A method for studying the interaction of a new pollutant with DNA

Abstract

The application discloses a research method for interaction between a new pollutant and DNA, and the method comprises the following steps: using a DFT method in quantum chemistry calculation software to optimize the molecular structure of the new pollutant and deoxy nucleoside; using Multiwfn software to obtain the contribution of each atom to a molecular orbital and atomic charge distribution; finding an electrophilic site of the new pollutant and a nucleophilic site of the deoxy nucleoside; using molecular docking software to perform docking calculation on the new pollutant and a double-stranded DNA fragment and search for an optimal combination position; using a UV-Vis ultraviolet spectrophotometer to perform ultraviolet spectrum titration on the interaction between the new pollutant and the double-stranded DNA; using a super high performance liquid chromatography-mass spectrometry method to perform gradient elution, detecting the deoxy nucleoside through a multiple reaction detection method, calculating a reaction rate, and identifying a deoxyribose unit prone to adduct reaction with the new pollutant; and analyzing the detection result.

Description

Technical Field

[0001] This invention relates to the field of research on the toxic effects of novel pollutants, and in particular to a research method for studying the interaction between novel pollutants and DNA. Background Technology

[0002] Emerging pollutants refer to a class of toxic and hazardous chemical substances that, once introduced into the environment, exhibit strong biotoxicity, high environmental persistence, and easy bioaccumulation, thus posing significant risks to the ecological environment and human health. Emerging pollutants are diverse, including pharmaceuticals and personal care products, drinking water disinfection byproducts (such as halogenated benzoquinones), endocrine disruptors, persistent organic pollutants, and microplastics. Compared to traditional pollutants, which are widely recognized and strictly controlled, emerging pollutants are unique in that their environmental and health risks have only recently gained attention, and existing environmental monitoring and management systems have not yet been able to comprehensively and effectively cover and control them; hence the term "emerging" pollutants. Emerging pollutants constitute new challenges in the field of environmental protection. They enter the water, soil, and atmosphere through various pathways such as industrial emissions, agricultural activities, and domestic sewage, and are characterized by wide distribution, low concentration levels (typically in the ng / L to μg / L range), environmental persistence, bioaccumulation, and potential biotoxicity.

[0003] DNA, as the core genetic material of living organisms, carries the genetic information necessary for almost all life activities. The structural integrity and sequence accuracy of DNA molecules are crucial for normal cellular physiological functions, 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 main categories: non-covalent binding and covalent binding. Non-covalent binding mainly includes electrostatic attraction, hydrophobic interactions, van der Waals forces, and binding to DNA through intercalation and groove binding. Although non-covalent interactions do not directly change the primary structure of DNA, they may affect DNA conformation, replication, and transcription. Covalent binding refers to the formation of stable chemical bonds between pollutants or their metabolically activated products and nucleophilic sites on DNA bases (such as the N7 and O6 sites of guanine, and the N1, N3, and N7 sites of adenine), forming DNA adducts. The formation of DNA adducts is one of the key initiation steps in the induction of mutations and tumors by chemical carcinogens. It can interfere with the normal replication and repair processes of DNA, leading to gene mutations, chromosomal aberrations, and even apoptosis or carcinogenesis.

[0004] Therefore, in-depth research into the interaction mechanisms between new pollutants and DNA, elucidating their binding modes, identifying key reaction sites, and assessing their ability and efficiency in forming DNA adducts are of vital scientific significance and practical application value for revealing the genotoxicity, carcinogenic potential, and health risks of new pollutants.

[0005] Currently, the main methods for studying the interaction between chemicals and DNA include: (1) computational simulation methods, such as molecular docking and quantum chemical calculations, which can predict the possible sites, binding energies and molecular conformations of the interaction from a theoretical perspective. However, 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, and circular dichroism spectroscopy, which are often used to study non-covalent interactions. By monitoring changes in spectral parameters, information such as binding modes and binding constants can be inferred. However, it is difficult to directly provide evidence of covalent binding and structural information; (3) chromatography-mass spectrometry, especially liquid chromatography-tandem mass spectrometry (LC-MS / MS), is 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. However, the equipment is expensive, the sample pretreatment is relatively complex, and the professional skills required of the analysts are high; (4) other biological methods, such as gel electrophoresis, comet experiments, and γ-H2AX assays, can be used to assess the degree of damage to DNA or chromosomes.

[0006] However, existing research methods often have limitations. For example, single computational simulations or spectroscopic methods cannot comprehensively reflect the full picture of the interaction between novel pollutants and DNA, especially in simultaneously assessing the preliminary screening of non-covalent interactions and the precise identification of covalent adducts. Methods relying on large-scale instruments, such as complex biological experimental platforms, face problems such as high equipment costs, cumbersome operation, and long experimental cycles, hindering rapid and economical high-throughput screening and preliminary risk assessment of large numbers of novel pollutants. Furthermore, in real-world environmental exposure, organisms are often exposed to multiple low-dose pollutants simultaneously, and their interactions with DNA may be more complex. Therefore, there is an urgent need to develop research methods that are accurate, economical, and comprehensive to effectively assess the potential genotoxicity of novel pollutants. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies in studying the interaction between new pollutants and DNA, such as high reliance on simulation calculations, high costs due to performance computing resources, and difficulty in economically and comprehensively examining non-covalent and covalent interactions simultaneously through experimental analysis. This invention proposes a research method for the interaction between new pollutants and DNA.

[0008] The objective of this invention is achieved through the following technical solution: a research method for the interaction between a novel pollutant and DNA, comprising the following steps:

[0009] (1) In the quantum chemical calculation software Gaussian, the molecular structure of the new pollutant and deoxynucleosides was optimized using density functional theory, and the optimized result file was 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 it in tabular form;

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

[0012] (4) The molecular docking software AutoDock Vina was used to perform multiple docking calculations on the new pollutant and the double-stranded DNA fragment, and the optimal binding position was searched based on the docking score of the molecular docking software AutoDock Vina.

[0013] (5) The interaction between the new pollutant and double-stranded DNA was titrated using a UV-Vis spectrophotometer;

[0014] (6) Gradient elution was performed using ultra-high performance liquid chromatography-mass spectrometry. Deoxyribonucleotides were detected by multiple reaction detection using a liquid chromatography column. The reaction rate of deoxyribonucleotides with new pollutants was calculated after 12 hours. Deoxyribose units that are prone to addition reactions with new pollutants were identified.

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

[0016] Furthermore, in step (1), when optimizing the molecular structure of the new pollutant and deoxynucleosides using density functional theory, the density functional theory condition is the functional method of the B3LYP / 6-31G** basis set.

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

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

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

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

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

[0022] Further, in step (6), the reaction conditions for the deoxynucleosides are as follows: take 1 mL of a 1 g / L dimethyl sulfoxide solution of the four new pollutants and 100 μL of a 1 g / L dimethyl sulfoxide solution of the deoxynucleosides in test tubes, and react them in a water bath at 37°C for 12 hours.

[0023] Furthermore, in step (6), the specific conditions for gradient elution are as follows:

[0024] Within 0-2 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid decreased from 95:5 to 80:20.

[0025] Within 2-6 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid and the acetonitrile solution containing 0.1% formic acid 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 and 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] Within 11-12 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid and the acetonitrile solution containing 0.1% formic acid was maintained at 95:5.

[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 specific conditions of the multiple reaction detection method are shown in the table below:

[0031]

[0032] Compared with existing technologies, the advantages of this invention are as follows: This invention integrates low-cost computational simulation with cost-effective experimental verification methods; the simulation calculation part does not require large computer servers, significantly reducing the equipment threshold and equipment costs; the experimental analysis part simultaneously covers two interaction mechanisms: computational simulation, non-covalent binding (UV spectral characterization), and covalent addition (UPLC-MS / MS detection), enabling economical and rapid comprehensive detection and characterization of the two main interaction modes of new pollutants and DNA, without relying on expensive dedicated computers and complex testing platforms, thus achieving comprehensive detection of the interaction modes of new pollutants and DNA; this invention provides a simple, cost-effective, and reliable technical approach for the study of new pollutant toxicity mechanisms and environmental risk assessment, and has broad application prospects in environmental science, analytical chemistry, and pollutant toxicity mechanism research. Attached Figure Description

[0033] Figure 1 This is a flowchart illustrating the research method for the interaction between the novel pollutant and DNA according to the present invention;

[0034] Figure 2 A schematic diagram of the chemical structure of the new pollutants halobenzoquinones and deoxynucleosides;

[0035] Figure 3 Atom numbering for new pollutants and deoxynucleosides in the Gaussian calculation process;

[0036] Figure 4A schematic diagram showing the optimal binding site after the new pollutant docks with the double-stranded DNA molecule;

[0037] Figure 5 A schematic diagram of the results of ultraviolet spectroscopic titration of a new pollutant with double-stranded DNA;

[0038] Figure 6 The detection signal intensity and chromatogram of four deoxynucleosides were obtained after using an elution gradient with a total duration of 12 min in liquid chromatography-tandem mass spectrometry.

[0039] Figure 7 This is a schematic diagram illustrating the degree of reaction between monodeoxynucleosides and new pollutants. Detailed Implementation

[0040] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this application as detailed in the appended claims. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit this application.

[0041] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The singular forms “a,” “the,” and “the” used in this application and the appended claims are also 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 includes any or 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, such information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, without departing from the scope of this application, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "when," "in response to determination," or "includes." Moreover, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process or method that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process or method. Without further limitations, an element defined by the phrase "comprising a..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0043] The present invention will now be described in detail with reference to the accompanying drawings. Unless otherwise specified, the features of the following embodiments and implementations can be combined with each other.

[0044] The research method for studying the interaction between novel pollutants and DNA in this invention first utilizes computer simulation technology, specifically employing the quantum chemical calculation software Gaussian to optimize the molecular structures of the novel pollutants and deoxynucleosides and predict their most likely reaction sites. Then, the molecular docking software Autodock Vina is used to simulate the interaction process and binding mode between the novel pollutants and DNA molecules on a conventional computer. Secondly, at the experimental level, ultraviolet spectroscopy is used to comprehensively detect the non-covalent interaction characteristics between the novel pollutants and double-stranded DNA. Finally, ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS) in multiple reaction monitoring (MRM) mode is used to detect the solution after the reaction of the novel pollutants and deoxynucleosides, thereby determining the reaction rate and identifying deoxyribose units that readily undergo addition reactions with the novel pollutants.

[0045] See Figure 1 The method for studying the interaction between novel pollutants and DNA according to the present invention specifically includes the following steps:

[0046] (1) In the quantum chemical calculation software Gaussian, the molecular structure of the new pollutant and deoxynucleosides was optimized using the density functional theory (DFT) method, and the optimized result file was exported.

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

[0048] Furthermore, when using the DFT method to optimize the molecular structure of new pollutants and deoxynucleosides, the DFT conditions are 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 it in a table for easy comparison.

[0050] Specifically, Multiwfn is a powerful, open-source quantum chemical wavefunction analysis software. It supports the output files of various quantum chemical calculation programs, provides a wealth of wavefunction analysis tools, and is widely used in fields such as electronic structure analysis, chemical bond research, and molecular property prediction. Therefore, the result file exported in step (1) is first imported into Multiwfn. Then, the electrostatic potential surface and average local ionization energy of each atom in the result file are viewed. The contribution of each atom to the molecular orbital is then analyzed and calculated using the three methods in Multiwfn: Mulliken, C-squared Population Analysis (SCPA), and Hirshfeld. The atomic charge distribution is then analyzed and obtained using the three methods in Multiwfn: Hirshfeld, AtomicDipole Corrected Hirshfeld Atomic Charge (ADCH), and Merz-Kollman (MK). Finally, the contribution of each atom to the molecular orbital and the atomic charge distribution are visualized and organized into a table for easy comparison.

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

[0052] It should be understood that LUMO is a key concept in molecular orbital theory, referring to the lowest energy unoccupied molecular orbital under ground state (unexcited electrons) conditions. 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 were used in step (2) to obtain the contribution of atoms to molecular orbitals and the atomic charge distribution, the tendency of each site may be different due to different reaction types. Therefore, when searching for electrophilic sites and nucleophilic sites in step (3), the determination is 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, SCPA method, or Hirshfeld method, and the nucleophilic site is determined based on the atomic charge distribution analyzed by the Hirshfeld method, ADCH method, or MK method.

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

[0055] Specifically, chemical structure information of four novel pollutants was retrieved from the PubChem database, and X-ray diffraction structure data of DNA was obtained from the RCSB Protein Data Bank (PDB 3IXN). Preprocessing was performed to remove existing water molecules, replenish the DNA molecules with complete hydrogen atoms, and designate the preprocessed DNA molecules as the acceptor for the docking process. The prepared acceptor and ligand molecule files were selected from the menu, and the grid options dialog box was opened to set the grid box size to 60×60×100, the grid spacing to the default value, and the grid center to DNA. The program path, name, and log file were also set. The program was then run to perform docking calculations and export the results. After the molecular docking calculations were completed, the exported results file was imported into PyMOL molecular visualization software, and the conformation of the resulting docking complex was observed and analyzed. The docking score in Autodock Vina software is Affinity; a smaller value indicates a stronger binding force, indicating the optimal binding site.

[0056] (5) The interaction between the new pollutant and double-stranded DNA was titrated using a UV-Vis spectrophotometer.

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

[0058] Further, in step (5), the ultraviolet spectral 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 different final concentrations of new pollutants, incubating at 160 rpm, pH 7.0 and 25 °C for 2 hours, and then taking the sample and measuring the absorbance in the wavelength range of 250 to 270 nm using a UV-1800 spectrometer.

[0059] (6) Gradient elution was performed using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS). Deoxyribonucleotides were detected by multiple reaction detection (MRM) using a liquid chromatography column. The reaction rate of deoxyribonucleotides with new pollutants was calculated after 12 hours, and deoxyribose units that are prone to addition reactions with new pollutants were identified.

[0060] Further, in step (6), the reaction conditions for deoxynucleosides are as follows: 1 mL of 1 g / L dimethyl sulfoxide (DMSO) solution of each of the four new pollutants and 100 μL of 1 g / L dimethyl sulfoxide (DMSO) solution of deoxynucleosides are placed in test tubes, and the mixture is reacted in a water bath at 37°C for 12 hours. There are a total of 16 combinations of new pollutants and deoxynucleosides. Each combination is tested in triplicate.

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

[0062] Within 0-2 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid decreased from 95:5 to 80:20.

[0063] Within 2-6 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid and the acetonitrile solution containing 0.1% formic acid 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 and 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] Within 11-12 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid and the acetonitrile solution containing 0.1% formic acid was maintained at 95:5.

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

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

[0069] Table 1: Setup conditions for multiple reaction monitoring methods

[0070]

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

[0072] In summary, this invention first optimizes the structures of novel pollutants and deoxynucleosides using Gaussian 09W software to predict the most likely reaction sites for their interactions; second, it simulates the interaction between the novel pollutants and DNA using Autodock Vina docking software. Subsequently, it characterizes the interaction pattern between the novel pollutants and double-stranded DNA using ultraviolet spectroscopy. Finally, it detects the deoxynucleosides after the reaction using multiple reaction monitoring (MRM) mode of ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), calculates their reaction rate with the novel pollutants, and identifies deoxynucleosides that readily undergo addition reactions.

[0073] The following detailed description of the research method for the interaction between the novel pollutant and DNA according to the present invention, based on the embodiments, will make the purpose and effects of the present invention more apparent.

[0074] Example 1

[0075] This embodiment examines the sites where halogenated benzoquinones, a common and highly cytotoxic disinfection byproduct, readily react with four typical deoxynucleosides. The specific steps include the following:

[0076] (1) First, draw the following in Gaussian 09W software: Figure 2 The chemical structures of the novel pollutants halobenzoquinones and deoxyribonucleotides 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 Multiwfn software to view the electrostatic potential surface and average local ionization energy of each atom in the compound. Further analyze the results using Multiwfn software and visualize them accordingly to find the electrophilic sites in the new pollutant and the nucleophilic sites in the deoxynucleosides. 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 HirshfeldAtomic Charge (ADCH), and Merz-Kollman (MK). The atom numbering in the calculation process of the new pollutant and deoxynucleosides in Gaussian software is as follows: Figure 3 As shown.

[0078] The calculated contributions of each atom on the molecular orbital and the atomic charge distribution 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) to the molecular orbital and the four deoxyribonucleotides are shown in Tables 2 to 9.

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

[0080]

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

[0082]

[0083]

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

[0085]

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

[0087]

[0088]

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

[0090]

[0091]

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

[0093]

[0094]

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

[0096]

[0097]

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

[0099]

[0100]

[0101] (3) Results Analysis: Based on the visualizations in Tables 1-9 above, 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 novel pollutant halogenated benzoquinone is the most readily accepting orbital for foreign electrons, i.e., the most electrophilic molecular orbital. Therefore, when interacting with nucleophiles (such as nucleophilic sites on DNA that may be involved in this invention), this LUMO orbital is usually the primary target for the nucleophile to provide electrons, such as... Figure 2 The C1 and C2 positions in the DNA. Further analysis shows that when a DNA molecule interacts with a reagent having multiple electrophilic reaction centers (or when the initial adduct itself still has multiple electrophilic sites), it readily forms a stable cyclic adduct structure between spatially adjacent nitrogen atoms on the bases. For example, Figure 2 The reactivity of guanine bases with nucleophilic reactions is typically between the N1 and N2 sites, cytosine bases with nucleophilic sites with the N3 and N4 sites, and adenine bases with the N6 and N1 sites. In contrast, thymine bases, due to the lack of amino functional groups in their structure that can provide lone pairs of electrons as nucleophilic reaction sites, generally exhibit extremely low reactivity with electrophilic reagents.

[0102] Example 2

[0103] This embodiment examines the docking results of the novel pollutant halobenzoquinone with double-stranded DNA molecules, specifically including the following steps:

[0104] (1) First, chemical structure information of four new pollutants was retrieved from the PubChem database, specifically 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). After obtaining the initial structures of the above new pollutants, their three-dimensional spatial structures were geometrically optimized using the MM2 molecular force field built into Chem3D software (CambridgeSoft, MA, USA) under simulated physiological pH 7.0 conditions.

[0105] (2) X-ray crystallography (XRD) structure data of deoxyribonucleic acid (DNA) were obtained from the RCSB Protein Data Bank, with the selected PDB accession number being 3IXN. This 3IXN structure represents a typical B-type DNA double helix conformation, which is consistent with the conformation of the model DNA used in the subsequent experimental studies of this invention.

[0106] (3) Before performing docking calculations, the DNA macromolecule structure was pretreated as necessary. The pretreatment included removing the original water molecules in the structure, replenishing the DNA molecule with complete hydrogen atoms, and setting the treated DNA molecule as the acceptor for the docking process.

[0107] (4) The molecular docking simulation calculation of the halobenzoquinone compound molecule and the DNA acceptor was performed using the molecular docking software AutoDock Vina. Select the previously prepared acceptor and ligand molecule files from the menu, open the grid options dialog box, set the grid box size to 60×60×100, the grid spacing to the default value, set the grid center to DNA, set the program path and name, and the record file, etc., and run the program for calculation.

[0108] (5) After the molecular docking calculations were completed, the conformation of the resulting docking complex was observed and analyzed using PyMOL molecular visualization software. The docking score in Autodock Vina software is Affinity; the smaller the value, the stronger the binding force. The binding energy data obtained from the experiment are shown in Table 10.

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

[0110]

[0111] (6) A schematic diagram showing the optimal binding sites of the new pollutants after docking with double-stranded DNA molecules is shown below. Figure 4 As shown in the diagram, docking results analysis revealed that in all the docking simulations of the novel pollutants with DNA, the optimal binding site of the novel pollutant molecules to DNA was invariably located within the grooves of the DNA double helix structure, and all of them bound to deoxyguanosine. This result suggests that novel pollutant molecules tend to interact with DNA through a groove-bound binding mechanism, where the planar quinone ring structure portion of the novel pollutant molecule further embeds into the minor groove region of the DNA. Specifically, they form a "sandwich"-like stacked structure.

[0112] Example 3

[0113] This embodiment involved ultraviolet spectral titration of a novel pollutant with double-stranded DNA and analysis of the detection 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, this ctDNA solution was mixed with different volumes of 500 μg / L halobenzoquinone (HBQ) stock solution in several reaction tubes. By precisely controlling the volume of the added HBQ 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) Place the prepared mixed solution in a constant temperature shaker and incubate it for 2 hours at a shaking rate of 160 rpm under the conditions of pH 7.0 and temperature 25°C to ensure that the DNA and HBQs interact fully.

[0117] (4) After incubation, 2 mL of the reaction mixture was transferred 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 ultraviolet-visible spectrophotometer (Shimadzu Corporation). The measurement was performed under precisely controlled temperature conditions, i.e., (25±0.1)℃, and the scanning wavelength range was set between 250nm and 270nm.

[0119] (6) Schematic diagram of the UV spectral titration results of the new pollutant with double-stranded DNA. Figure 5As shown in the results, analysis revealed that as the concentrations of different novel pollutants (specifically 2,6-DCBQ, 2,6-DBBQ, 2,5-DCBQ, and 2,5-DBBQ) gradually increased from 0 μg / L to 116 μg / L, the absorbance of DNA at the characteristic absorption peak at 260 nm exhibited different trends for each novel pollutant. Under the influence of lower concentrations of specific HBQs (e.g., when the concentration range of 2,6-DCBQ was 0–11 μg / L and the concentration range of 2,6-DBBQ was 0–25 μg / L), the DNA molecule's double helix structure may have become more compact or ordered due to electrostatic binding or base planar intercalation of the novel pollutants, thus exhibiting a hypochromic effect in the spectrum. However, when the concentration of the new contaminant increased to a high level (e.g., ≥16 μg / L), a significant hyperchromic effect was observed in the absorption spectrum of DNA. This phenomenon indicates that the exposure of DNA bases increased in the presence of higher concentrations of the new contaminant, which may be related to a certain degree of unwinding or perturbation of the local DNA structure.

[0120] Example 4

[0121] This embodiment establishes a method for the detection of deoxyribonucleosides based on ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS) multiple reaction detection mode. The reaction rate of deoxyribonucleosides with a novel contaminant after 12 hours is calculated, deoxyribose units that readily undergo addition reactions with the novel contaminant are identified, and the results of the interaction between the novel contaminant halobenzoquinone and DNA are comprehensively analyzed. The specific steps include:

[0122] (1) An aqueous solution containing 0.1% formic acid and an acetonitrile solution containing 0.1% formic acid were used as the mobile phases for liquid chromatography-tandem mass spectrometry (LC-MS / MS). Gradient elution was performed for 12 minutes. The concentrations of the four deoxyribonucleotides were separated and quantified using a BEH C18 (2.1 mm × 150 mm, Waters) spectrometer in multiple reaction monitoring mode. The specific gradient elution conditions were as follows:

[0123] Within 0-2 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid decreased from 95:5 to 80:20.

[0124] Within 2-6 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid and the acetonitrile solution containing 0.1% formic acid 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 and 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] Within 11-12 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid and the acetonitrile solution containing 0.1% formic acid was maintained at 95:5.

[0128] (2) The detection signal intensity and chromatogram of the four deoxynucleosides obtained by liquid chromatography-tandem mass spectrometry detection using an elution gradient with a total duration of 12 min are shown in the figure below. Figure 6 As shown, the four chromatographic peaks from left to right correspond to 2'-deoxycytidine dC, 2'-deoxyadenosine dA, 2'-deoxyguanosine dG, and β-thymidine dT, respectively, with retention times of 1.682, 1.924, 2.204, and 3.163 minutes. Standard curves were constructed using concentration gradients for the four deoxynucleotides, and the linear correlation coefficients r² were all greater than 0.996. The standard curves allow for the quantification of deoxynucleotide concentrations in the system, facilitating comparison of deoxynucleotide consumption after the reaction.

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

[0130] (4) The nucleoside reaction rate after 12 hours of reaction between the new pollutant and deoxynucleosides is as follows: Figure 7 As shown, the results indicate that all four novel halogenated benzoquinone pollutants react with deoxynucleosides to some extent, with dG exhibiting the strongest reaction. The reaction 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 other three novel pollutants and the other three deoxynucleosides showed weaker and lower reaction rates: dC showed a reaction rate of 0.0%–35.0%, dA a reaction rate of 6.1%–10.8%, and dT a reaction rate of 5.7%–10.3%. dG was the deoxynucleoside with the highest reaction rate with the novel pollutants.

[0131] (5) Based on the experimental results and literature analysis in this case, there are two pathways for the reaction between halobenzoquinones and deoxynucleosides: one is through self-redox to generate hydroxyl radicals that attack deoxynucleosides to form oxidized nucleosides, and the other is through a direct addition reaction. However, in this reaction system, the solvent DMSO is a strong solvent for hydroxyl radicals, and the amount of deoxynucleosides produced by the oxidation reaction can be ignored. Therefore, the reduction in deoxynucleosides can be considered as an addition reaction with HBQs.

[0132] (6) A comprehensive analysis of the results of Examples 1-4 shows that the new pollutants can interact with DNA through non-covalent interactions such as groove interaction, electrostatic binding and planar embedding, as well as covalent interactions that directly form adducts, and they tend to interact more closely with the deoxyguanosine moiety.

[0133] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for studying the interaction of a new pollutant with DNA, characterized in that, Includes the following steps: (1) In the quantum chemical calculation software Gaussian, the molecular structure of the new pollutant and deoxynucleosides was optimized using density functional theory, and the optimized result file was exported; the new pollutant is a halobenzoquinone; In step (1), when using density functional theory to optimize the molecular structure of new pollutants and deoxynucleosides, the condition for density functional theory is the functional method of the B3LYP / 6-31G** basis set. (2) Import the result file exported in step (1) into the Multiwfn software to obtain the contribution of each atom to the molecular orbital and the atomic charge distribution, and visualize it in tabular form; (3) Based on the contribution of atoms to molecular orbitals and the distribution of atomic charges in tabular form, the potential sites in new pollutants are found by searching for the top M atoms that contribute the most to the lowest unoccupied molecular orbitals and the top N atoms with the largest positive charge. At the same time, the nucleophilic sites in deoxynucleosides are found by searching for the top P atoms that contribute the most to the highest occupied molecular orbitals and the top Q atoms with the largest negative charge. (4) The molecular docking software AutoDock Vina was used to perform multiple docking calculations on the new pollutant and the double-stranded DNA fragment, and the optimal binding position was searched based on the docking score of the molecular docking software AutoDock Vina. (5) Use a UV-Vis spectrophotometer to perform ultraviolet spectral titration of the interaction between the new pollutant and double-stranded DNA; In step (5), the concentration of the double-stranded DNA solution is 100 mg / L; the final concentration gradient of the new pollutants is 0, 1, 2, 4, 7, 11, 16, 20, 25, 34, 42, 54, 70, 84, 101, 116 μg / L. In step (5), the ultraviolet spectral titration specifically includes: The double-stranded DNA solution was mixed with different volumes of new pollutant solution to obtain a total volume of 20 mL and a final concentration of new pollutant of different gradients. The mixture system was incubated at 160 rpm, pH 7.0 and 25℃ for 2 hours. Then, the absorbance of the sample was measured in the wavelength range of 250~270 nm using a UV-1800 spectrometer. (6) Gradient elution was performed using ultra-high performance liquid chromatography-mass spectrometry. Deoxyribonucleotides were detected by multiple reaction detection using a liquid chromatography column. The reaction rate of deoxyribonucleotides with new pollutants was calculated after 12 hours. Deoxyribose units that are prone to addition reactions with new pollutants were identified. In step (6), the reaction conditions for the deoxynucleosides are as follows: take 1 mL of a 1 g / L dimethyl sulfoxide solution of four halobenzoquinones and 100 μL of a 1 g / L dimethyl sulfoxide solution of four deoxynucleosides in a test tube and react them in a water bath at 37°C for 12 hours. In step (6), the specific conditions for gradient elution are as follows: Within 0-2 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid to the acetonitrile solution containing 0.1% formic acid decreased from 95:5 to 80:

20. Within 2-6 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid and the acetonitrile solution containing 0.1% formic acid 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 and 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. Within 11-12 minutes, the volume ratio of the aqueous solution containing 0.1% formic acid and the acetonitrile solution containing 0.1% formic acid was maintained at 95:5; (7) Analyze the detection results obtained in steps (3) to (6).

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

3. The method for investigating the interaction of a new pollutant with DNA according to claim 1, characterized in that, 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.

4. The method for investigating the interaction of a new pollutant with DNA according to claim 1, characterized in that, The specific conditions for the multiple reaction detection method in step (6) are shown in the table below: 。

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