technology in studying the interaction between new pollutants and DNA, such as high reliance on simulation calculations, high costs caused by high-performance computing resources, and difficulty in economically and comprehensively conducting experimental analysis to simultaneously examine non-covalent and covalent interactions.

[0008] The object of the present invention is achieved through the following technical solution: a method for studying the interaction between a new pollutant and DNA, comprising the following steps:

[0009] (1) Using density functional theory in the quantum chemistry calculation software Gaussian, the molecular structures of the new pollutants and deoxynucleosides were optimized, and the optimized result files were exported;

[0010] (2) Import the result file exported in step (1) into Multiwfn software to obtain the contribution of each atom to the molecular orbital and the atomic charge distribution, and visualize them in a table form;

[0011] (3) Based on the tabular atomic contributions to molecular orbitals and atomic charge distribution, the electrophilic sites in the new pollutants are found by searching for the top M atoms that contribute most to the lowest unoccupied molecular orbital and the top N atoms with the largest positive charge and the largest charge number in the new pollutants. At the same time, the nucleophilic sites in the deoxynucleoside are found by searching for the top P atoms that contribute most to the highest occupied molecular orbital and the top Q atoms with the largest negative charge and the largest charge number in the deoxynucleoside.

[0012] (4) Using the molecular docking software AutoDock Vina, multiple docking calculations were performed on the new pollutants and double-stranded DNA fragments, and the optimal binding position was searched based on the docking scores of the molecular docking software AutoDock Vina;

[0013] (5) UV-Vis spectrophotometer was used to perform UV spectrophotometric titration on the interaction between the new pollutant and double-stranded DNA;

[0014] (6) Using ultra-high performance liquid chromatography-mass spectrometry for gradient elution, deoxynucleosides were detected using a liquid chromatography column with a multiple reaction detection method, and the reaction rate of deoxynucleosides with the new pollutants after 12 hours was calculated to identify the deoxyribose units that are prone to adduction reactions with the new pollutants;

[0015] (7) Analyze the test results obtained in steps (3) to (6).

[0016] Furthermore, in the step (1), when the density functional theory method is used to optimize the molecular structures of the new pollutant and the deoxynucleoside, the density functional theory condition is a functional method of the B3LYP / 6-31G** basis set.

[0017] Furthermore, the step (2) specifically includes:

[0018] First, import the result file exported in step (1) into the Multiwfn software, then check the electrostatic potential surface and average local ionization energy of each atom in the compound in the result file, and then use the Mulliken, C-squared Population Analysis and Hirshfeld methods in the Multiwfn software to analyze and calculate the contribution of each atom to the molecular orbital. Use the Hirshfeld, Atomic Dipole Corrected Hirshfeld Atomic Charge and Merz-Kollman methods in the Multiwfn software to analyze and obtain the atomic charge distribution. Finally, visualize the contribution of each atom to the molecular orbital and the atomic charge distribution in a table.

[0019] Furthermore, in step (5), the concentration of the double-stranded DNA solution is 100 mg / L; the final concentration gradient of the new pollutant is 0, 1, 2, 4, 7, 11, 16, 20, 25, 34, 42, 54, 70, 84, 101, and 116 μg / L.

[0020] Furthermore, in step (5), the ultraviolet spectroscopic titration specifically includes:

[0021] The double-stranded DNA solution was mixed with different volumes of the new contaminant solution to obtain a total volume of 20 mL and a mixture system with different gradients of the final concentration of the new contaminant. The system was incubated at 160 rpm, pH 7.0 and 25°C for 2 h, and then the samples were taken and the absorbance was measured using a UV-1800 spectrometer in the wavelength range of 250 to 270 nm.

[0022] Furthermore, in step (6), the reaction conditions of the deoxynucleoside are specifically as follows: 1 mL of 1 g / L dimethyl sulfoxide solution of the four new pollutants and 100 μL of 1 g / L dimethyl sulfoxide solution of deoxynucleoside are placed in a test tube, and reacted in a water bath at 37° C. for 12 hours.

[0023] Furthermore, in step (6), the conditions of the gradient elution are specifically:

[0024] Within 0-2 min, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid was reduced from 95:5 to 80:20;

[0025] Within 2-6 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid was increased from 80:20 to 99.9:0.1;

[0026] Within 6-10 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid was maintained at 99.9:0.1;

[0027] Within 10-11 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid decreased from 99.9:0.1 to 95:5;

[0028] The volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid was maintained at 95:5 within 11-12 minutes.

[0029] Furthermore, in step (6), the liquid chromatography column used is a BEH C18 liquid chromatography column with an inner diameter of 2.1 mm and a column length of 150 mm.

[0030] Furthermore, in step (6), the conditions of the multiple reaction detection method are specifically shown in the following table:

[0031]

[0032] Compared with the existing technology, the beneficial effects of the present invention are as follows: the present invention integrates low-cost computational simulation and economical and efficient experimental verification means; the simulation calculation part does not require the help of large computer servers, which significantly lowers the equipment threshold and significantly reduces the equipment cost; the experimental analysis part covers both computational simulation, non-covalent binding (ultraviolet spectroscopy characterization) and covalent addition (UPLC-MS / MS detection) interaction mechanisms, and can economically and quickly perform relatively comprehensive detection and characterization of the two main modes of action of new pollutants with DNA, namely non-covalent binding and covalent addition, without relying on expensive dedicated computers and building complex test platforms, to achieve comprehensive detection of the new pollutant-DNA interaction mode; the present invention provides a simple, cost-effective and reliable technical approach for the study of the toxicity mechanism of new pollutants and environmental risk assessment, and has broad application prospects in the fields of environmental science, analytical chemistry and research on the toxicity mechanism of pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 A flow chart of the method for studying the interaction between the novel pollutants and DNA of the present invention;

[0034] Figure 2 Schematic diagram of the chemical structure of the new pollutants halogenated benzoquinone and deoxynucleoside;

[0035] Figure 3 Number the atoms of the new contaminants and deoxynucleosides during Gaussian calculations;

[0036] Figure 4This is a schematic diagram of the optimal binding position after the new pollutant is docked with the double-stranded DNA molecule;

[0037] Figure 5 Schematic diagram of UV spectrometric titration results of new pollutants and double-stranded DNA;

[0038] Figure 6 The detection signal intensity and chromatographic peaks of the four deoxynucleosides after a total elution gradient of 12 minutes in liquid chromatography tandem mass spectrometry detection;

[0039] Figure 7 Schematic diagram of the reaction degree between monodeoxynucleosides and new pollutants. DETAILED DESCRIPTION

[0040] Exemplary embodiments will be described in detail herein, with examples illustrated in the accompanying drawings. When the following description refers to the drawings, identical numbers in different figures represent identical or similar elements, unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present application. Rather, they are merely examples of apparatus and methods consistent with certain aspects of the present application, as detailed in the appended claims. It should be understood that the foregoing general description and the detailed description that follows are exemplary and illustrative only and do not limit the present application.

[0041] The terms used in this application are for the purpose of describing specific embodiments only and are not intended to limit this application. As used in this application and the appended claims, the singular forms "a," "an," "the," and "the" are intended to include the plural forms, unless the context clearly indicates otherwise. It should also be understood that the term "and / or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0042] It should be understood that although the terms first, second, third, etc. may be used in this application to describe various information, these information should not be limited to these terms. These terms are only used to distinguish information of the same type from each other. For example, without departing from the scope of this application, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Depending on the context, the word "if" as used herein may be interpreted as "at the time of..." or "when..." or "in response to determination." Moreover, the term "comprises," "comprising," or any other variant thereof is intended to cover non-exclusive inclusion, so that the process or method comprising a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process or method. In the absence of further restrictions, the elements defined by the statement "comprising a..." do not exclude the presence of other identical elements in the process, method, article, or device comprising the elements.

[0043] The present invention will be described in detail below with reference to the accompanying drawings. Unless there is any conflict, the features of the following embodiments and implementations may be combined with each other.

[0044] The present invention's method for studying the interaction between new pollutants and DNA first uses computer simulation technology, specifically the quantum chemical calculation software Gaussian, to optimize the molecular structures of the new pollutants and deoxynucleosides and predict their most likely reaction sites. The molecular docking software Autodock Vina is then used to simulate the interaction process and binding mode between the new pollutants and DNA molecules on an ordinary computer. Secondly, at the experimental level, ultraviolet spectroscopy is used to comprehensively detect the non-covalent interaction characteristics between the new pollutants and double-stranded DNA. Finally, ultra-performance liquid chromatography-mass spectrometry (UPLC-MS / MS) in multiple reaction monitoring (MRM) mode is used to detect the solution after the reaction between the new pollutants and deoxynucleosides to determine their reaction rate and identify the deoxyribose units that are susceptible to adduction reactions with the new pollutants.

[0045] See also Figure 1 The method for studying the interaction between the novel pollutant and DNA of the present invention specifically comprises the following steps:

[0046] (1) In the quantum chemical calculation software Gaussian, the density functional theory (DFT) method was used to optimize the molecular structures of the new pollutants and deoxynucleosides, and the optimized result files were exported.

[0047] It should be understood that DFT is a method for studying the electronic structure of multi-electron systems. DFT has a wide range of applications in physics and chemistry, particularly in studying the properties of molecules and condensed matter. It is one of the most commonly used methods in condensed matter physics, computational materials science, and computational chemistry.

[0048] Furthermore, when using the DFT method to optimize the molecular structures of new pollutants and deoxynucleosides, the DFT conditions were functional methods based on the B3LYP / 6-31G** basis set.

[0049] (2) Import the result file exported in step (1) into Multiwfn software to obtain the contribution of each atom to the molecular orbital and the atomic charge distribution, and visualize them in a table form for easy comparison.

[0050] Specifically, Multiwfn software is a powerful, open-source quantum chemical wave function analysis software that supports the output files of a variety of quantum chemical calculation programs and provides a wealth of wave function analysis tools. It is widely used in the fields of electronic structure analysis, chemical bond research, molecular property prediction, etc. Therefore, first import the result file exported in step (1) into Multiwfn software, then check the electrostatic potential surface and average local ionization energy of each atom of the compound in the result file, and then use the Mulliken, C-squared Population Analysis (SCPA) and Hirshfeld methods in Multiwfn software to analyze and calculate the contribution of each atom to the molecular orbital. Use the Hirshfeld, AtomicDipole Corrected Hirshfeld Atomic Charge (ADCH) and Merz-Kollman (MK) methods in Multiwfn software to analyze and obtain the atomic charge distribution. Finally, visualize the contribution of each atom to the molecular orbital and the atomic charge distribution obtained in a table for easy comparison.

[0051] (3) Based on the tabular contributions of atoms to molecular orbitals and the atomic charge distribution, the electrophilic sites in the new pollutants are found by searching for the top M atoms that contribute most to the lowest unoccupied molecular orbital (LUMO) and the top N atoms with the largest positive charge and the largest charge number. At the same time, the nucleophilic sites in the deoxynucleoside are found by searching for the top P atoms that contribute most to the highest occupied molecular orbital (HOMO) and the top Q atoms with the largest negative charge and the largest charge number.

[0052] It should be understood that LUMO is a key concept in molecular orbital theory, referring to the lowest energy unoccupied molecular orbital in the ground state (unexcited electrons). Together with HOMO, it determines the electronic properties, chemical reactivity, and optical characteristics of a molecule.

[0053] It should be noted that, since three methods are used to obtain the contribution of atoms to molecular orbitals and the atomic charge distribution in step (2), the tendency of each site may be different due to different reaction types. When searching for electrophilic sites and nucleophilic sites in step (3), they are determined based on one of the reaction trends. For example, the electrophilic site is determined based on the contribution of atoms to molecular orbitals analyzed by the Mulliken method, the SCPA method, or the Hirshfeld method, and the nucleophilic site is determined based on the atomic charge distribution analyzed by the Hirshfeld method, the ADCH method, or the MK method.

[0054] (4) The molecular docking software AutoDock Vina was used to perform multiple docking calculations on the new pollutants and double-stranded DNA fragments and the optimal binding position was searched based on the docking scores of the molecular docking software AutoDock Vina.

[0055] Specifically, the chemical structures of four new contaminants were retrieved from the PubChem database. X-ray crystallographic structure data for DNA were obtained from the RCSB Protein Data Bank (PDB ID 3IXN). Preprocessing was performed to remove existing water molecules from the structure, complete the hydrogen atoms in the DNA molecule, and set the treated DNA molecule as the receptor for the docking process. The previously prepared receptor and ligand molecule files were selected from the menu. The grid options dialog box was opened to set the grid box. The grid size was set to 60×60×100, the grid spacing to the default value, and the grid center to DNA. The program path and name, as well as the log file, were set. The program was run to perform the docking calculation and export the result file. After the molecular docking calculation was completed, the result file was imported using PyMOL molecular visualization software to observe and analyze the resulting docked complex conformation. The docking score in Autodock Vina software is based on Affinity. A smaller value indicates stronger binding, indicating the optimal binding position.

[0056] (5) Use UV-Vis spectrophotometer to perform ultraviolet spectral titration on the interaction between new pollutants and double-stranded DNA.

[0057] Furthermore, in step (5), the concentration of the double-stranded DNA solution is 100 mg / L; the final concentration gradient of the new pollutant is 0, 1, 2, 4, 7, 11, 16, 20, 25, 34, 42, 54, 70, 84, 101, and 116 μg / L.

[0058] Furthermore, in step (5), the UV spectroscopic titration specifically includes: mixing the double-stranded DNA solution with different volumes of new pollutant solution to obtain a mixture system with a total volume of 20 mL and a final concentration of the new pollutant in different gradients, incubating the system at 160 rpm, pH 7.0 and 25° C. for 2 hours, and then taking a sample and measuring the absorbance using a UV-1800 spectrometer in the wavelength range of 250 to 270 nm.

[0059] (6) Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS) was used for gradient elution, and deoxynucleosides were detected by liquid chromatography column using multiple reaction monitoring (MRM). The reaction rate of deoxynucleosides with new pollutants after 12 hours was calculated to identify the deoxyribose units that are prone to adduction reactions with new pollutants.

[0060] Furthermore, in step (6), the reaction conditions for the deoxynucleosides are as follows: 1 mL of a 1 g / L dimethyl sulfoxide (DMSO) solution of each of the four new pollutants and 100 μL of a 1 g / L dimethyl sulfoxide (DMSO) solution of the deoxynucleosides are placed in a test tube and reacted in a 37°C water bath for 12 hours. There are a total of 16 combinations of new pollutants and deoxynucleosides. Three replicates are set up for each combination.

[0061] Furthermore, in step (6), the conditions for gradient elution are specifically:

[0062] Within 0-2 min, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid was reduced from 95:5 to 80:20;

[0063] Within 2-6 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid was increased from 80:20 to 99.9:0.1;

[0064] Within 6-10 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid was maintained at 99.9:0.1;

[0065] Within 10-11 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid decreased from 99.9:0.1 to 95:5;

[0066] The volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid was maintained at 95:5 within 11-12 minutes.

[0067] Furthermore, in step (6), the liquid chromatography column used is a BEH C18 liquid chromatography column with an inner diameter of 2.1 mm and a column length of 150 mm.

[0068] Furthermore, in step (6), the conditions of the multiple reaction detection method are specifically shown in Table 1:

[0069] Table 1: Multiple reaction monitoring method setup conditions

[0070]

[0071] (7) Analyze the test results obtained in steps (3) to (6).

[0072] In summary, the present invention first uses Gaussian 09W software to optimize the structures of the new pollutant and deoxynucleoside to predict the most likely reaction sites for their interaction. Second, Autodock Vina docking software is used to simulate the interaction between the new pollutant and DNA. Subsequently, the interaction pattern between the new pollutant and double-stranded DNA is characterized by ultraviolet spectroscopy. Finally, ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) in multiple reaction monitoring mode (MRM) is used to detect the post-reaction deoxynucleoside, calculate its reaction rate with the new pollutant, and identify deoxynucleosides that are prone to adduction reactions.

[0073] The method for studying the interaction between the novel pollutants and DNA of the present invention will be described in detail below with reference to the examples, and the purpose and effect of the present invention will become more apparent.

[0074] Example 1

[0075] This example investigates the sites where a common and highly cytotoxic disinfection byproduct, namely a new pollutant, halobenzoquinone, easily reacts with four typical deoxynucleosides, and specifically includes the following steps:

[0076] (1) First, draw the following in Gaussian 09W software: Figure 2 The chemical structures of the new pollutants halogenated benzoquinones and deoxynucleosides are shown, and the structural models are optimized under the B3LYP / 6-31G** basis set.

[0077] (2) Import the result file obtained in step (1) into the Multiwfn software, view the electrostatic potential surface and average local ionization energy of each atom of the compound, further analyze the results with the Multiwfn software and visualize the results accordingly, and find the electrophilic sites in the new pollutant and the nucleophilic sites in the deoxynucleoside. When analyzing the contribution of each atom to the molecular orbital, three calculation methods were selected: Mulliken, C-squared Population Analysis (SCPA) and Hirshfeld; when analyzing the atomic charge distribution, three methods were selected: Hirshfeld, Atomic Dipole Corrected Hirshfeld Atomic Charge (ADCH) and Merz-Kollman (MK). The atomic numbers of the new pollutants and deoxynucleosides in the calculation process in Gaussian software are as follows: Figure 3 shown.

[0078] The calculated contributions of each atom to the molecular orbitals and atomic charge distribution values ​​of the four new pollutants 2,6-dichlorobenzoquinone (2,6-DCBQ), 2,6-dibromobenzoquinone (2,6-DBBQ), 2,5-dichlorobenzoquinone (2,5-DCBQ) and 2,5-dibromobenzoquinone (2,5-DBBQ) and the four deoxynucleosides are shown in Tables 2 to 9.

[0079] Table 2: Contribution of each atom to the molecular orbital and atomic charge distribution values ​​on 2,6-DCBQ

[0080]

[0081] Table 3: Contribution of each atom to the molecular orbital and atomic charge distribution values ​​on 2,5-DCBQ

[0082]

[0083]

[0084] Table 4: Contribution of each atom to the molecular orbital and atomic charge distribution values ​​on 2,6-DBBQ

[0085]

[0086] Table 5: Contribution of each atom to the molecular orbital and atomic charge distribution values ​​on 2,5-DBBQ

[0087]

[0088]

[0089] Table 6: Contribution of each atom on 2'-deoxyguanosine to the molecular orbital and the atomic charge distribution value

[0090]

[0091]

[0092] Table 7: Contribution of each atom to the molecular orbital and atomic charge distribution values ​​on 2'-deoxyadenosine

[0093]

[0094]

[0095] Table 8: Contribution of each atom to the molecular orbital and atomic charge distribution values ​​on 2'-deoxycytidine

[0096]

[0097]

[0098] Table 9: Contribution of each atom to the molecular orbital and atomic charge distribution values ​​on β-thymidine

[0099]

[0100]

[0101] (3) Result analysis: Based on the above-mentioned visualized Tables 1 to 9, the results were analyzed according to basic and well-known chemical principles. From the perspective of molecular orbital theory, the lowest unoccupied molecular orbital (LUMO) of the new pollutant halobenzoquinone is the orbital in its molecule that is most receptive to external electrons, that is, the most electrophilic molecular orbital. Therefore, when interacting with a nucleophile (such as the nucleophilic site on DNA that may be involved in the present invention), the LUMO orbital is usually the main target for the nucleophile to provide electrons, such as Figure 2 Further analysis shows that when a DNA molecule interacts with a reagent having multiple electrophilic reaction centers (or the formed preliminary adduct itself still has multiple electrophilic sites), a stable cyclic adduct structure is easily formed between the nitrogen atoms adjacent to each other on the base. For example, Figure 2 The reactivity of thymine with electrophiles is generally very low, as it lacks an amino group that can provide a lone pair of electrons as a nucleophilic reaction site.

[0102] Example 2

[0103] This example investigates the docking results of new pollutant halobenzoquinone with double-stranded DNA molecules, specifically including the following steps:

[0104] (1) First, the chemical structures of four new pollutants, including 2,6-dichlorobenzoquinone (2,6-DCBQ), 2,6-dibromobenzoquinone (2,6-DBBQ), 2,5-dichlorobenzoquinone (2,5-DCBQ), and 2,5-dibromobenzoquinone (2,5-DBBQ), were retrieved and obtained from the PubChem database. After obtaining the initial structures of the above new pollutants, the MM2 molecular force field built into Chem3D software (CambridgeSoft, MA, USA) was used to perform geometric optimization of their three-dimensional spatial structures under conditions simulating physiological pH 7.0.

[0105] (2) X-ray crystallographic structure data for deoxyribonucleic acid (DNA) were obtained from the Protein Data Bank (PDB) using the PDB accession number 3IXN. The 3IXN structure represents a typical B-form DNA double helix conformation and is conformationally consistent with the model DNA used in subsequent experimental studies of the present invention.

[0106] (3) Before performing the docking calculation, the DNA macromolecular structure is subjected to necessary preprocessing, which includes: removing the original water molecules in the structure, supplementing the DNA molecule with complete hydrogen atoms, and setting the processed DNA molecule as the receptor of the docking process.

[0107] (4) The molecular docking software AutoDock Vina was used to perform molecular docking simulation calculations of the halobenzoquinone compound molecule and the DNA receptor. The previously prepared receptor molecule and ligand molecule files were selected from the menu. The grid options dialog box was opened to set the grid box. The grid size was set to 60 × 60 × 100, the grid spacing was the default value, the grid center was set to DNA, the program path and name, and the record file were set, and the program was run for calculation.

[0108] (5) After the molecular docking calculations were completed, the resulting docked complex conformation was observed and analyzed using PyMOL molecular visualization software. The docking score in the Autodock Vina software is Affinity, with smaller values ​​indicating stronger binding. The experimentally obtained binding energy data are shown in Table 10.

[0109] Table 10: Binding energies of the first seven conformations of four new pollutants after docking with DNA molecules

[0110]

[0111] (6) Schematic diagram of the optimal binding position after docking of the new pollutant with the double-stranded DNA molecule. Figure 4 As shown. Analysis of the docking results showed that in all the docking simulations of new pollutants and DNA investigated, the optimal position for the new pollutant molecules to bind to DNA was, without exception, located within the groove of the DNA double helix structure, and all bound to deoxyguanosine. This result suggests that the new pollutant molecules tend to interact with DNA in a groove-bound manner, in which the planar quinone ring structure of the new pollutant molecules is further embedded in the minor groove region of the DNA. Specifically, they form a stacking structure similar to a "sandwich".

[0112] Example 3

[0113] This example performs UV spectrophotometric titration of new pollutants and double-stranded DNA and analyzes the test results, specifically including the following steps:

[0114] (1) First, prepare a calf thymus DNA solution with a concentration of 100 mg / L.

[0115] (2) Subsequently, the ctDNA solution was mixed with different volumes of 500 μg / L halobenzoquinone (HBQs) stock solution in several reaction tubes. By precisely controlling the volume of the added HBQs stock solution, the final working concentrations of HBQs in each independent reaction system were 0 μg / L, 1 μg / L, 2 μg / L, 4 μg / L, 7 μg / L, 11 μg / L, 16 μg / L, 20 μg / L, 25 μg / L, 34 μg / L, 42 μg / L, 54 μg / L, 70 μg / L, 84 μg / L, 101 μg / L, and 116 μg / L, respectively. The total volume of each reaction system was adjusted to 20 mL.

[0116] (3) The prepared mixed solution was placed in a thermostatic shaker and incubated at a set pH of 7.0 and a temperature of 25°C at a shaking rate of 160 rpm for 2 hours to ensure sufficient interaction between DNA and HBQs.

[0117] (4) After incubation, 2 mL of the reaction mixture was taken from each concentration gradient sample and injected into a 1 cm quartz cuvette for spectral analysis.

[0118] (5) Finally, the absorbance of each sample placed in a quartz cuvette was measured using a UV-1800 UV-visible spectrophotometer (Shimadzu). The measurement process was carried out at a precisely controlled temperature of (25 ± 0.1) °C, and the scanning wavelength range was set between 250 nm and 270 nm.

[0119] (6) Schematic diagram of UV spectrophotometric titration results of new pollutants and double-stranded DNA. Figure 5As shown in the figure, the results showed that the absorbance of DNA at the characteristic absorption peak at 260 nm exhibited different trends for each new pollutant (specifically 2,6-DCBQ, 2,6-DBBQ, 2,5-DCBQ, and 2,5-DBBQ) as the concentration gradually increased from 0 μg / L to 116 μg / L. At lower concentrations of specific HBQs (for example, when the concentration of 2,6-DCBQ ranged from 0 to 11 μg / L and the concentration of 2,6-DBBQ ranged from 0 to 25 μg / L), the double helix structure of DNA molecules became more compact or ordered, possibly due to electrostatic binding or base intercalation of the new pollutants, resulting in a spectral hypochromic effect. However, when the concentration of new pollutants increased to a higher level (e.g., ≥16 μg / L), the absorption spectrum of DNA was observed to show a significant hyperchromic effect. This phenomenon indicates that in the presence of higher concentrations of new pollutants, the exposure of DNA bases increased, which may be related to a certain degree of unwinding or disturbance of the local structure of DNA.

[0120] Example 4

[0121] This example establishes a deoxynucleoside detection method based on ultra-performance liquid chromatography-mass spectrometry (UPLC-MS / MS) multiple reaction detection mode. The method calculates the reaction rate of deoxynucleosides with new pollutants after 12 hours, identifies deoxyribose units that are prone to adduction reactions with new pollutants, and comprehensively analyzes the results of the interaction between the new pollutant halobenzoquinone and DNA. The method specifically includes the following steps:

[0122] (1) Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was performed using a BEH C18 (2.1 mm × 150 mm, Waters) mobile phase with a 0.1% formic acid aqueous solution and a 0.1% formic acid acetonitrile solution as the mobile phases, with a 12-minute gradient elution. The concentrations of the four deoxynucleosides were separated and quantified in multiple reaction monitoring mode. The gradient elution conditions were as follows:

[0123] Within 0-2 min, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid was reduced from 95:5 to 80:20;

[0124] Within 2-6 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid was increased from 80:20 to 99.9:0.1;

[0125] Within 6-10 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid was maintained at 99.9:0.1;

[0126] Within 10-11 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid decreased from 99.9:0.1 to 95:5;

[0127] The volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid was maintained at 95:5 within 11-12 minutes.

[0128] (2) The detection signal intensities and chromatographic peaks of the four deoxynucleosides obtained in the liquid chromatography tandem mass spectrometry detection were as follows: Figure 6 As shown in the figure, the four chromatographic peaks from left to right correspond to 2'-deoxycytidine dC, 2'-deoxyadenosine dA, 2'-deoxyguanosine dG, and β-thymidine dT, with retention times of 1.682, 1.924, 2.204, and 3.163 minutes, respectively. A concentration gradient was set for the four deoxynucleosides to create a standard curve, and the linear correlation coefficients r² were all greater than 0.996. This standard curve allows for the quantitative determination of the deoxynucleoside concentrations in the system, facilitating comparison of deoxynucleoside consumption after the reaction.

[0129] (3) The reaction conditions for the four deoxynucleosides were as follows: 1 mL (1 g / L) of each of the four new pollutants in DMSO and 100 μL (1 g / L) of a deoxynucleoside in DMSO were placed in a test tube and reacted in a 37°C water bath for 12 hours. There were 16 combinations of new pollutants and deoxynucleosides. Three replicates were set up for each combination.

[0130] (4) After 12 hours of reaction between the new pollutant and deoxynucleoside, the nucleoside reaction rate is as follows: Figure 7 As shown, the results indicate that all four new halobenzoquinone contaminants react to some extent with deoxynucleosides, with dG showing the highest reactivity. The reactivity rates of dG with 2,5-DBBQ, 2,5-DCBQ, 2,6-DBBQ, and 2,6-DCBQ were 72.4%, 94.5%, 79.8%, and 91.4%, respectively. The reactivity rates of the other three new contaminants with the other three deoxynucleosides were relatively low, with reactivity rates of 0.0%–35.0% for dC, 6.1%–10.8% for dA, and 5.7%–10.3% for dT. dG was the deoxynucleoside with the highest reactivity with the new contaminants.

[0131] (5) Combining the experimental results in this case and literature analysis, there are two pathways for the reaction of halogenated benzoquinones with deoxynucleosides: one is the self-oxidation and reduction to generate hydroxyl radicals that attack the deoxynucleosides to form oxidized nucleosides, and the other is a direct addition reaction between the two. However, the solvent DMSO in this reaction system is a strong solvent for hydroxyl radicals, and the deoxynucleosides in the oxidation reaction can be ignored. Therefore, the reduced deoxynucleosides can be considered to have undergone an addition reaction with HBQs.

[0132] (6) Comprehensive analysis of the results of Examples 1-4 shows that the new pollutants will not only undergo non-covalent interactions with DNA, such as groove interaction, electrostatic binding, and planar embedding, but also undergo covalent interactions that directly form adducts, and are more inclined to interact more closely with the deoxyguanosine portion.

[0133] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for studying the interaction between a new pollutant and DNA, characterized in that: The following steps are involved: (1) Using density functional theory in the quantum chemistry calculation software Gaussian, the molecular structures of the new pollutants and deoxynucleosides were optimized, and the optimized result files were exported; (2) Import the result file exported in step (1) into Multiwfn software to obtain the contribution of each atom to the molecular orbital and the atomic charge distribution, and visualize them in a table form; (3) Based on the tabular atomic contributions to molecular orbitals and atomic charge distribution, the electrophilic sites in the new pollutants are found by searching for the top M atoms that contribute most to the lowest unoccupied molecular orbital and the top N atoms with the largest positive charge and the largest charge number in the new pollutants. At the same time, the nucleophilic sites in the deoxynucleoside are found by searching for the top P atoms that contribute most to the highest occupied molecular orbital and the top Q atoms with the largest negative charge and the largest charge number in the deoxynucleoside. (4) Using the molecular docking software AutoDock Vina, multiple docking calculations were performed on the new pollutants and double-stranded DNA fragments, and the optimal binding position was searched based on the docking scores of the molecular docking software AutoDock Vina; (5) UV-Vis spectrophotometer was used to perform UV spectrophotometric titration on the interaction between the new pollutant and double-stranded DNA; (6) Using ultra-high performance liquid chromatography-mass spectrometry for gradient elution, deoxynucleosides were detected using a liquid chromatography column with a multiple reaction detection method, and the reaction rate of deoxynucleosides with the new pollutants after 12 hours was calculated to identify the deoxyribose units that are prone to adduction reactions with the new pollutants; (7) Analyze the test results obtained in steps (3) to (6).

2. The method for studying the interaction between a novel pollutant and DNA according to claim 1, characterized in that: In the step (1), when the density functional theory method is used to optimize the molecular structures of the new pollutant and the deoxynucleoside, the density functional theory condition is a functional method of the B3LYP / 6-31G** basis set.

3. The method for studying the interaction between a novel pollutant and DNA according to claim 1, characterized in that: The step (2) specifically includes: First, import the result file exported in step (1) into the Multiwfn software, then check the electrostatic potential surface and average local ionization energy of each atom in the compound in the result file, and then use the Mulliken, C-squared Population Analysis and Hirshfeld methods in the Multiwfn software to analyze and calculate the contribution of each atom to the molecular orbital. Use the Hirshfeld, Atomic Dipole Corrected Hirshfeld Atomic Charge and Merz-Kollman methods in the Multiwfn software to analyze and obtain the atomic charge distribution. Finally, visualize the contribution of each atom to the molecular orbital and the atomic charge distribution in a table.

4. The method for studying the interaction between a novel pollutant and DNA according to claim 1, characterized in that: In the step (5), the concentration of the double-stranded DNA solution is 100 mg / L; the final concentration gradient of the new pollutant is 0, 1, 2, 4, 7, 11, 16, 20, 25, 34, 42, 54, 70, 84, 101, and 116 μg / L.

5. The method for studying the interaction between new pollutants and DNA according to claim 1, characterized in that: In the step (5), the ultraviolet spectroscopic titration specifically comprises: The double-stranded DNA solution was mixed with different volumes of the new contaminant solution to obtain a total volume of 20 mL and a mixture system with different gradients of the final concentration of the new contaminant. The system was incubated at 160 rpm, pH 7.0 and 25°C for 2 h, and then the samples were taken and the absorbance was measured using a UV-1800 spectrometer in the wavelength range of 250 to 270 nm.

6. The method for studying the interaction between a novel pollutant and DNA according to claim 1, characterized in that: In step (6), the reaction conditions of the deoxynucleoside are specifically as follows: 1 mL of 1 g / L dimethyl sulfoxide solution of the four new pollutants and 100 μL of 1 g / L dimethyl sulfoxide solution of deoxynucleoside are respectively taken in a test tube, and the mixture is reacted in a water bath at 37° C. for 12 hours.

7. The method for studying the interaction between a new pollutant and DNA according to claim 1, characterized in that: In step (6), the conditions for the gradient elution are specifically: Within 0-2 min, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid was reduced from 95:5 to 80:20; Within 2-6 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid was increased from 80:20 to 99.9:0.1; Within 6-10 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid was maintained at 99.9:0.1; Within 10-11 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid decreased from 99.9:0.1 to 95:5; The volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid was maintained at 95:5 within 11-12 minutes.

8. The method for studying the interaction between new pollutants and DNA according to claim 1, characterized in that: In the step (6), the liquid chromatography column used is a BEH C18 liquid chromatography column with an inner diameter of 2.1 mm and a column length of 150 mm.

9. The method for studying the interaction between a new pollutant and DNA according to claim 1, characterized in that: In step (6), the conditions of the multiple reaction detection method are specifically shown in the following table:

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