A method for analyzing non-covalent nucleic acid intercalators in complex mixtures

Abstract

This invention belongs to the field of analytical chemistry and discloses a method for analyzing non-covalent nucleic acid intercalating agents in complex mixed solutions. The method includes the following steps: (a) preparing the complex mixed solution to be tested; (b) constructing double-stranded nucleic acid molecules; (c) mechanically stretching the double-stranded nucleic acid molecules and measuring their length; (d) applying the complex mixed solution to be tested to the double-stranded nucleic acid molecules, and detecting the length change of the double-stranded nucleic acid molecules by mechanical stretching. Based on the length change, it can be determined whether a non-covalently intercalated nucleic acid-binding compound exists in the complex mixed solution. This invention is the first to use single-molecule manipulation technology to detect complex mixed solutions and identify small molecule compounds with non-covalently intercalated double-stranded nucleic acids. This method is particularly useful for screening the bioactivity of microbial nucleic acid-binding compounds, enabling the discovery of new nucleic acid intercalating compounds from microorganisms, animals, and plants.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, and more specifically, relates to a method for analyzing non-covalent nucleic acid intercalating agents in complex mixed solutions. Background Technology

[0002] Nucleic acids are important targets for anti-tumor chemotherapy drugs. Clinically used chemotherapeutic drugs, such as daunorubicin, kill tumor cells by intercalating into and binding to DNA, affecting DNA replication and transcription, and causing DNA damage. Furthermore, drugs that covalently bind to DNA, such as cisplatin, cause tumor cell death by cross-linking DNA. Traditional nucleic acid-targeting compounds face various problems such as toxic side effects and drug resistance. In addition to anti-tumor drug development, small molecule compounds that bind to RNA became the first marketed small molecule compound for the treatment of spinal muscular atrophy (SMA). This also demonstrates the potential significance of developing novel nucleic acid-binding compounds in the treatment of various diseases.

[0003] Natural products have always been an important source of innovative drugs. According to the latest statistics from Newman, DJ, & Cragg, GM, etc., over 60% of small molecule drugs launched globally from 1981 to 2019 are directly or indirectly related to natural products. Especially in anti-tumor drugs, 70.8% of drugs are derived from natural products (Journal of Natural Products 2020, 83, 770-803). Drugs that intercalate and bind to DNA, such as doxorubicin, daunorubicin, camptothecin, vinblastines, podophyllotoxin, and berberine, are all derived from natural products. Doxorubicin and daunorubicin are derived from metabolites of *Streptomyces bosère*. Camptothecin, vinblastines, podophyllotoxin, and berberine are derived from plant extracts. Screening for new compounds that intercalate and bind to nucleic acids from natural extracts can provide opportunities for developing novel anti-tumor drugs and other new drugs targeting nucleic acids, offer unique insights into modifying existing drug structures, reveal the structure-activity relationship of DNA-binding compounds, and thus optimize the design of existing anticancer drugs.

[0004] Traditional analytical methods for detecting nucleic acid-binding compounds include ultraviolet absorption spectroscopy, fluorescence spectroscopy, DNA affinity chromatography, dialysis, gel electrophoresis, surface plasmon resonance, mass spectrometry, and nuclear magnetic resonance. However, these methods are difficult to directly detect the presence of nucleic acid-binding compounds in complex mixed solutions containing multiple components. Often, multi-step purification processes (such as extraction, chromatographic purification, crystallization, resin adsorption, and distillation) are required to obtain a detailed breakdown of soluble components before detecting the interaction between these compounds and nucleic acids. Because the secondary metabolites of microorganisms and plants and animals are diverse, and certain secondary metabolic reactions only occur in specific species, organs, or tissues under specific environmental and temporal conditions, coupled with the difficulty in enriching some secondary metabolites through solvent extraction, identifying the presence of nucleic acid-intercalated compounds in microorganisms and plants before achieving separation and purification is extremely challenging.

[0005] Currently, the main methods for separating and purifying compounds from natural products are chromatographic methods, including adsorption column chromatography, partition chromatography, membrane separation, gel chromatography, and ion exchange chromatography. After separation and purification, the components are then analyzed using techniques such as fluorescence, ultraviolet, and mass spectrometry to determine whether they contain DNA-binding compounds. However, these methods rely on the separation of single compounds; if compounds interfere with the analysis of the spectra or mass spectrometry signals, they cannot be analyzed. Furthermore, the separation and purification process requires large quantities of samples, involves cumbersome purification steps, and some unstable compounds may react during the purification process. Therefore, developing methods for directly detecting complex mixtures such as bacterial cultures and plant and animal extracts would facilitate the rapid identification of biological samples that produce nucleic acid adducts, provide rapid and accurate guidance for compound separation, and accelerate the discovery of novel nucleic acid-binding compounds.

[0006] Single-molecule manipulation techniques have been widely used to detect interactions between small molecules and nucleic acids. In 1995, Perkins et al. observed that the insertion of the DNA intercalator ethidium bromide into DNA caused an increase in the length of double-stranded DNA using a stretching experiment with a single DNA molecule (Science, 1995, 268, 83-87). Vladesc et al. measured the force-extension curve of double-stranded DNA using single-molecule manipulation experiments to quantify the dissociation constant of the DNA intercalator and DNA (Nature Methods, 2007, 4, 517-522). According to Biebricher et al. (Nature Communications, 2015, 6(1), 1-12), each intercalation of a compound molecule causes an increase in the outline length of DNA of 0.34 nanometers. We previously developed a method (CN15166131A) for detecting covalently bound intercalation adducts between small molecule compounds and DNA using single-molecule mechanical manipulation. This method mainly quantifies the ability of purified single compounds to covalently bind nucleic acids under enzyme catalysis. It detects covalently bound intercalation adducts by washing away non-covalently bound compounds. However, it does not target compounds that do not covalently bind nucleic acids, nor does it identify the presence of nucleic acid intercalators that do not covalently bind nucleic acids in complex samples.

[0007] It is worth noting that previous single-molecule analyses were mainly used to analyze the binding affinity of known single compounds to nucleic acid intercalations (Nature Methods, 2007, 4, 517-522). This was particularly relevant for the analysis and detection of simple solution systems, i.e., solution samples with simple background components, such as chemically synthesized compounds and single compounds extracted and isolated from natural products. Currently, effective analytical methods are still lacking for identifying the presence of non-covalently bound nucleic acid intercalators in extracts, metabolites, secondary metabolites, and natural soil and water samples from microorganisms, plants, and animals. For trace amounts of nucleic acid intercalators in complex solution systems, the ability to detect them using single-molecule methods without purification remains a challenging issue. Summary of the Invention

[0008] To address the aforementioned deficiencies or improvement needs of existing technologies, the present invention aims to provide a method for analyzing nucleic acid intercalating agents in complex mixed solutions. This method, for the first time, applies single-molecule manipulation technology to detect complex mixed solutions (which may originate from, for example, extracts, metabolites, and secondary metabolites from microorganisms, plants, or animals; natural soil samples or natural water samples) to detect the presence of small molecule compounds with non-covalently intercalated double-stranded nucleic acids. This method is particularly suitable for the field of natural organic chemistry, especially for screening the bioactivity of microbial nucleic acid-binding compounds, and for discovering new nucleic acid intercalating compounds from microorganisms, animals, and plants. Furthermore, this method can also be applied in the pharmaceutical field for drug analysis, specifically for the pharmacokinetic analysis, pharmacological and toxicological analysis of non-covalently intercalated nucleic acid drugs.

[0009] To achieve the above objectives, according to one aspect of the present invention, a method for detecting and analyzing non-covalent nucleic acid intercalating agents in complex mixed solutions using single-molecule manipulation is provided, characterized by comprising the following steps:

[0010] (a) Prepare a complex mixture solution to be tested, said complex mixture solution being tested being derived from at least one of the following: microbial culture medium, microbial extract, microbial secondary metabolites; animal body fluids, animal and plant tissues, animal and plant tissue extracts, animal and plant tissue metabolites; natural soil samples, natural water samples;

[0011] (b) Fixing a double-stranded nucleic acid molecule between a first surface and a second surface, wherein the first surface remains in a fixed position and the second surface is a movable surface;

[0012] (c) Measure the length of the double-stranded nucleic acid molecule by mechanically stretching it;

[0013] (d) Apply the complex mixed solution to be tested to the double-stranded nucleic acid molecule and use the same mechanical conditions as the mechanical stretching in step (c). Detect the length change of the double-stranded nucleic acid molecule by mechanical stretching, and then determine whether there is a non-covalently intercalated nucleic acid compound in the complex mixed solution to be tested based on the length change.

[0014] Furthermore, the method also satisfies at least one of the following conditions:

[0015] Condition 1: In step (a), the complex mixed solution to be tested does not contain any enzyme components, or the complex mixed solution to be tested is treated by at least one of heating, sonication, adding a protein denaturant, adding an enzyme inhibitor, or adding a metal ion chelating agent to inactivate the potential enzymes in the system.

[0016] Condition 2: In step (d), the time from the application of the complex mixed solution to be tested to the start of detecting the length change of the double-stranded nucleic acid molecule is less than 10 minutes.

[0017] As a further preferred embodiment of the present invention, in step (a), the complex mixed solution to be tested is obtained by centrifuging at least one of microbial culture medium, microbial extract, microbial secondary metabolites, animal body fluids, animal and plant tissue extracts, and animal and plant tissue metabolites and taking the supernatant; or, it is obtained by dissolving and dispersing at least one of animal and plant tissues and soil in a liquid medium and taking the supernatant.

[0018] The animal body fluid is preferably at least one of blood, urine, digestive juice, and tissue fluid.

[0019] As a further preferred embodiment of the present invention, in step (a), the complex mixed solution to be tested is not purified, but is a clear liquid obtained by removing insoluble components only through filtration and / or centrifugation.

[0020] As a further preferred embodiment of the present invention, in step (b), the double-stranded nucleic acid molecule is selected from: double-stranded DNA molecules, double-stranded RNA molecules, and DNA / RNA hybrid molecules;

[0021] The length of the double-stranded nucleic acid molecule is in the range of 100 base pairs to 100,000 base pairs;

[0022] Preferably, the double-stranded nucleic acid molecule is a double-stranded DNA molecule.

[0023] As a further preferred embodiment of the present invention, in step (b), the fixation involves directly or indirectly attaching at least one base of one strand of the double-stranded nucleic acid to the first surface, and directly or indirectly attaching at least one base of the other strand of the double-stranded nucleic acid to the second surface.

[0024] Alternatively, the fixation involves attaching at least one base of one strand of a double-stranded nucleic acid directly or indirectly to a first surface, and at least one base at different positions of the strand directly or indirectly to a second surface.

[0025] As a further preferred embodiment of the present invention, in step (b), the first surface and the second surface are independently selected from glass surface, plastic surface, quartz surface, graphene surface, metal surface, and ceramic surface;

[0026] Furthermore, the second surface is a particle surface or a probe surface; the size of the particle or the probe is in the range of 10 nanometers to 100 micrometers;

[0027] Preferably, the second surface is a superparamagnetic particle surface.

[0028] As a further preferred embodiment of the present invention, in step (c), the external force applied by the mechanical stretching is in the range of 0.1 piconewtons to 100 piconewtons and is applied by a permanent magnet, electromagnet, centrifuge, acoustics, ultrasound, laser beam, fluid motion, fluid buoyancy or gravity.

[0029] Preferably, the mechanical stretching is applied using a single-molecule magnetic tweezers device;

[0030] More preferably, the external force applied by the mechanical tension is 1-60 piconewtons.

[0031] As a further preferred embodiment of the present invention, in steps (c) and (d), the mechanical stretching is performed by applying an external force that varies with time and recording the length change of the double-stranded nucleic acid molecule with mechanical parameters.

[0032] As a further preferred embodiment of the present invention, when the complex mixed solution to be tested contains a single non-covalent nucleic acid intercalating agent, and the type of the non-covalent nucleic acid intercalating agent is known, the method further includes the step of:

[0033] (e) Prepare standards of different concentrations of the non-covalent nucleic acid intercalating agent, replace the complex mixed solution to be tested with each standard, and use the same operation as in steps (b)-(d) under the same mechanical tensile stress conditions to measure the length change of double-stranded nucleic acid molecules after adding intercalating agent standards at different concentration gradients, as a standard curve; by comparing the length change in the standard curve with the length change caused by the complex mixed solution to be tested obtained in step (d), quantify the concentration of the single non-covalent nucleic acid intercalating compound contained in the complex mixed solution to be tested;

[0034] Preferably, the external force applied during mechanical tension is 1-60 piconewtons.

[0035] According to another aspect of the present invention, the present invention provides the application of the above method in the preparation or screening of drugs to determine whether a complex mixed solution contains a non-covalent nucleic acid intercalation compound;

[0036] Preferably, the complex mixed solution to be tested is derived from any one of the following: microbial culture medium, microbial extract, microbial secondary metabolites; plant tissue, plant tissue extract.

[0037] According to another aspect of the present invention, the present invention provides the application of the above method in the preparation or screening of drugs for pharmacokinetic analysis of drugs, characterized in that the complex mixed solution to be tested is derived from animal body fluids, animal tissues, animal tissue extracts and / or animal tissue metabolites of animals after drug administration.

[0038] According to another aspect of the invention, the invention provides the application of the above method in drug toxicology analysis during drug preparation or drug screening, characterized in that the complex mixed solution to be tested is derived from animal body fluids, animal tissues, animal tissue extracts and / or animal tissue metabolites of animals after drug administration.

[0039] As a further preferred embodiment of the present invention, the method further includes the following steps:

[0040] (e) Prepare non-covalent nucleic acid intercalation drug solution standards with different concentration gradients, replace the complex mixed solution to be tested with each standard, and use the same operation as in steps (b)-(d) under the same mechanical tensile stress conditions to measure the length change of double-stranded nucleic acid molecules after adding nucleic acid intercalation drug solution standards with different concentration gradients as a standard curve;

[0041] By comparing the length change in the standard curve with the length change caused by the complex mixed solution to be tested obtained in step (d), the concentration of non-covalent nucleic acid intercalated drug contained in the complex mixed solution to be tested is quantified.

[0042] Compared with the prior art, the present invention reports for the first time the direct detection of compounds that non-covalently intercalate and bind double-stranded nucleic acids in mixed solutions such as bacterial supernatant, plant extracts and animal blood samples using single-molecule manipulation experiments. The prior art cannot directly identify DNA intercalators in mixed solutions. It requires extraction of the product from several liters or kilograms of microbial culture and fermentation using a large amount of solvent to obtain active compounds. Then, it requires multi-step separation and purification using different chromatographic techniques to obtain relatively pure compounds before they can be detected by spectroscopy or mass spectrometry. Compared with traditional methods, the present invention has the following advantages: (1) The sample to be tested does not require complicated separation and purification. Only simple filtration and / or centrifugation are needed to remove insoluble components (for example, in Examples 2-3 below, only simple centrifugation was performed to obtain the supernatant). There is no need to purify or subdivide soluble components, avoiding cumbersome multi-step purification processes (such as extraction, chromatographic purification, crystallization, resin adsorption, distillation, etc.). (2) The required sample volume is small, only microliters of sample volume is needed. (3) High detection sensitivity; furthermore, by applying external force (preferably 1-60 piconewtons) to both ends of the double-stranded nucleic acid, the intercalation binding of the compound can be enhanced, further improving the detection sensitivity. This invention can also be used to measure the concentration using gradient concentration standards when there is only one non-covalent nucleic acid intercalating agent and the type of this non-covalent nucleic acid intercalating agent is known. Taking Examples 1 and 5 below as examples, this invention proposes a method for quantifying the concentration of the intercalating agent in a mixed solution by measuring a standard curve of DNA elongation after adding different concentrations of DNA intercalating agent standards. Examples of this invention demonstrate that detection under stress on double-stranded DNA can increase the detection limit for the DNA intercalating agent daunorubicin from micromoles per liter to nanomoles per liter.

[0043] The method of this invention can both inactivate the enzyme in the complex mixed solution to be tested (e.g., heating, sonication, addition of protein denaturing agents, addition of enzyme inhibitors, addition of metal ion chelating agents), and control the duration of application of the complex mixed solution to be tested to the double-stranded nucleic acid molecules (less than 10 minutes) to ensure that the length change of the double-stranded nucleic acid molecules comes from non-covalently intercalated nucleic acid compounds, avoiding the influence of covalent intercalation (of course, for unknown complex samples to be tested that do not contain enzyme components, enzyme inactivation treatment can also be omitted).

[0044] This invention is applicable to the detection and analysis of complex mixed solutions, capable of analyzing and detecting the presence of small molecule compounds that non-covalently intercalate and bind double-stranded DNA (or double-stranded RNA, or DNA / RNA hybrid strands) in the sample. This invention is applicable to complex mixed solutions derived from microbial culture media, microbial extracts, microbial secondary metabolites; animal body fluids, animal and plant tissues, animal and plant tissue extracts, animal and plant tissue metabolites; natural soil samples, and natural water samples, with complex compositions (e.g., microbial secondary metabolites are samples derived from microorganisms after simple water dissolution, filtration, and centrifugation; animal body fluids include blood, urine, saliva, serum, and fluids derived from cells or tissues after simple water dissolution, filtration, and centrifugation; plant tissue fluids are mixed solutions of plant tissues after disruption and uniform dispersion in one or more liquid media); it can also be used to detect environmental samples, such as soil and water samples.

[0045] Taking microbial culture media, microbial extracts, and microbial secondary metabolites as examples, the tissue fluids, metabolites, and secondary metabolites of microorganisms, animals, and plants contain complex components, including proteins, nucleic acids, sugars, lipids, and various organic and inorganic small molecules. These complex components often interfere with detection. Whether traditional methods can detect small molecule compounds that can intercalate and bind to double-stranded nucleic acids in these complex solution systems remains a challenging issue. Furthermore, due to the influence of nucleic acid-binding compounds themselves on cellular nucleic acid metabolism, the concentration of nucleic acid intercalation-binding compounds in microbial, animal, and plant tissue fluids, metabolites, and secondary metabolites is not high. How to improve detection sensitivity using single-molecule manipulation experiments still requires the development of new quantitative methods. This invention demonstrates for the first time that these complex components do not interfere with the measurement of force-tension curves in single-molecule manipulation experiments, and proves that by applying tension to nucleic acids, the binding constant of small molecule compounds intercalating into double-stranded nucleic acids can be increased, thereby improving the detection limit (the lowest concentration at which nucleic acid intercalation can be detected) for trace amounts of nucleic acid intercalators. This allows for efficient and highly sensitive detection of whether complex mixed solutions contain nucleic acid intercalators (i.e., compounds capable of intercalating into nucleic acids).

[0046] Based on the method of this invention, for the sample to be tested, the sample can be collected and pretreated to dissolve the target analyte in an aqueous solution or uniformly disperse it in a liquid medium to obtain a mixed solution to be tested. For example, when the sample to be tested is derived from animals, plants, microorganisms, environmental samples, or chemically synthesized samples, the pretreatment method can be one or more operations such as water dissolution, dilution, sonication, filtration, centrifugation, etc., to dissolve the target analyte in an aqueous solution or uniformly disperse it in a liquid medium (preferably a solvent miscible with water, such as ethanol), to obtain, for example, supernatant of microbial liquid culture medium, supernatant of animal blood, urine, digestive fluid, extracts and metabolites of animals and plants, supernatant of chemically synthesized compound solutions, etc., which can be used as complex mixed solutions to be tested. The method of this invention only requires simple dissolution pretreatment to detect and analyze whether complex samples contain nucleic acid intercalating agents. For example, when the sample is a liquid sample such as a culture medium, extract, or body fluid, the sample pretreatment method can be centrifugation to obtain the supernatant; when the sample is a solid sample such as animal or plant tissue, soil, etc., the pretreatment method can be dissolving and dispersing it in a liquid medium and then obtaining the supernatant.

[0047] This invention utilizes single-molecule manipulation technology to detect the increase in double-stranded nucleic acid length caused by nucleic acid intercalation agents by applying external force to double-stranded nucleic acids, and particularly improves the detection sensitivity of nucleic acid intercalation compounds through external force. This invention can be used for screening microbial bioactivity, and also for detecting blood and tissue drug concentrations of nucleic acid intercalation agent drugs. For example, in one embodiment, nucleic acid intercalation compounds were detected in the metabolites of *Streptomyces cerevisiae*, in another embodiment, nucleic acid intercalation compounds were detected in the aqueous extract of the medicinal plant *Polygonum multiflorum*, and in yet another embodiment, daunorubicin was detected in the plasma of mice after injection of daunorubicin, with the ability to further determine blood drug concentrations using standard curves. This invention can be applied to new drug development (e.g., screening new drugs) to determine whether a sample contains target functional components such as nucleic acid intercalation compounds. For example, the method of this invention can be used to screen novel nucleic acid intercalation compounds from microorganisms and plants, and to screen new drugs. This invention is also applicable to the detection and analysis of whether other traditional Chinese medicine materials contain nucleic acid intercalation compounds, thereby providing a preliminary evaluation of whether a particular traditional Chinese medicine material is worth purifying to obtain its functional components.

[0048] This invention utilizes single-molecule manipulation technology to record the length changes of double-stranded nucleic acid molecules before and after the addition of a sample solution during a single-molecule mechanical tensile test. This data is used to determine the ability of compounds in the sample to intercalate and bind to nucleic acids. The effect of solution ionic conditions on the elongation of double-stranded nucleic acids primarily affects the persistence length. Under a certain external force, the end-to-end distance of a double-stranded nucleic acid is close to its counter length. Solution ionic conditions do not lead to a significant increase in the counter length of nucleic acids (Biophysical Journal 2019, 116, 196-204). Most proteins binding to and encapsulating double-stranded nucleic acids reduce their length or increase their persistence length, but do not cause an increase in length under stress. Therefore, the increase in double-stranded nucleic acid length measured under stress is a unique characteristic of nucleic acid intercalating agents.

[0049] Taking the detection of nucleic acid intercalators in microbial metabolites using double-stranded DNA as an example, the present invention alters the properties of the force-extension curve of DNA after the binding of bacterial nucleic acids and proteins to double-stranded DNA, including: (i) protein binding alters the persistence length of DNA, leading to an increase or decrease in the length of double-stranded nucleic acids under low force (less than 10 piconewtons), but no significant increase in the length of double-stranded nucleic acids under high force. (ii) proteins, such as histones, bend or entangle double-stranded DNA after binding, resulting in a decrease in the end-to-end distance of the DNA under stretching. Both of these changes are significantly different from the changes in the double-stranded DNA stretching curve caused by small-molecule nucleic acid intercalators. Therefore, the present invention demonstrates that the presence of nucleic acid intercalation compounds in complex mixed solutions can be directly identified by analyzing the length of nucleic acids under stress without separating a single nucleic acid intercalator compound. After using the purified compound to measure the force-extension curve as a standard curve, the present invention can also be used to quantify the concentration of compounds contained in blood and tissue fluid.

[0050] This invention is the first to report the use of double-stranded nucleic acid molecules (e.g., double-stranded DNA) in single-molecule manipulation experiments to detect compounds embedded with nucleic acids in mixed solutions such as bacterial supernatant, plant extracts, and animal blood samples. By introducing complex mixed solutions into single-molecule stretching experiments, this method can not only evaluate the ability of compounds to embed nucleic acids in mixed solutions, but also analyze the nucleic acid embedding ability of crude bacterial extracts, plant extracts, blood samples, and subsequent single-component products obtained through separation and extraction.

[0051] Furthermore, this invention also discovered that increasing the external force can increase the detection limit of nucleic acid intercalation compounds. Taking Example 1 below as an example, this invention demonstrates that only 100 nanomoles per liter of daunorubicin can be detected at 1 piconewton, but when the external force at both ends of the double-stranded DNA is increased to 60 piconewtons, the binding constant of daunorubicin intercalation into the DNA increases by several orders of magnitude, thereby enabling the detection of daunorubicin at a concentration of 10 nanomoles per liter in solution. Therefore, based on this invention, the single-molecule manipulation method can detect nanomolar levels of daunorubicin in trace solutions (10 μL), thus enabling its application to the detection of samples including trace amounts.

[0052] This invention is also applicable to various types of samples, including microbial colonies, plant tissues, and animal blood. Soluble components, including the target analyte, can be dissolved in an aqueous solution or uniformly dispersed in a liquid medium through operations such as water dissolution, dilution, sonication, filtration, and centrifugation, while removing insoluble components. No purification is required (i.e., no further subdivision of soluble components). This successfully enables the detection of samples from multiple sources without isolating a single compound. Furthermore, by applying an external force of 1-60 piconewtons, the detection limit is several orders of magnitude higher than in the case of no force in solution. As demonstrated in Examples 2-5 below, the complex components in microbial, plant, and animal samples do not cause a significant increase in the length of double-stranded DNA between 1 and 60 piconewtons (length increase <10%). The increase in double-stranded nucleic acid length measured under force is a unique characteristic of nucleic acid intercalators, allowing this invention to detect the presence of compounds intercalating and binding nucleic acids in complex samples without isolating a single intercalator compound. Furthermore, the detection limit of nucleic acid intercalators can be increased by applying external force, making it possible to detect trace amounts of nucleic acid intercalators in complex solutions.

[0053] In summary, this invention uses a single-molecule method to identify the presence of non-covalently intercalated nucleic acid-binding small molecule compounds in complex solutions. This invention is universally applicable to tissue fluids, metabolites, and secondary metabolites of microorganisms, animals, and plants, and can analyze both known and unknown intercalated nucleic acid compounds. Examples of this invention demonstrate its applicability to the detection of microbial, plant, and animal samples, capable of detecting any compound that binds to double-stranded nucleic acids and causes an increase in the length of both ends of the double-stranded nucleic acid. In the embodiments of this invention, complex components in bacterial supernatants, plant aqueous extracts, and plasma do not interfere with the length of the double-stranded nucleic acid ends under stress. This invention requires no compound separation or special labeling, and is suitable for screening microorganisms and plants based on nucleic acid binding activity. Combined with microfluidics and other methods, this invention is also suitable for high-throughput screening of nucleic acid-binding compounds, which is of great significance for developing new nucleic acid-binding natural products and discovering new anticancer active molecules. In the field of pharmacology and toxicology research, this invention can also be used to analyze the drug concentration of purified single nucleic acid intercalated drugs in animals. Attached Figure Description

[0054] Figure 1 This is a schematic diagram and operational flowchart illustrating the detection of DNA-embedded compounds in a mixed solution using single-molecule magnetic tweezers, as described in an embodiment of the present invention. Figure 1 The diagram in Figure 'a' corresponds to the detection method. The nucleic acid intercalating agent containing an aromatic planar loop interacts with DNA and intercalates into the bound DNA, thus increasing the length of the double-stranded DNA. Figure 1 The diagram in step b corresponds to the following operation process: First, collect the test samples (such as microbial culture medium, extracts and secondary metabolites; animal body fluids, plant tissue fluids and metabolites, etc.), and process them to obtain the mixed solution to be tested. Then, set up a device to measure the sample, connect a magnetic ball to the double-stranded DNA on the surface of a glass slide, and measure the height of the magnetic ball. Next, add the mixed solution to be tested, and measure the height of the magnetic ball again. By comparing the height before and after sample addition, the degree of change in height can be used to determine the strength of the compound embedded in the DNA in the mixed solution.

[0055] Figure 2 This is the standard curve for daunorubicin in an embodiment of the present invention. Figure 2 The 'a' in the formula corresponds to the chemical structural formula of daunorubicin. Figure 2In the figure, b corresponds to the intercalation binding of daunorubicin to double-stranded DNA under concentration gradients measured by force-stretch curves (external force conditions were 1 piconewton, 10 piconewton, 20 piconewton, 30 piconewton, 40 piconewton, 50 piconewton, 60 piconewton, 65 piconewton, 70 piconewton, and 75 piconewton). At a force of 60 piconewton, an increase in double-stranded DNA elongation and microsphere height was observed due to the intercalation of 2 nanomoles per liter of daunorubicin. At a force of 1 piconewton, an increase in double-stranded DNA elongation and microsphere height was observed due to the intercalation of 200 nanomoles per liter of daunorubicin. Figure 2 The 'c' in the figure corresponds to the standard curves of DNA elongation at different concentrations. Standard curves measured at 1 piconewton, 20 piconewton, 40 piconewton, and 60 piconewton are shown. The curves are fitted using a Hill function. Applying external force can increase the detection limit for small molecule compounds embedded in double-stranded DNA.

[0056] Figure 3 The image shows the results of a single-molecule stretching experiment performed on the solution obtained by culturing *Streptomyces cerevisiae* producing daunorubicin in an embodiment of the present invention (external force conditions were 1 piconewton, 10 piconewton, 20 piconewton, 30 piconewton, 40 piconewton, 50 piconewton, 60 piconewton, 65 piconewton, 70 piconewton, and 75 piconewton). Specifically, after obtaining single clones of *Streptomyces cerevisiae* through plate culture, the selected colonies were soaked in 1 ml of water and centrifuged to obtain the supernatant. After injecting 5 μL of the supernatant, the length of the double-stranded DNA (triangle diagram) was significantly increased compared to the DNA length measured at 0-60 piconewtons without injection (circle diagram), indicating that *Streptomyces cerevisiae* colonies secrete compounds embedded in DNA.

[0057] Figure 4 This image shows the results of a single-molecule stretching experiment performed on the supernatant obtained by centrifuging *Streptomyces cerevisiae* and *Escherichia coli* in an embodiment of the present invention. Figure 4 The figure for 'a' corresponds to the results of *Streptomyces cerevisiae* (external force conditions are 1 piconewton, 10 piconewton, 20 piconewton, 30 piconewton, 40 piconewton, 50 piconewton, 60 piconewton, 65 piconewton, 70 piconewton, and 75 piconewton). Specifically, after culturing *Streptomyces cerevisiae* culture and centrifuging to obtain the supernatant, the length of double-stranded DNA measured at 0-60 piconewtons (square diagram) after injecting 5 μL of the supernatant was significantly increased compared to the DNA without injection (circle diagram), indicating that *Streptomyces cerevisiae* culture secreted and formed a strong DNA-intercalating compound. Figure 4In the diagram, b corresponds to the results of E. coli (external conditions are the same as above). Specifically, after culturing E. coli culture and centrifuging to obtain supernatant, the length of double-stranded DNA (diamond diagram) after injecting 5 μL of supernatant did not increase significantly compared to DNA without injection (circle diagram) (length increase <10%), indicating that the E. coli culture did not secrete or secreted a small amount of DNA-intercalating compounds. Figure 4 The 'c' in the figure corresponds to the HPLC-MS / MS result of the crude extract of *Streptomyces cerevisiae* bacterial suspension. Specifically, the *Streptomyces cerevisiae* bacterial suspension was extracted with an organic solvent, the organic layer was evaporated to obtain the crude extract, which was then dissolved and injected into the HPLC-MS / MS instrument; daunorubicin [M+H] was extracted from the results. + The chromatographic peak is located at the gray peak indicated by the arrow in the upper spectrum. The lower spectrum is the mass spectrum corresponding to the chromatographic peak, with [M+H] in the figure. + The corresponding peak is the characteristic peak of daunorubicin, and its value is the molecular mass of daunorubicin plus the mass of hydrogen atoms.

[0058] Figure 5 The figures shown are from an embodiment of the present invention, illustrating the results of single-molecule stretching experiments on the processed Polygonum multiflorum tissue extract (external force conditions were 1 piconewton, 10 piconewton, 20 piconewton, 30 piconewton, 40 piconewton, 50 piconewton, 60 piconewton, 65 piconewton, 70 piconewton, and 75 piconewton). Specifically, Polygonum multiflorum was sliced, dried, and ground to obtain plant tissue powder. The powder was then extracted with boiling water and filtered to obtain a filtrate of the tissue extract. After injecting 5 μL of the filtrate, the length of double-stranded DNA (triangular diagram) under 0-60 piconewton conditions showed a significant increase compared to the DNA without injection (circular diagram), indicating that the present invention can detect emodin-like DNA intercalation compounds in Polygonum multiflorum tissue.

[0059] Figure 6 This diagram shows the results of single-molecule detection of daunorubicin, a DNA-intercalated drug, in mouse serum in an embodiment of the present invention (external force conditions were 1 piconewton, 10 piconewton, 20 piconewton, 30 piconewton, 40 piconewton, 50 piconewton, 60 piconewton, 65 piconewton, 70 piconewton, and 75 piconewton). Specifically, daunorubicin hydrochloride solution or an equal volume of physiological saline was injected into mice via tail vein injection. After waiting for 5 minutes, blood was collected from the eyeballs and centrifuged to obtain plasma samples. After injecting 100 μL of plasma, the length of double-stranded DNA (triangle diagram) in the daunorubicin hydrochloride group (0-60 piconewtons) was significantly increased compared to the DNA without injection (circle diagram). The length of double-stranded DNA (triangle diagram) in the physiological saline group was essentially unchanged compared to the DNA without injection (circle diagram), indicating that the present invention can detect daunorubicin in mouse blood. Detailed Implementation

[0060] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0061] In summary, this invention applies the relatively mature single-molecule manipulation technology in the prior art to detect the presence of known or unknown nucleic acid intercalating agents in complex mixed solutions. Specifically, it utilizes single-molecule manipulation technology to detect the presence of small molecule compounds intercalated with double-stranded nucleic acids in complex mixed solutions. This technology can be applied to: microbial culture media, extracts, and secondary metabolites; animal body fluids (blood, urine, digestive juices, tissue fluids), animal and plant extracts, and metabolites; soil and water samples to be tested; and to detect the presence of small molecule compounds intercalated with bound double-stranded nucleic acids.

[0062] Taking double-stranded DNA as an example (the same applies to double-stranded RNA and DNA / RNA hybrid strands), based on this invention, double-stranded nucleic acids can be fixed between a fixed interface (i.e., the first surface) and a movable interface (i.e., the second surface). The length of the double-stranded nucleic acid is measured using single-molecule manipulation techniques (i.e., by applying mechanical force to stretch the double-stranded nucleic acid molecule and measuring its length). Then, a sample to be tested (e.g., microbial culture medium, extract, and secondary metabolites; animal body fluids, plant tissue fluids, and metabolites; natural soil samples, and natural water samples) is added. Under the same mechanical conditions, the double-stranded nucleic acid molecules are stretched by applying mechanical force. By measuring the change in length between the two ends of the double-stranded nucleic acid molecule before and after sample addition, the degree of small molecule compound embedding into DNA in the sample can be determined. For samples containing only a single type of nucleic acid intercalator, the end-to-end length change caused by binding a standard of that single nucleic acid intercalator can be used as a reference to determine the concentration of the nucleic acid intercalator in the sample.

[0063] In actual operation, such as Figure 1 As shown, the following steps may be included:

[0064] a. Fixation of double-stranded DNA molecules between a glass slide surface and a magnetic ball: The method of this invention is not limited to magnetic tweezers; any device capable of stretching DNA by external force and simultaneously measuring its length change can implement the method of this invention. For example, double-stranded DNA molecules can be fixed between two microspheres in optical tweezers, between the sample surface and microspheres in acoustic tweezers, or between the sample surface and the tip in an atomic force microscope.

[0065] b. Use single-molecule manipulation experiments to measure the length of double-stranded DNA under stress;

[0066] c. Provide the sample to be tested (e.g., culture medium of microorganisms, plants, animals, or tissue extract) to the system;

[0067] d. Measure the change in length of double-stranded DNA under stress relative to the length of the original DNA under the same stress conditions.

[0068] In step a, the double-stranded DNA molecule used can be a random sequence or a double-stranded DNA with a specific nucleic acid sequence. It can be a PCR product, a plasmid fragment, or an extracted genomic DNA fragment. When generating double-stranded DNA using PCR, biotin and thiol-modified oligonucleotide primers can be used to introduce biotin and thiol into the 5' end of the double-stranded DNA (Nucleic Acids Research. 2014, 42(13), 8789-8795). When using genomic DNA, restriction endonucleases can be used to digest the genomic DNA, and ligases can be used to ligate the modified oligonucleotides to the ends of the nucleic acids. Preferably, this invention uses sulfosuccinimide 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (Sulfo-SMCC) to ligate the thiol groups of the nucleic acids to the glass-modified amino groups.

[0069] Step b involves single-molecule manipulation experiments. The method of this invention can be implemented using magnetic tweezers. However, the implementation of the method is not limited to the aforementioned equipment; any device that allows for the measurement of the force-extension curve of double-stranded nucleic acids can be used to implement the method. For example, optical tweezers, atomic force microscopy, or acoustic tweezers can be used. Of course, magnetic tweezers, compared to optical tweezers and atomic force microscopy, are more conducive to parallel measurement of multiple molecules for high-throughput measurements.

[0070] Step d involves comparing the length of the double-stranded nucleic acid obtained under the external force with the result obtained in step b to determine the change in length. Based on the degree of change in nucleic acid length after the addition of the target analyte, it is determined whether the mixed solution contains a compound that embeds double-stranded nucleic acid. If it contains a compound that embeds double-stranded nucleic acid, there will be a significant change in nucleic acid length.

[0071] Taking the supernatant of *Streptomyces cerevisiae* bacterial culture as the test sample as an example, in actual operation, the method for detecting DNA-intercalated compounds released extracellularly by *Streptomyces cerevisiae* during growth using single-molecule manipulation technology based on the present invention can include the following specific steps:

[0072] 1) Immobilize double-stranded DNA molecules in a microfluidic detection cell and magnetic beads;

[0073] 2) Apply external force to double-stranded DNA molecules and measure the length of double-stranded DNA under different forces;

[0074] 3) Provide the supernatant of Streptomyces cerevisiae (blue-red) to the detection pool;

[0075] 4) Apply an external force to the double-stranded DNA molecule and measure the change in the length of the double-stranded DNA after sample addition.

[0076] To determine whether a sample (i.e., the supernatant of *Streptomyces cerevisiae*) contains small molecule compounds capable of intercalating into DNA, this invention, based on its method, involves adding the target mixed solution to a microfluidic detection cell and comparing the length changes caused by the small molecule compound intercalating into double-stranded DNA, thereby screening out mixed solutions exhibiting significant DNA intercalation. Furthermore, by utilizing the negative feedback system of a single-molecule magnetic tweezers device to reduce instrument drift, this invention enables precise measurement of DNA length changes as small as a few nanometers (~5 nanometers) even after a hundredfold volume change in the microfluidic channel.

[0077] In the following examples, thiol groups at both ends are first prepared via PCR. Then, in a microfluidic detection cell, one end of the double-stranded DNA is immobilized on a glass slide via a coupling reaction, and the other end is immobilized on a magnetic bead. A schematic diagram and flowchart of the measurement process are shown below. Figure 1 As shown, the length of double-stranded DNA without the test solution was measured using a magnetic tweezers single-molecule manipulation system. Specifically, the height of the permanent magnet was adjusted to control the magnetic field strength, and the height of the magnetic ball was measured under different magnetic forces. The intercalation of the compound caused an increase in the length of the double-stranded DNA. The DNA intercalation capability of the small molecule compound in the test solution was determined by measuring the amount of increase in double-stranded DNA length caused by the compound intercalation. In addition, the mechanical stretching of the double-stranded nucleic acid molecule and the detection of its length in the following embodiments were performed automatically by the instrument using a program. Data for one detection point under different external force conditions typically takes 5-6 seconds to obtain. After the detection is completed, the process immediately switches to another external force condition to obtain data for another detection point.

[0078] The following are specific examples:

[0079] Example 1

[0080] Single-molecule stretching measurements of daunorubicin intercalation into DNA leading to double-stranded DNA elongation

[0081] Anthracycline compounds such as daunorubicin are typical DNA intercalation agents. In this embodiment, the end-to-end length increase of double-stranded DNA caused by daunorubicin intercalation into double-stranded DNA was detected by single-molecule magnetic tweezers experiment. Simultaneously, a standard curve for measuring daunorubicin concentration in complex solution systems was obtained based on the amount of double-stranded DNA length increase caused by daunorubicin at different concentration gradients (which can be achieved by preparing standards of known concentrations of daunorubicin).

[0082] 1.1 Preparation of Double-Stranded DNA Samples

[0083] PCR amplification of 6618 base pairs of end-modified double-stranded DNA was used for single-molecule stretching experiments. The reaction system included Ex Taq enzyme (catalog number: RR001A, Beijing Baoriyi Biotechnology Co., Ltd.) and 10×Ex Taq Buffer (Mg... 2+ Plus (Catalog No.: 9152A, Beijing Baoriyi Biotechnology Co., Ltd.), 2.5 mmol dNTP Mixtures (Catalog No.: 4030Q, Beijing Baoriyi Biotechnology Co., Ltd.), 2 μg λ-DNA template (Catalog No.: SD0011, Shanghai Thermo Fisher Scientific Co., Ltd.), 1 μm 5' biotin primer (Shanghai Sangon Biotech Co., Ltd.), and 1 μM 5' thiol primer (Shanghai Sangon Biotech Co., Ltd.). PCR products were purified and recovered using the kit.

[0084] 1.2 Assemble the microfluidic detection cell

[0085] Amino-modified glass surfaces were used to construct microfluidic detection cells. 24×54 mm and 22×22 mm glass slides (Leibus, Shanghai) were placed on supports and immersed in a 20% Decon90 cleaning agent (Decon, USA), ultrasonically cleaned, and dried. A 24×54 mm glass slide was soaked overnight in a 10 mol / L potassium hydroxide solution, rinsed with ultrapure water, ultrasonically cleaned again, and dried. Another 24×54 mm slide was immersed in a 1% APTES (3-aminopropyltriethoxysilane, CAS No.: 919-30-2, Cool Chemical Technology (Beijing) Co., Ltd.) methanol solvent in the dark for 30 minutes to modify the amino groups, then rinsed twice with methanol, washed 6–8 times with ultrapure water, and dried. The 24×54 mm and 22×22 mm glass slides were then bonded together using double-sided adhesive to construct a microfluidic detection cell. Figure 1 The microfluidic detection cell shown in b.

[0086] 1.3 Immobilizing double-stranded DNA on a glass surface and attaching streptavidin-coated superparamagnetic spheres

[0087] A chemical reaction was used to couple the terminal thiol groups of double-stranded DNA to the amino groups on a glass surface. 1 mg / mL of Sulfo-SMCC solution (sulfosuccinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate, Thermo Fisher Scientific, USA, catalog number 22322) was added to the microfluidic detection cell to activate the glass surface. After reacting for 25 minutes, unreacted Sulfo-SMCC was eluted with 500 μL of 1× phosphate buffer. The prepared DNA sample was added and covalently reacted with the glass surface for 45 minutes. Blocking buffer (prepared by mixing 0.1 g bovine serum albumin, 5 μL β-mercaptoethanol, and 10 mL 1× phosphate buffer) was then added, and blocking was performed for at least 4 hours. Finally, streptavidin-coated superparamagnetic beads (Dynabeads) were immobilized on the double-stranded DNA. TM M-280 Part Number: 35136 (Shanghai Thermo Fisher Scientific Co., Ltd.)

[0088] 1.4 Force-stretch curve and double-stranded DNA elongation-drug concentration standard curve after the addition of daunorubicin to double-stranded DNA

[0089] Daunorubicin was added to a microfluidic detection cell at concentration gradients ranging from 2 nanomoles per liter to 200 micromoles per liter. A jump test was used to measure the height of magnetic spheres bound to double-stranded DNA under different forces, ranging from 1 piconewton to 75 piconewtons. By subtracting the curves of the height of the magnetic spheres at different concentrations from those of double-stranded DNA without the compound under the same external force, the elongation of the double-stranded DNA caused by the small molecule compound insertion under different forces could be obtained. The dissociation constant Kd of dunorubicin binding to double-stranded DNA under different forces was fitted using the Hill equation (Formula 1).

[0090]

[0091] In formula (1), x saturate -x pure DNA The difference in elongation between DNA and the control group after daunorubicin binds to DNA at saturated concentration is represented by c, where c is the concentration of daunorubicin, Kd is the dissociation constant, and n is the Hill coefficient. After obtaining the standard curve, the concentration of daunorubicin in the solution can be determined by measuring the elongation caused by the complex solution under test. This example demonstrates that under stress conditions, the intercalation-binding constant of daunorubicin and double-stranded DNA increases by two orders of magnitude. At 1 piconewton, the lowest detectable concentration of daunorubicin bound to double-stranded DNA is on the order of 1 micromoles per liter. Under a stress of 60 piconewtons, the concentration of daunorubicin bound to double-stranded DNA that can be detected by this method reaches 10 nanomoles per liter (e.g., ...). Figure 2(As shown in c). This is because the external force lowers the dissociation constant of daunorubicin intercalating into DNA, reducing the detection limit from micromoles per liter to nanomoles per liter. While conventional analytical methods may also achieve detection limits in the micromole per liter range (e.g., chromatographic analysis), the binding of this compound to double-stranded DNA often cannot be detected in the same assay procedure. Compared to conventional analytical methods for DNA under stress-free conditions, this method improves the detection of DNA intercalators by two orders of magnitude.

[0092] Example 2: Single-molecule detection of daunorubicin produced by a single colony of *Streptomyces cerevisiae*

[0093] 2.1 Culture of single colonies of Streptomyces cerevisiae

[0094] A single colony of *Streptomyces coerleorubidus* (purchased from the China Industrial Microbial Culture Collection Center, strain number: CICC 11043) was inoculated onto agar plates. The *Streptomyces coerleorubidus* strain was dissolved in 1 mL of sterile water, mixed thoroughly, and then streaked onto prepared agar plates using an inoculation loop. The agar plate composition consisted of 0.4 g glucose, 0.4 g yeast extract, 1 g malt extract, and 15 g agar dissolved in 100 mL of water. After streaking, the plates were transferred to a 30°C incubator and incubated for 7 days until the colonies were largely established.

[0095] 2.2 Soaking in water to dissolve DNA intercalating agent secreted by a single colony

[0096] Individual colonies were soaked in water along with the culture medium below to obtain the DNA-intercalated compound secreted by the colonies. The individual colonies were picked out along with the culture medium below, weighed, and then soaked in 1 ml of sterile water. They were ultrasonically cleaned for 30 minutes, followed by centrifugation at 12,000 rpm for 5 minutes. 300 μL of the supernatant was collected into another clean centrifuge tube.

[0097] 2.3 Single-molecule magnetic tweezers measurement of DNA length changes caused by intercalated DNA compounds secreted by single colonies

[0098] The steps for measuring the embedded DNA compounds secreted by a single colony in this protocol are as follows: Figure 3 As shown, firstly, a single-molecule magnetic tweezers device was used to measure the force-stretch curve of the magnetic spheres and double-stranded DNA within the microfluidic detection cell. Figure 3 (As shown in the dotted diagram), then 5 μL of the supernatant obtained from the water solution was added to the microfluidic detection cell (timing started immediately after sample addition; at this time, Time = 0). One minute after adding the supernatant (at this time, Time = 1 min), the force-stretch curve of the double-stranded DNA was measured. Figure 3(As shown in the triangle diagram), the detection is completed within 2 minutes (at this time, Time = 3min).

[0099] like Figure 3 As shown, the results demonstrate that when an external force of 1-60 pN is applied to the detection chamber, the supernatant obtained from soaking colonies in water can intercalate into DNA, causing an increase in double-strand length exceeding 10% of the double-stranded DNA length. In the embodiments of this invention, an increase in double-stranded DNA length caused by the secretions of a single colony of *Streptomyces cerevisiae* can be detected, indicating that the single-molecule method can detect trace amounts of DNA-intercalating compounds produced by a single colony.

[0100] Example 3 Single-molecule detection of DNA intercalating agent in the supernatant of Streptomyces cerevisiae var. cyanide.

[0101] 3.1 Culture the bacterial culture and collect the supernatant.

[0102] Streptomyces coerleorubidus (purchased from China Industrial Microbial Culture Collection Center, strain number: CICC11043) and Escherichia coli (E. coli BL21(DE3) Competent Cells, Sangon Biotech (Shanghai) Co., Ltd.) were inoculated and cultured in liquid culture medium. The supernatant was obtained by centrifugation. The Streptomyces culture medium consisted of 0.4 g glucose, 0.4 g yeast extract, and 1 g malt extract dissolved in 100 mL of water; the E. coli culture medium consisted of 0.5 g yeast extract, 1 g tryptone, and 1 g sodium chloride dissolved in 100 mL of water. After preparation, the medium was sterilized and allowed to cool naturally to room temperature. The bacterial cultures were then inoculated into the liquid culture medium and incubated at a constant temperature. After incubation, the medium was heated to 95°C for 10 minutes, centrifuged at 12,000 rpm for 5 minutes, and 300 μL of supernatant was collected.

[0103] 3.2 Single-molecule magnetic tweezers measurement of DNA length changes caused by DNA-intercalating compounds produced in bacterial cultures

[0104] The steps for measuring the DNA-intercalated compounds produced by bacterial culture in this protocol are as follows: Figure 4 As shown, the force-stretch curve of DNA without bacterial culture was first measured using a single-molecule magnetic tweezers device as a control. Figure 4 (See the dotted diagrams a and b in the image). Then, add 5 μL of the supernatant obtained from centrifugation to the microfluidic detection chamber. Within 1 minute of adding the Streptomyces azure-red supernatant, begin measuring the force-stretch curve of the double-stranded DNA. Figure 4 (See the block diagram shown in Figure a). The detection was completed within 2 minutes. As a control experiment, we also tested the force-stretch curve of double-stranded DNA in the E. coli supernatant after the same treatment. Figure 4(The rhombus illustration shown in b).

[0105] Depend on Figure 4 Comparing a and b in the figures, it can be seen that the increase in double-stranded DNA length in the force-stretch curve after applying the Streptomyces cerevisiae supernatant is much greater than 10% of the 2250 nm double-stranded DNA length. This is completely different from the force-stretch curve of double-stranded DNA after applying the negative control Escherichia coli supernatant. This indicates that there are DNA-intercalating compounds in the Streptomyces cerevisiae supernatant, while there are no DNA-intercalating compounds in the Escherichia coli supernatant (or the content of DNA-intercalating compounds is below the detection limit). To further verify this result, the following operations were also performed in this embodiment:

[0106] 3.3 High performance liquid chromatography-mass spectrometry (HPLC-MS) was used to identify daunorubicin, a DNA-intercalated compound produced by bacterial culture.

[0107] First, 300 mL of ethyl acetate was mixed with 50 mL of bacterial culture by inverting, followed by extraction under sonication for 30 minutes. The ethyl acetate layer was separated and evaporated to dryness to obtain the crude bacterial extract, which was then dissolved in methanol, centrifuged at 12000 rpm for 10 minutes, and added to a high-performance liquid chromatography-mass spectrometry (HPLC-MS) instrument. After analysis, the HPLC chromatogram was obtained (e.g., ...). Figure 4 The upper spectrum shown in c) and the mass spectrum corresponding to the chromatographic peak containing daunorubicin ( Figure 4 (The lower part of the diagram shown in c).

[0108] like Figure 4 As shown in a, the results have demonstrated that when the supernatant obtained from centrifuged Streptomyces cerevisiae solution is added to the detection cell, DNA-intercalating compounds in the solution can intercalate into DNA, causing an increase in double-strand length; as shown in a, ... Figure 4 As shown in b, the results have demonstrated that when the supernatant obtained from centrifuged E. coli culture was added to the detection tank, there was no significant change in the double-stranded DNA. Figure 4 As shown in c, the results demonstrate that the crude extract of *Streptomyces cerevisiae* culture analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) showed the presence of daunorubicin in the crude extract. This example demonstrates the ability to detect DNA intercalators in bacterial cultures without isolating a single compound, and this method can be widely used to screen microorganisms that produce DNA intercalators.

[0109] Example 4: Detection of anthraquinone DNA intercalation compounds in Polygonum multiflorum aqueous extract

[0110] It is known in existing technology that the medicinal plant *Polygonum multiflorum* contains anthraquinone compounds such as emodin, chrysophanol, and rhein methyl ether, which can intercalate and bind to DNA. This embodiment uses a single-molecule magnetic tweezers experiment to detect whether the aqueous extract of *Polygonum multiflorum* contains compounds that intercalate and bind to DNA.

[0111] 4.1 Extraction of water-soluble compounds from the tuberous roots of Polygonum multiflorum

[0112] The roots of *Polygonum multiflorum* were sequentially sliced, dried, ground, and subjected to boiling water extraction to obtain water-soluble compounds. One root was sliced ​​and dried. It was then ground to obtain *Polygonum multiflorum* powder. 1g of the powder was added to 50ml of boiling water and boiled for 1 hour, adding approximately 60ml of water halfway through. After the extraction, the mixture was allowed to cool naturally to room temperature. The crude extract was filtered using defatted cotton as a filter. Then, 1-2ml of the filtrate was filtered through a 0.22-micron filter membrane into a clean centrifuge tube.

[0113] 4.2 Single-molecule magnetic tweezers measurement of DNA length changes caused by DNA-intercalated compounds in Polygonum multiflorum aqueous extract

[0114] The steps for measuring the DNA-intercalated compounds produced by bacterial culture in this protocol are as follows: Figure 5 As shown, the force-stretch curve of DNA without extraction solution was first measured using a single-molecule magnetic tweezers device as a control. Figure 5 (Dot diagram), then 5 μL of filtered Polygonum multiflorum aqueous extract was added to the microfluidic detection cell. The force-stretch curve of double-stranded DNA was measured within 1 minute of adding the Polygonum multiflorum aqueous extract. Figure 5 (Triangle diagram), the test can be completed within 2 minutes. Figure 5 The results demonstrate that this embodiment can detect anthraquinone compounds that intercalate and bind to DNA in the medicinal plant *Polygonum multiflorum* through simple water extraction. This method can be widely used to screen plants that produce DNA intercalation agents.

[0115] Example 5: Single-molecule detection of daunorubicin concentration in mouse serum

[0116] 5.1 Mice were injected with daunorubicin hydrochloride via the tail vein and blood was collected.

[0117] Mice were injected intravenously with daunorubicin hydrochloride at a dose of 10 mg / kg. After 5 minutes, blood was collected from the eye, yielding approximately 500 μL of blood. The blood was then centrifuged at 14,000 g / min for 5 minutes, and 100 μL of the supernatant was collected for analysis. Control mice were injected with the same volume of saline, and the same procedure was used to obtain the blood supernatant.

[0118] 5.2 Single-molecule magnetic tweezers measurement of changes in DNA length induced by daunorubicin in mouse blood supernatant

[0119] The steps for measuring daunorubicin in mouse blood supernatant in this embodiment are as follows: Figure 6 As shown, the force-stretch curve of DNA without sample was first measured using a single-molecule magnetic tweezers device as a control. Figure 6(See the dotted diagram shown). Then, 100 μL of mouse blood supernatant was added to the microfluidic detection chamber. The force-stretch curve of the double-stranded DNA was measured within 1 minute of adding the mouse blood supernatant. Figure 6 (As shown in the triangular diagram), the test is completed within 2 minutes. The presence of DNA intercalators in the serum is determined by analyzing the increase in DNA length.

[0120] Depend on Figure 6 A comparison of the force-stretch curves of double-stranded DNA obtained from the blood supernatant obtained by injecting "daunorubicin hydrochloride" and the blood supernatant obtained by injecting "physiological saline" shows that there are no DNA-intercalated compounds in the blood supernatant obtained by injecting "physiological saline", while DNA-intercalated compounds are present in the blood supernatant obtained by injecting "daunorubicin hydrochloride".

[0121] Furthermore, the following operations can also be performed:

[0122] 5.3. Calculation of free daunorubicin concentration in mouse blood supernatant based on single-molecule magnetic tweezers measurement results

[0123] according to Figure 6 Substitute the elongation of double-stranded DNA at 60 piconewtons in the experiment. Figure 2 The standard curve of double-stranded DNA elongation at different drug concentrations was fitted using a Hill function (Formula 1). The difference in elongation between the DNA and the control group at saturated concentrations of daunorubicin after binding to DNA is represented by x. saturate -x pure DNA The nanometer size is 917.5 nm, the dissociation constant Kd is 0.085 μmol / L, and the Hill factor n is 0.455. Figure 6 In the experiment, after adding mouse blood supernatant, the double-stranded DNA elongation at 60 piconewtons was measured to be 415 nanometers, thus the concentration of daunorubicin in the mouse blood supernatant of the daunorubicin hydrochloride group was determined to be 56 nanomoles per liter.

[0124] like Figure 6 As shown, the results demonstrate that when mouse blood supernatant injected with daunorubicin hydrochloride is added to the detection cell, the DNA-intercalating compound in the solution can intercalate into DNA, causing an increase in double-strand length, while mouse blood supernatant injected with physiological saline does not cause a significant change in DNA length. The embodiments of this invention can be used to detect the blood concentration of daunorubicin from small amounts of blood. Under nucleic acid stress, the single-molecule method can achieve a detection limit of nanomolar per liter. This method can be widely used for the pharmacokinetic detection of DNA-intercalated drugs.

[0125] The above embodiments are merely examples. In addition to those exemplified above, for unknown complex samples to be tested, to ensure that the length variation of the double-stranded nucleic acid molecule originates from non-covalently intercalated nucleic acid compounds and avoids the influence of covalent intercalation, this invention can either perform enzyme inactivation treatment on the complex mixed solution to be tested (e.g., heating, sonication, addition of protein denaturing agents, addition of enzyme inhibitors, or addition of metal ion chelating agents commonly used in the prior art), or control the duration of application of the complex mixed solution to be tested to the double-stranded nucleic acid molecule for detection (less than 10 minutes). Of course, for unknown complex samples to be tested that do not contain enzyme components, enzyme inactivation treatment can be omitted. Furthermore, for single-molecule manipulation techniques, for aspects not described in detail in this invention, please refer to existing technologies (e.g., Chinese patent application CN115166131A entitled "Method for Single-Molecular Manipulation Detection of DNA Adducts Catalyzed by In Vitro Drug Metabolism Enzymes").

[0126] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting and analyzing non-covalent nucleic acid intercalating agents in complex mixed solutions using single-molecule manipulation, characterized in that, Includes the following steps: (a) Prepare a complex mixture solution to be tested, said complex mixture solution being tested being derived from at least one of the following: microbial culture medium, microbial extract, microbial secondary metabolites; Animal body fluids, animal and plant tissues, extracts from animal and plant tissues, metabolic products of animal and plant tissues; soil samples from nature, water samples from nature; (b) Fixing a double-stranded nucleic acid molecule between a first surface and a second surface, wherein the first surface remains in a fixed position and the second surface is a movable surface; (c) Measure the length of the double-stranded nucleic acid molecule by mechanically stretching it; (d) Apply the complex mixed solution to be tested to the double-stranded nucleic acid molecule and use the same mechanical conditions as the mechanical stretching in step (c). Detect the length change of the double-stranded nucleic acid molecule by mechanical stretching, and then determine whether there is a non-covalently intercalated nucleic acid compound in the complex mixed solution to be tested based on the length change. Furthermore, the method also satisfies at least one of the following conditions: Condition 1: In step (a), the complex mixed solution to be tested does not contain any enzyme components, or the complex mixed solution to be tested is treated by at least one of heating, sonication, adding a protein denaturant, adding an enzyme inhibitor, or adding a metal ion chelating agent to inactivate the potential enzymes in the system. Condition 2: In step (d), the time from the application of the complex mixed solution to be tested to the start of detection of the length change of the double-stranded nucleic acid molecule is less than 10 minutes.

2. The method as described in claim 1, characterized in that, In step (a), the complex mixed solution to be tested is obtained by centrifuging at least one of the following: microbial culture medium, microbial extract, microbial secondary metabolites, animal body fluids, animal and plant tissue extracts, and animal and plant tissue metabolites, and collecting the supernatant. Alternatively, it can be obtained by dissolving and dispersing at least one of the animal or plant tissues or soil in a liquid medium and then taking the supernatant.

3. The method as described in claim 2, characterized in that, The animal body fluid is at least one of blood, urine, digestive juices, and tissue fluid.

4. The method as described in claim 1, characterized in that, In step (a), the complex mixed solution to be tested has not undergone purification treatment, but is a clear liquid obtained by only filtering and / or centrifuging to remove insoluble components.

5. The method as described in claim 1, characterized in that, In step (b), the double-stranded nucleic acid molecule is selected from: double-stranded DNA molecules, double-stranded RNA molecules, and DNA / RNA hybrid molecules; The length of the double-stranded nucleic acid molecule is in the range of 100 base pairs to 100,000 base pairs.

6. The method as described in claim 5, characterized in that, The double-stranded nucleic acid molecule is a double-stranded DNA molecule.

7. The method as described in claim 1, characterized in that, In step (b), the fixation involves attaching at least one base of one strand of the double-stranded nucleic acid directly or indirectly to the first surface, and attaching at least one base of the other strand of the double-stranded nucleic acid directly or indirectly to the second surface. Alternatively, the fixation involves attaching at least one base of one strand of a double-stranded nucleic acid directly or indirectly to a first surface, and at least one base at different positions of the strand directly or indirectly to a second surface.

8. The method as described in claim 1, characterized in that, In step (b), the first surface and the second surface are independently selected from glass surface, plastic surface, quartz surface, graphene surface, metal surface, and ceramic surface; Furthermore, the second surface is a particle surface or a probe surface; the size of the particle or the probe is in the range of 10 nanometers to 100 micrometers.

9. The method as described in claim 8, characterized in that, The second surface is a superparamagnetic particle surface.

10. The method as described in claim 1, characterized in that, In step (c), the external force applied by the mechanical stretching is in the range of 0.1 piconewtons to 100 piconewtons and is applied by a permanent magnet, electromagnet, centrifuge, acoustics, ultrasound, laser beam, fluid motion, fluid buoyancy or gravity.

11. The method as described in claim 10, characterized in that, The mechanical stretching is applied using a single-molecule magnetic tweezers device.

12. The method as described in claim 10, characterized in that, The external force applied during the mechanical tension is 1-60 piconewtons.

13. The method as described in claim 1, characterized in that, In steps (c) and (d), mechanical stretching is achieved by applying an external force that varies with time and recording the length change of the double-stranded nucleic acid molecule as a function of mechanical parameters.

14. The method as described in claim 1, characterized in that, When the complex mixed solution to be tested contains only one non-covalent nucleic acid intercalating agent, and the type of the non-covalent nucleic acid intercalating agent is known, the method further includes the step of: (e) Prepare standards of different concentrations of the non-covalent nucleic acid intercalating agent, replace the complex mixed solution to be tested with each standard, and use the same operation as in steps (b)-(d) under the same mechanical tensile stress conditions to measure the length change of double-stranded nucleic acid molecules after adding intercalating agent standards at different concentration gradients, as a standard curve; by comparing the length change in the standard curve with the length change caused by the complex mixed solution to be tested obtained in step (d), quantify the concentration of the single non-covalent nucleic acid intercalating compound contained in the complex mixed solution to be tested.

15. The method as described in claim 14, characterized in that, The external force applied during mechanical tension is 1-60 piconewtons.

16. The application of the method according to any one of claims 1-15 in the preparation or screening of drugs, for determining whether a complex mixed solution contains a non-covalent nucleic acid intercalation compound.

17. The application as described in claim 16, characterized in that, The complex mixture to be tested is derived from any of the following: microbial culture medium, microbial extract, microbial secondary metabolites; plant tissue, plant tissue extract.

18. The application of the method according to any one of claims 1-15 in drug pharmacokinetic analysis, characterized in that, The pharmacokinetic analysis of the drug is conducted during the drug preparation or screening stage, and the complex mixture to be tested is derived from animal body fluids, animal tissues, animal tissue extracts and / or animal tissue metabolites from animals after drug administration.

19. The application of the method according to any one of claims 1-15 in drug toxicology analysis, characterized in that, The drug toxicology analysis is conducted during the drug preparation or screening stage, and the complex mixture to be tested is derived from animal body fluids, animal tissues, animal tissue extracts, and / or animal tissue metabolites from animals after drug administration.

20. The application as described in claim 18 or 19, characterized in that, The method also includes the following steps: (e) Prepare non-covalent nucleic acid intercalation drug solution standards with different concentration gradients, replace the complex mixed solution to be tested with each standard, and use the same operation as in steps (b)-(d) under the same external force applied by mechanical stretching to measure the length change of double-stranded nucleic acid molecules after adding nucleic acid intercalation drug solution standards with different concentration gradients as a standard curve; By comparing the length change in the standard curve with the length change caused by the complex mixed solution to be tested obtained in step (d), the concentration of non-covalent nucleic acid embedded drug contained in the complex mixed solution to be tested is quantified.

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