Application of fluorescent fusion proteins in DNA damage repair detection

Abstract

The application relates to the technical field of molecular biology detection, and particularly discloses application of a fluorescent fusion protein in DNA damage repair detection. The application comprises the following steps: incubating the fluorescent fusion protein and a sample to be detected, and then performing fluorescence imaging to obtain the damage repair condition of the DNA to be detected in the sample to be detected; the sample to be detected contains the DNA to be detected; and the fluorescent fusion protein is formed by fusing a single-strand DNA binding protein and a fluorescent protein. The application can in-situ detect any DNA damage generating single-strand DNA exposure, and the detection result is accurate and quantifiable. In addition, the application is not limited by species, and can provide efficient visualization and quantitative analysis for DNA damage detection of various organisms.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection technology, and more specifically, to the application of a fluorescent fusion protein in the detection of DNA damage repair. Background Technology

[0002] DNA damage is a core threat to maintaining genome stability, widely present in all living organisms and triggered by endogenous metabolites (such as reactive oxygen species, ROS) and exogenous stresses (such as ultraviolet radiation, high temperatures, chemical mutagens, and ionizing radiation). Its main types include base damage (BD), single-strand breaks (SSB), and double-strand breaks (DSB). If not repaired in time, this damage can lead to replication errors, gene mutations, apoptosis, and even tumorigenesis or abnormal organismal development. Therefore, accurate, in-situ, and quantifiable detection of DNA damage, especially in exposed single-stranded DNA regions, is a key scientific issue in assessing genome stability, stress tolerance, and drug toxicity in organisms.

[0003] Common existing methods for detecting DNA damage include: comet assays, γ-H2AX focus analysis, base oxidation and modification damage detection, quantitative detection of abase desaturation sites (AP sites), nucleic acid gel electrophoresis and fragmentation detection, and high-throughput sequencing. However, these methods still have significant limitations in terms of species universality, operational complexity, signal specificity, quantitative accuracy, and experimental cost. Specifically:

[0004] 1) The comet assay (single-cell gel electrophoresis) can only provide the degree of DNA fragmentation of the overall cell population, but cannot locate the specific cell or subcellular location where the damage occurred, and cannot distinguish the damage type (such as SSB vs. AP site). It has problems such as being difficult to use for in situ tissue analysis, low throughput, and high subjectivity.

[0005] 2) γ-H2AX immunofluorescence shows almost no response to single-strand DNA damage (SSB, base excision intermediates, replication fork arrest) and is mostly used to determine severe DNA breakage. This method is highly dependent on species-specific antibodies—in non-model organisms (such as soybean, wheat, and insect cells), antibody cross-reactivity is poor, background is high, costs are high, and effective antibodies may not even be available, severely limiting its application in agricultural, ecotoxicological, and non-model animal studies.

[0006] 3) Base oxidation and modification damage detection only reflects the global oxidative stress level and cannot locate the cell or tissue region where damage occurs. It also does not respond to non-oxidative SSBs (such as replication fork unwinding, transcription vesicles, and recombination intermediates) and cannot capture single-strand exposure events during the dynamic repair process.

[0007] 4) While quantitative detection of abase sites (AP sites) can specifically detect abase sites generated during base excision repair (BER), it is limited to intermediate products of the BER pathway and cannot reflect the extensive single-stranded DNA regions generated by replication, transcription, recombination, or DSB end excision, resulting in a narrow coverage.

[0008] 5) Nucleic acid gel electrophoresis and fragmentation detection are mainly used to detect apoptosis-related high molecular weight DNA breaks, and are completely insensitive to local, non-apoptotic SSB or transcription-related single-strand exposures at the single-cell level.

[0009] 6) High-throughput sequencing technologies (such as END-seq, XR-seq, and Comet-Seq) can locate DNA damage at the whole-genome level, but they rely on DNA extraction, disrupt cell structure, lose spatial information, and cannot achieve single-cell resolution and dynamic repair tracking. More importantly, these methods require extremely high sample sizes (usually ≥10). 6 Furthermore, its application in non-model species (such as soybeans, wheat, and insects) is limited by the conservation of reference genomes and repair enzymes, making it difficult to promote its use.

[0010] In summary, it is of great significance to establish a highly specific, highly sensitive, and cross-species applicable in situ detection method for DNA damage. Summary of the Invention

[0011] The main objective of this invention is to provide an application of a fluorescent fusion protein in the detection of DNA damage repair, so as to solve the problem that existing technologies cannot accurately detect DNA damage in situ.

[0012] To achieve the above objectives, according to a first aspect of the present invention, an application of a fluorescent fusion protein in DNA damage repair detection is provided, the application comprising: incubating the fluorescent fusion protein and a test sample and then performing fluorescence imaging to obtain the damage repair status of the DNA to be tested in the test sample; wherein the test sample contains the DNA to be tested; wherein the fluorescent fusion protein is formed by the fusion of a single-stranded DNA-binding protein and a fluorescent protein.

[0013] Furthermore, the samples to be tested are selected from any one of the following: cell samples or tissue samples.

[0014] Furthermore, before incubating the fluorescent fusion protein and the test sample, the application also includes fixing and permeabilizing the test sample.

[0015] Furthermore, when the sample to be tested is a tissue sample, after the permeabilization process described above, the application also includes slicing the sample to be tested.

[0016] Furthermore, the above sectioning process can be performed using any of the following methods: frozen sectioning or paraffin sectioning.

[0017] Furthermore, the samples to be tested are derived from any of the following organisms: plants, animals, bacteria, fungi, archaea, or protists.

[0018] Furthermore, the above-mentioned fluorescence imaging was performed using a laser confocal microscope or a fluorescence microscope.

[0019] Furthermore, the aforementioned single-stranded DNA-binding proteins originate from prokaryotes or viruses.

[0020] Furthermore, the prokaryotes mentioned above are selected from any one of the following: Escherichia coli, bacteria, actinomycetes, or cyanobacteria.

[0021] Furthermore, the aforementioned viruses are selected from any one of the following: bacteriophages, tobacco mosaic virus, or tulip mosaic virus.

[0022] Furthermore, the fluorescent protein mentioned above is selected from any one of the following: StayGold, EGFP, mNeonGreen, sfGFP, mClover3, mScarlet-I, or mWasabi.

[0023] Furthermore, the fluorescent fusion protein described above has any one of the following amino acid sequences: SEQ ID NO: 1 or SEQ ID NO: 3.

[0024] The present invention utilizes the technical solution of fusing a fluorescent protein and a single-stranded DNA-binding protein to form a fluorescent fusion protein. This fluorescent fusion protein retains the single-stranded DNA-binding protein's ability to bind to single-stranded DNA while also possessing fluorescent labeling functionality. After incubating the fluorescent fusion protein with a test sample, fluorescence imaging is performed to obtain the damage repair status of the test DNA in the test sample; wherein the test sample contains the test DNA; and wherein the fluorescent fusion protein is formed by the fusion of a single-stranded DNA-binding protein and a fluorescent protein.

[0025] The method of this invention enables in-situ detection of DNA damage repair, providing intuitive results that are accurate and quantifiable. It is also not limited by species and can provide efficient visualization and quantitative analysis for DNA damage detection in various organisms. Attached Figure Description

[0026] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0027] Figure 1The image shows a gel electrophoresis pattern of the expressed and purified P5SG protein according to an embodiment of the present invention.

[0028] Figure 2 The specificity of P5SG in DNA damage affinity imaging detection according to an embodiment of the present invention is shown, wherein the P5SG signal substantially overlaps with the γ-H2A.X immunofluorescence signal.

[0029] Figure 3 The diagram shows the detection of DNA damage in Arabidopsis root tip tissue induced by high temperature stress using P5SG according to an embodiment of the present invention.

[0030] Figure 4 The diagram shows the detection of DNA damage in soybean root tips induced by DNA damaging agents and high temperature stress using P5SG according to an embodiment of the present invention.

[0031] Figure 5 The diagram shows the detection of DNA damage in the root tip meristem of adzuki beans and soybeans induced by heat stress using P5SG according to an embodiment of the present invention.

[0032] Figure 6 The image shows a gel electrophoresis diagram of the expressed and purified SSB-SG protein according to an embodiment of the present invention.

[0033] Figure 7 The specificity of SSB-SG in DNA damage affinity imaging detection according to an embodiment of the present invention is shown, wherein the SSB-SG signal substantially overlaps with the γ-H2A.X immunofluorescence signal.

[0034] Figure 8 The diagram shows the detection of DNA damage in Arabidopsis root tip tissue induced by high temperature stress using SSB-SG according to an embodiment of the present invention.

[0035] Figure 9 The invention illustrates the detection of heat stress-induced DNA damage in soybean root tips using SSB-SG, with imaging observation performed using laser confocal microscopy.

[0036] Figure 10 The invention illustrates the detection of heat stress-induced DNA damage in soybean W82 root tips using SSB-SG according to an embodiment of the present invention, wherein imaging observation is performed using a conventional fluorescence microscope.

[0037] Figure 11 The diagram shows the detection of DNA damage in insect cells Hi5 treated with DNA damaging agents using P5SG and SSB-SG according to an embodiment of the present invention.

[0038] Figure 12A spectrum of the P5SG vector according to an embodiment of the present invention is shown.

[0039] Figure 13 A spectrum of the SSB-SG vector according to an embodiment of the present invention is shown.

[0040] Figure 14 The diagram shows the detection of DNA damage in soybean pollen tubes induced by high temperature stress using P5SG and SSB-SG according to an embodiment of the present invention. Detailed Implementation

[0041] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0042] Terminology Explanation:

[0043] DNA damage refers to abnormal changes in the structure of DNA molecules, mainly caused by harmful substances produced by the cell's own metabolism or by external factors such as radiation and chemicals. If it is not corrected in time by the cell's repair system, this damage will be fixed as a gene mutation during DNA replication, becoming a key cause of diseases such as aging and cancer. There are many types of DNA damage, including base damage (BD), single-strand breaks (SSB), and double-strand breaks (DSB).

[0044] Single-stranded DNA exposure is itself a form of DNA damage / replication stress. SSB / RPA (single-strand binding protein) binding to single-stranded DNA is a key upstream step in damage recognition and repair initiation, and is part of the damage repair pathway. Although not entirely equivalent, they are highly coupled and occur sequentially.

[0045] DNA damage repair mechanism: It is the core defense system for cells to maintain the integrity of the genome. It accurately identifies, removes and correctly replaces damaged DNA fragments through a series of highly coordinated pathways (such as direct reversal, base excision repair, nucleotide excision repair, mismatch repair and double strand break repair, etc.), thereby effectively correcting various types of damage caused by internal and external factors, preventing the accumulation of mutations, and is the key to ensuring the normal function of life, delaying aging and inhibiting the occurrence of cancer.

[0046] Single-stranded DNA-binding proteins (SSBs / SSBPs) are a class of proteins that specifically bind to single-stranded DNA regions, playing a role in protecting the single-stranded structure during DNA replication, recombination, and repair. Structurally, SSBs consist of an oligonucleotide / oligosaccharide-binding (OB) fold and a flexible C-terminal tail. The OB fold of the SSB binds to single-stranded DNA with high affinity, while the C-terminal tail plays a crucial role in regulating the activity of other interacting proteins. When an SSB binds to single-stranded DNA (ssDNA), it forms an SSB–ssDNA complex, which completes DNA replication, recombination, and repair processes by binding, removing, pushing, or rebinding to single-stranded DNA.

[0047] SSB (Single-Strand Break): refers to the breakage of the phosphodiester bond in one strand of the DNA double helix, resulting in a discontinuous strand while the complementary strand remains intact. This structural damage can create single-strand sites.

[0048] Replication Fork: During DNA replication, double-stranded DNA is unwound and opened to form a Y-shaped structure containing the leading strand and lagging strand synthesis regions, in which the template strand exists briefly as a single strand after unwound.

[0049] Transcription bubble: During transcription, the DNA region bound by RNA polymerase locally unwinds, forming a dynamic structure where a single-stranded DNA template is read and the newly generated RNA pairs with the template.

[0050] DSB end nick (referring to the initial cutting / trimming state of the DSB end): After a DNA double-strand break, nucleases cut or trim the broken ends, especially the 5' end, where an initial nick is cut out to provide a starting site for subsequent 5'→3' excision, ultimately forming a single-stranded overhang.

[0051] Invading strand: In homologous recombination repair, the 3' single-stranded DNA tail generated by trimming the DSB ends invades the homologous double-stranded DNA under the action of recombinase and pairs with the complementary strand, while displacing the single-stranded DNA of the template strand to form the D-loop.

[0052] An AP site (Apurinic / Apyrimidinic site) is a site on the DNA strand where a base (purine or pyrimidine) of a nucleotide has been lost (either spontaneously or by glycosidase), leaving only the deoxyribose and phosphate backbone. It does not directly create a single-stranded site; it only lacks a base, the phosphodiester bond remains intact, and the double-stranded structure is preserved. However, if cleaved by an AP endonuclease, it is converted to SSB, indirectly producing ssDNA.

[0053] Comet assay (single-cell gel electrophoresis): This assay consists of two systems: neutral and alkaline. Neutral electrophoresis is mainly used to identify DNA double-strand breaks; alkaline high-salt environment can amplify the detection range, enabling the simultaneous detection of DNA single-strand breaks, alkaline-sensitive sites, abase-degrading sites (AP sites), and localized chain instability structures caused by base oxidation damage. It is currently the most commonly used classic method in scientific research for screening various types of single-strand DNA damage, and can visually reflect the degree of nucleic acid fragmentation at the single-cell level.

[0054] γ-H2AX immunofluorescence: This technique utilizes specific antibodies to recognize and bind to the modified form (γ-H2AX) formed by phosphorylation of histone H2AX at serine 139, and then uses a fluorescently labeled secondary antibody to image and quantify intracellular γ-H2AX foci under a fluorescence microscope. γ-H2AX is an early and specific molecular marker of DNA double-strand breaks (DSBs). It rapidly aggregates at the break site to form visible intranuclear fluorescent foci, with each foci typically corresponding to a DSB event. This technique is widely used in research on radiation damage, the effects of chemotherapy drugs, off-target effects of gene editing, and DNA repair defects (such as ataxia-telangiectasia), and is one of the gold standard methods for assessing genome stability and DNA damage responses.

[0055] Detection of base oxidation and modification damage: Using 8-OHdG as a typical biomarker, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) are employed to detect the content of guanine oxidation products in genomic DNA, in order to evaluate oxidative stress-induced endogenous single-stranded DNA damage. Similar detection targets also include lipid peroxidation-associated DNA adducts, alkylated base modifications, and other chronic damage types.

[0056] Quantitative detection of abase removal sites (AP sites): Cells easily generate a large number of abase removal regions during base excision repair, and these structures are typical examples of single-strand instability damage. The number of AP sites within the genome can be quantitatively counted using specific aldehyde reaction colorimetry, biotin-labeled probe binding, and biochemical colorimetric kits, reflecting persistent base damage and localized nucleic acid strand breakage.

[0057] Nucleic acid gel electrophoresis and fragmentation detection: including conventional agarose gel DNA ladder electrophoresis and large-fragment genomic DNA separation electrophoresis, used to detect large-scale nucleic acid breaks accompanying apoptosis; at the same time, alkali denaturing gel electrophoresis can be used to separate double-stranded and single-stranded nucleic acids, and the overall level of single-stranded breaks in the sample can be judged based on the degree of band diffusion.

[0058] As mentioned in the background section, existing methods for detecting DNA damage in organisms suffer from poor accuracy and the inability to perform in-situ detection. In this invention, the inventors attempt to utilize the ability of single-stranded DNA-binding proteins to bind to single-stranded DNA sites and the fluorescent labeling capability of fluorescent proteins to fuse these two proteins together, forming a fluorescent fusion protein. This fluorescent fusion protein can in-situ label exposed single-stranded DNA sites in DNA damage, and based on the intensity of the fluorescence signal, it can quantitatively detect the degree of DNA damage repair in cell or tissue samples. Therefore, the protection scheme of this invention is proposed.

[0059] In a first typical embodiment of the present invention, an application of a fluorescent fusion protein in DNA damage repair detection is provided. The application includes: incubating the fluorescent fusion protein and a sample to be tested and then performing fluorescence imaging to obtain the damage repair status of the DNA to be tested in the sample to be tested; wherein the sample to be tested contains the DNA to be tested; and the fluorescent fusion protein is formed by fusing a single-stranded DNA-binding protein and a fluorescent protein.

[0060] After incubation with the sample, the fluorescent fusion protein captures single-stranded DNA sites under the action of single-stranded DNA-binding proteins. When exposed to excitation light, the specific chromophore within the fluorescent protein (such as GFP) absorbs light energy of a specific wavelength, causing electrons to transition to an excited state. When these electrons return to the ground state, they release longer-wavelength emitted light. This specific light absorption and emission characteristic allows the fluorescent protein, which is attached to the target molecule or cellular structure, to be captured by a microscope, thus achieving fluorescence imaging.

[0061] The above applications do not rely on DNA damage-induced post-translational modifications or species-specific antibodies. They achieve visualization solely through the physical binding of exogenously added fusion proteins to the damage site. They are suitable for in situ detection of single-stranded DNA in samples from various biological sources. The detection signal directly reflects the presence and abundance of single-stranded DNA. These applications not only enable in situ observation and preserve the complete microenvironment of DNA damage repair, but also enable quantitative detection of the degree of DNA damage based on the intensity of fluorescence.

[0062] The aforementioned fluorescent protein retains the single-stranded DNA binding properties of single-stranded DNA binding proteins while also possessing fluorescent labeling functionality. Using this fluorescent protein, in-situ and quantitative detection of DNA damage repair can be achieved. Any type of damage capable of exposing single-stranded DNA can be detected using the method described in this application, such as SSB, replication forks, transcription vesicles, DSB terminal nicks, or recombination intrusion strands.

[0063] In some specific embodiments, the aforementioned fluorescent fusion protein is used to detect plant DNA damage, which has multiple key implications in the fields of agriculture and plant science. Its technical solution can be widely applied in various scenarios such as germplasm resource evaluation, stress resistance breeding, cultivation management, environmental monitoring, and variety protection, providing direct data support and decision-making basis for precision agriculture and stress resistance breeding. Specifically, it includes, but is not limited to:

[0064] 1) Cultivation optimization and stress management: This can be used to assess the impact of different cultivation practices on plant DNA stability. For example, by detecting the level of DNA damage repair in crops under different water and fertilizer management and regulator treatments, cultivation schemes that can effectively reduce damage and improve stress resistance can be screened. In facility agriculture, environmental parameters can be adjusted based on damage monitoring data to achieve precise cultivation management under stress conditions.

[0065] 2) Environmental pollutant biomonitoring and ecological risk assessment: Using model plants or indicator crops as materials, by detecting their DNA damage levels, biotoxicity monitoring of pollutants such as heavy metals and pesticide residues in soil, water and atmosphere can be carried out, assessing the potential harm of polluted environment to plant genome, and providing biological indicators for environmental governance and ecological restoration.

[0066] 3) Early screening and functional verification of stress-resistant breeding materials: In gene editing, transgenic and mutagenesis breeding, the level of DNA damage can be used as an early screening indicator to quickly verify the stress resistance function of the target gene, screen out improved materials with significantly improved genome stability, avoid the high cost of later field screening, and accelerate the breeding process of new stress-resistant varieties.

[0067] 4) The detection and quantitative analysis of plant DNA damage can serve as an indicator for evaluating the tolerance traits of biological germplasm to various stresses. It is suitable for assessing the degree of environmental stress tolerance of organisms and studying DNA damage repair mechanisms.

[0068] Furthermore, the detection of DNA damage (especially single-strand DNA damage) is also of crucial significance in animal model construction, drug development, toxicological evaluation, and preclinical studies. It can provide core quantitative indicators for drug safety assessment, efficacy verification, and toxicity mechanism analysis. The application scenarios and advantages of its technical solutions are as follows:

[0069] 1) Evaluation of drug genotoxicity and mutagenicity: In preclinical safety evaluation of drugs, the potential genotoxicity of candidate drugs can be assessed by detecting the level of DNA damage in animal tissues / cells. Direct detection of DNA damage can more intuitively reflect the damage effects induced by the drug in vivo, such as single-strand breaks, base oxidation, and chain cross-linking. This provides early and direct biological evidence for determining whether a drug has a mutagenic risk, avoiding later-stage development failures due to genotoxicity issues.

[0070] 2) Screening and Mechanism of Action of Antitumor Drugs: Most chemotherapeutic drugs exert their killing effect by inducing DNA damage in tumor cells (especially single-strand and double-strand breaks). By detecting the level of DNA damage in tumor cells or tumor-bearing animal models under different drug treatments, the damaging effect of drugs on the tumor cell genome can be quantified, and candidate drugs with DNA damage-inducing activity can be rapidly screened. At the same time, by combining damage repair pathway indicators, the drug's target and drug resistance mechanisms can be analyzed, providing a basis for the design of combination therapy regimens.

[0071] It should be noted that DNA damage and repair detection are a complementary technical system. The DNA damage repair detection method of this application can detect both DNA damage and DNA damage repair. Specifically, this application can determine whether DNA damage has occurred and its extent by comparing the fluorescence intensity before and after stress treatment. Correspondingly, this application can also determine whether DNA damage has been repaired and its extent by comparing the fluorescence intensity before and after repair treatment.

[0072] Fluorescent proteins serve as fluorescent labeling agents in this application. Any type of fluorescent protein capable of fluorescent labeling is suitable for this invention. The excitation wavelengths of different fluorescent proteins may differ, and those skilled in the art can determine the excitation wavelength based on the specific type of fluorescent protein. In a preferred embodiment of this invention, the fluorescent protein is selected from any of the following: StayGold, EGFP, mNeonGreen, sfGFP, mClover3, mScarlet-I, or mWasabi. In a preferred embodiment of this invention, the fluorescent protein is selected from StayGold.

[0073] StayGold is a recently developed ultra-stable green fluorescent protein with extremely high photostability and an extremely slow photobleaching rate. It can maintain its fluorescence signal for a long time even under strong laser irradiation, making it very suitable for long-term live-cell dynamic imaging and phototoxicity-sensitive experiments.

[0074] EGFP (Enhanced Green Fluorescent Protein) is the most commonly used enhanced mutant of green fluorescent protein (GFP). By replacing multiple amino acids (such as F64L and S65T), it optimizes folding efficiency and fluorescence intensity, and is the gold standard probe for marker protein localization and expression in biological research.

[0075] mNeonGreen is a green fluorescent protein derived from echinoderms (chickentails). Although it is not a homolog of GFP, it has extremely high brightness and matures quickly. It performs well in mammalian cells and is often used as a powerful alternative to GFP for multicolor labeling and ultra-high resolution microscopy.

[0076] sfGFP (superfold GFP) is a "superfolded" green fluorescent protein mutant obtained through directed evolution. It can still fold correctly and emit fluorescence under denaturing conditions or when fused to proteins that are difficult to fold, which greatly improves its practicality as a fusion tag.

[0077] mClover3 is the third-generation Clover green fluorescent protein, a further optimized monomeric green fluorescent protein with better folding efficiency, brightness and photostability. It is often used in the construction of FRET (fluorescence resonance energy transfer) sensors and for precise quantitative imaging.

[0078] mScarlet-I is a high-performance monomeric red fluorescent protein (RFP) mutant derived from mCherry, exhibiting excellent brightness, maturation speed, and photostability.

[0079] mWasabi is a monomeric yellow / green fluorescent protein (based on sea anemone fluorescent protein) with an emission peak of approximately 509 nm (yellow-green light). It has high brightness and is often used as an intermediate color reporter gene with a spectrum between green and red for multicolor fluorescent labeling experiments.

[0080] Single-stranded DNA binding proteins (SDNA-binding proteins) function by binding to single-stranded DNA. The binding affinity of SDNA-binding proteins from different biological sources varies depending on the organism from which they originate. Generally, SDNA-binding proteins from a particular source have a stronger binding affinity to single-stranded DNA from the same source.

[0081] Single-stranded DNA binding proteins with binding sites on single-stranded DNA from any biological or non-biological source (e.g., artificially synthesized) are applicable to this application. In a preferred embodiment of the invention, the aforementioned single-stranded DNA binding protein is derived from prokaryotes or viruses. These derived single-stranded binding proteins exhibit high specificity for binding to single-stranded DNA, significantly improving the specificity and sensitivity of detection.

[0082] Any single-stranded DNA-binding protein derived from any prokaryote or bacteriophage capable of binding to single-stranded DNA sites is applicable to this application. In a preferred embodiment of the invention, the prokaryote is selected from any of the following: *Escherichia coli*, bacteria, actinomycetes, or cyanobacteria. In a preferred embodiment of the invention, the virus is selected from any of the following: bacteriophage, tobacco mosaic virus, or tulip mosaic virus.

[0083] It should be noted that in the fluorescent fusion protein of the present invention, the single-stranded DNA binding protein and the fluorescent protein can be directly linked or optionally linked via a linker. In a preferred embodiment of the present invention, the single-stranded DNA binding protein and the fluorescent protein are directly linked. Furthermore, the single-stranded DNA binding protein of the present invention can be fused to the N-terminus of the fluorescent protein or to the C-terminus of the fluorescent protein. In a preferred embodiment of the present invention, the single-stranded DNA binding protein and the fluorescent protein... In a more preferred embodiment of the present invention, the fluorescent fusion protein has any one of the following amino acid sequences: SEQ ID NO: 1 or SEQ ID NO: 3.

[0084] The above SEQ ID NO: 1 corresponds to the fusion sequence of bacteriophage Φ29 P5 protein and StayGold, and the above SEQ ID NO: 3 corresponds to the fusion sequence of Escherichia coli SSB protein and StayGold. Both have been verified in experiments to be successfully expressed and purified, and have achieved significant and specific fluorescent signals in a variety of biological samples. The accuracy of their amino acid sequences ensures the integrity of the fusion protein structure and the reproducibility of its function.

[0085] In a preferred embodiment of the present invention, the above-mentioned fluorescent fusion protein is obtained through heterologous expression and purification. Both the heterologous expression method and the purification method are conventional methods in the art. In a more preferred embodiment of the present invention, the above expression is performed using an *E. coli* heterologous expression system.

[0086] This application allows for the selection of appropriate sample types for testing based on the biological type of the sample to be tested and the experimental requirements. In a preferred embodiment of this invention, the sample to be tested is selected from any one of the following: cell samples or tissue samples.

[0087] In a preferred embodiment of the present invention, before incubating the fluorescent fusion protein and the test sample, the application further includes fixing and permeabilizing the test sample. The methods for fixing and permeabilizing the test sample in this application are all conventional methods in the art.

[0088] Immobilization aims to rapidly anchor biomolecules within cells or tissues using chemical cross-linking agents (such as paraformaldehyde), fixing the sample in its current physiological or damaged state to prevent structural collapse, molecular degradation, or positional drift, thus ensuring the authenticity of the signal.

[0089] Permeation, on the other hand, uses detergents (such as Triton X-100) to create micropores in the cell membrane, eliminating the barrier effect of the cell membrane and allowing subsequent exogenous probes (such as fluorescent fusion proteins and antibodies) to enter the cell and reach the target (such as DNA in the cell nucleus), thereby achieving specific binding and labeling. Together, these two processes provide a structurally stable and accessible detection environment for subsequent fluorescence imaging.

[0090] In a preferred embodiment of the present invention, when the sample to be tested is a tissue sample, after the permeabilization treatment, the application further includes sectioning the sample to be tested. In a preferred embodiment of the present invention, the sectioning is performed using any of the following methods: cryosectioning or paraffin sectioning. The method for sectioning the sample to be tested in this application is a conventional method in the art.

[0091] In a preferred embodiment of the present invention, after slicing the sample to be tested and before incubating it, the above application further includes a step of secondary fixation and sealing of the sample to be tested. In a preferred embodiment of the present invention, bovine serum albumin is used for sealing.

[0092] In a preferred embodiment of the present invention, the sample to be tested is derived from any of the following organisms: plants, animals, bacteria, fungi, archaea, or protozoa. It should be noted that when the method of this application is used to detect DNA damage in animals, it is not used for disease diagnosis.

[0093] Any microscope capable of fluorescence imaging is suitable for use in this invention. In a preferred embodiment of the invention, a laser confocal microscope or a fluorescence microscope is used for the aforementioned fluorescence imaging.

[0094] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0095] Example 1: Construction, expression, and purification of single-chain binding protein P5-fluorescent fusion protein

[0096] (1) Based on the sequences of the single-stranded binding protein P5 encoding gene and the fluorescent protein (StayGold) encoding gene of phage Φ29, the vector was constructed by Huada Pharmaceutical Co., Ltd., and the inserted fragment was located between 658bp and 1722bp. Figure 12 ).

[0097] (2) The successfully constructed expression vector P5 stay gold (hereinafter referred to as P5SG) was transformed into Escherichia coli competent cells BL21(DE3) for prokaryotic expression and inoculated in LB liquid medium containing 25 mg / L kanamycin at 37°C and 200 rpm. When the OD600 value was approximately 0.6-1.0, isopropyl thiogalactoside (IPTG) was added to the medium for overnight induction at 16°C and 170 rpm. The cells were then collected, the supernatant was discarded, and the cells were resuspended in buffer (25 mM Tris pH 8.0, 150 mM NaCl).

[0098] (3) The expressed fluorescent fusion protein was purified using protein purification technology: the bacterial cells were disrupted using an ultrasonic disruptor, precipitated after high-speed centrifugation, and the supernatant was collected and filtered. The supernatant sample of the target protein was slowly added to Ni-NTA for flow-through, allowing the target protein to fully bind with the nickel ion affinity medium.

[0099] Then, 1 M imidazole was diluted with buffer to prepare elution buffers of different gradients (10 mM, 20 mM, 35 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM, 300 mM). The elution buffers of different concentrations were collected and identified. The optimal concentration of imidazole was selected for elution of the target protein three times, and the elution buffers were collected for subsequent experiments.

[0100] (4) Slowly add the target protein sample to the ultrafiltration tube and centrifuge at 4℃ and 4000 rpm for 30 min until the sample is concentrated to about 2 mL. Repeat the above operation 3-4 times. Then take 20 µL of protein sample, add 6× Proteinloading buffer, boil and perform SDS-PAGE electrophoresis. After electrophoresis, remove the separating gel and place it in Coomassie Brilliant Blue staining solution for staining. Then add destaining solution for destaining. Finally, take pictures under a developing instrument and record the experimental results. Figure 1 The purified protein was then tested for concentration, with a final concentration of 1 mg / ml, and then stored at -80°C.

[0101] Example 2: Specificity validation of P5SG DNA damage affinity imaging detection technology

[0102] The P5SG (P5 StayGold) fusion protein utilizes the P5 domain to specifically bind to single-stranded regions exposed by DNA damage, enabling visualization of the damage site via GFP fluorescence signal. The amino acid sequence of the P5SG fusion protein is shown in SEQ ID NO: 1, where the first 124 amino acids are the amino acid sequence of the SSB protein; the nucleotide sequence of the P5SG fusion protein is shown in SEQ ID NO: 2. It should be noted that in this embodiment, the single-stranded DNA-binding protein is fused to the N-terminus of the fluorescent protein. In the sequence shown in SEQ ID NO: 1, the underlined sequence is the amino acid sequence of the fluorescent protein.

[0103] Among them, SEQ ID NO:1 is:

[0104] MENTNIVKATFDTETLEGQIKIFNAQTGGGQSFKNLPDGTIIEANAIAQYKQVSDTYGDAKEETVTTIFAADGSLYSAISKTVAEAASDLIDLVTRHKLETFKVKVVQGTSSKGNVFFSLQLSL MVSTGEELFTGVVPFKFQLK GTINGKSFTVEGEGEGNSHEGSHKGKYVCTSGKLPMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRH IQYKGDGSIHAKHQHFMKNGTYHNIVEFTGQDFKENSPVLTGDMNVSLPNDVQHIPRDDGVECPVTLLYPLLSDKS KCVEAHQNTICKPLHNQPAPDVPYHWIRKQYTQSKDDTEERDHICQSETLEAHL .

[0105] Among them, SEQ ID NO:2 is:

[0106] atggaaaacacaacatcgtaaaggctacttttgacacagaaactcttgaaggacaaatcaaaatctttaatgctcagacaggcggcggacaatcttttaaaaaccttccagatggaacaattatagaagccaacgccattgctcaatataagcaagtgtccgatacatacggggacgctaaggaa gaaacagttactactatttttgcggctgacgggtcgttatattccgctatctctaagactgtagcagaagccgcatctgacttaattgaccttgtgactcgtcataagcttgaaacgtttaaggttaaagtggttcaaggaacatctagtaaaggtaacgtattctttagcttacaactatcccta atggtgtctacaggcgaggagctgtttaccggcgtggtgcccttcaagttccagctgaagggcaccatcaacggca agagcttcaccgtggaaggcgagggcgagggcaatagccacgagggcagccacaaaggcaagtacgtgtgcaccag cggcaaactgccaatgtcttgggccgccctgggaactagcttcggctatggcatgaagtactacaccaagtacccc agcggcctgaagaactggttccacgaggtgatgcccgagggcttcacctacgacagacacatccagtacaagggcg acggcagcatccacgccaagcaccagcacttcatgaagaacggcacctaccacaacatcgtggagttcaccggcca ggacttcaaggagaacagccccgtgctgaccggcgacatgaacgtgagcctgcccaacgacgtgcagcacatccccagagatgacggcgtggagtgcccagtgaccctgctgtaccctctgctgagcgacaagagcaagtgcgtggaggccc accagaacaccatctgcaagcccctgcacaatcagccagcccccgatgtgccataccactggatcagaaagcagta cacccagagcaaggacgacaccgaggagagagaccacatctgccagagcgagaccctggaggcccacctgtaa .

[0107] To verify its specificity, the applicant compared the P5SG signal with the immunofluorescence signal of γH2AX, a classic marker of DNA damage repair, in meristematic cells of soybean root tips.

[0108] DNA double-strand breaks (DSBs) induce rapid phosphorylation of histone H2AX at the Ser139 site to form γH2AX. DSBs can be visualized and quantified using specific antibodies and fluorescent labels.

[0109] The results showed that the two signals highly overlapped (see...). Figure 2 This indicates that P5SG can replace γH2AX antibodies for precise labeling of DNA damage sites. Compared with traditional immunoassays, P5SG technology does not rely on species-specific antibodies, is simple to operate, and has wider applicability.

[0110] It should be noted that γH2AX immunofluorescence technology is mainly used to detect DNA double-strand breaks (DSB). However, during the repair process of double-strand damage, the double strand needs to be opened to allow single-strand invasion, forming a single-strand state. Single-strand DNA binding proteins will recognize these single-strand sites. Therefore, the P5SG technology of this application has a high degree of overlap with the immunofluorescence signal of γH2AX.

[0111] Furthermore, since there are various types of damage that can cause single-stranded DNA exposure, such as SSB, replication fork, transcription vesicle, DSB end nick, or recombination invasion strand, the fluorescent immunoprotein of this application can detect any damage that leads to single-stranded DNA exposure when detecting DNA damage repair, compared to the γH2AX immunofluorescence technique, which can only detect DNA double-strand breaks. This results in higher accuracy.

[0112] Example 3: Detection of high-temperature stress-induced DNA damage in plants using P5SG DNA damage affinity imaging technology.

[0113] 1) The applicant subjected Arabidopsis thaliana seedlings that had germinated for 5 days to a high-temperature treatment at 35℃ for 2 days, with 100µM DNA-damaging agent bleomycin as a positive control. The control group was cultured routinely at 23℃, with all other culture conditions remaining the same.

[0114] 2) After the stress treatment, root tip tissue was collected and fixed in 4% paraformaldehyde (PFA) fixative for 4 hours; it was washed twice with 1×PBS buffer, and then decolorized twice each with 100% methanol and 100% ethanol for 15 minutes each time; then it was transferred to 34% sucrose solution prepared with 1×PBS buffer for 1 hour for dehydration, and then transferred to a mixture of 34% sucrose solution and OCT embedding agent in a volume ratio of 1:1 for overnight treatment, and finally OCT embedding was performed.

[0115] 3) Incubation and binding of fluorescent fusion protein with cells: Place the OCT-embedded sample in a cryostat and cut tissue sections with a thickness of 5-10µm. Adhere the sections to an adhesion slide and air dry for 15 minutes. Add 100% methanol for secondary fixation, rinse with 1×PBS buffer, block with 1% BSA solution for 30 minutes to 1 hour, rinse with 1×PBS buffer, and set aside.

[0116] 4) Incubation of fluorescent fusion protein: Take the purified fluorescent fusion protein and dilute it with 1% BSA solution to a final concentration of 5µM as the working solution; add the working solution to the tissue section on the glass slide and incubate overnight at 4°C.

[0117] 5) Laser confocal microscopy observation: Place the incubated cell samples under a laser confocal microscope, select the excitation wavelength (500nm) and emission wavelength (515nm) corresponding to the fluorescent protein, and scan the samples to observe them. Collect fluorescence images of the experimental group and control group cells respectively, and record the observation data. It should be noted that steps 2)-5) are integrated into the "freeze-section combined with P5SG affinity imaging technology".

[0118] 6) Data statistics and analysis: It was found that the number of cells undergoing DNA damage repair in the high-temperature treatment group was significantly increased compared with the control, and the signal characteristics were similar to those in the Zeocin treatment group. Figure 3 This result indicates that this method can be used to accurately detect DNA damage induced by high-temperature stress in Arabidopsis root tip meristem cells.

[0119] Example 4: Detection of DNA damage in soybean root tip induced by DNA damaging agents and high temperature stress using P5SG DNA damage affinity imaging technology.

[0120] Soybean seeds were soaked in sterile water overnight and then inoculated into 1 / 2 MS medium and cultured for 3 days to obtain soybean seedling roots with uniform growth. The seedling roots were randomly divided into three groups. The experimental group was subjected to 35℃ high temperature stress treatment for 6 hours and 100 µM bleomycin treatment overnight. The control group was cultured at 25℃ under normal conditions. All other culture conditions were kept the same.

[0121] Using frozen sections combined with P5SG affinity imaging, it was found that the number of cells undergoing DNA damage repair under high-temperature treatment increased more than 4 times compared to the control. Figure 4 This study confirms that P5SG affinity imaging technology can be effectively applied to the detection of DNA damage in crops such as soybeans.

[0122] Example 5: The heat tolerance of soybean germplasm can be evaluated by detecting heat stress-induced DNA damage using P5SG DNA damage affinity imaging technology.

[0123] Soybean cultivation is widespread in my country, and the temperature adaptability of the main varieties varies in different regions. For example, compared with W82, which is more suitable for cultivation in the north, red soybean, which is suitable for cultivation in the south, exhibits shorter plants and lower yield when W82 is propagated in the south, suggesting that there may be differences in their heat tolerance.

[0124] The applicant used frozen sections combined with P5SG affinity imaging technology to compare the degree of DNA damage in the root tip meristem of two varieties under high temperature stress.

[0125] The results showed that the number of cells undergoing DNA damage repair and the P5SG fluorescence intensity in red beans were significantly lower than those in W82. Figure 5 The results indicate that red beans exhibit less DNA damage under high-temperature stress and demonstrate superior heat resistance compared to W82. This example demonstrates that this method can be used to evaluate the heat resistance of soybean germplasm by detecting heat stress-induced DNA damage.

[0126] Example 6: Expression, purification, and application of E. coli single-stranded binding protein fusion fluorescent protein in DNA damage detection

[0127] (1) Based on the sequences of the SSB protein-coding gene and the fluorescent protein (StayGold)-coding gene in Escherichia coli, the vector construction was completed by Huada Pharmaceutical Co., Ltd. Figure 13 The inserted fragment is located between 658bp and 1884bp;

[0128] (2) The successfully constructed expression vector SSB StayGold (SSB-SG) was transformed into Escherichia coli competent cells BL21(DE3) for prokaryotic expression. Positive clones were obtained by resistance screening. The positive clones were inoculated into the culture medium for culture to induce the expression of fluorescent fusion protein.

[0129] (3) The expressed fluorescent fusion protein was purified using protein purification technology to obtain a high-purity fluorescent fusion protein, which was then tested and verified for later use. Figure 6 ).

[0130] Example 7: Specificity validation of SSB-SG DNA damage affinity imaging detection technology

[0131] The SSB-SG fusion protein utilizes the SSB domain to specifically bind to single-stranded regions exposed by DNA damage, enabling visualization of the damage site via GFP fluorescence signal. The amino acid sequence of the SSB-SG fusion protein is shown in SEQ ID NO: 3, where the first 178 amino acids represent the amino acid sequence of the SSB protein; the nucleotide sequence of the SSB-SG fusion protein is shown in SEQ ID NO: 4.

[0132] It should be noted that in the SSB-SG fusion protein of this embodiment, the single-stranded DNA-binding protein is fused to the N-terminus of the fluorescent protein. In the sequence shown in SEQ ID NO: 3, the underlined sequence is the amino acid sequence of the fluorescent protein.

[0133] Among them, SEQ ID NO:3 is:

[0134] MASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQVYIEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMDFDDDIPF MVSTGEELFTGVVPFKFQLKGTINGKSFTVEGEGEGNSHEGS HKGKYVCTSGKLPMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGSIHAKHQHFMKNGTY HNIVEFTGQDFKENSPVLTGDMNVSLPNDVQHIPRDDGVECPVTLLYPLLSDKSKCVEAHQNTICKPLHNQPAPDV PYHWIRKQYTQSKDDTEERDHICQSETLEAHL .

[0135] SEQ ID NO: 4 is:

[0136]

[0137] To verify its specificity, the applicant compared the SSB-SG signal with the immunofluorescence signal of γH2AX, a classic marker of DNA damage repair, in the same cells.

[0138] The results showed that the two signals highly overlapped. Figure 7 This indicates that SSB-SG can replace γH2AX antibodies for precise labeling of DNA damage sites. Like traditional immunoassays, SSB-SG technology does not rely on species-specific antibodies and has broad applicability.

[0139] Example 8: Detection of DNA damage in Arabidopsis root tip induced by DNA damaging agents and high temperature stress using SSB-SG DNA damage affinity imaging technology.

[0140] The applicant subjected Arabidopsis thaliana seedlings that had germinated for 5 days to a high-temperature treatment at 35°C for 2 days, and used bleomycin, a DNA-damaging agent of 100µM, as a positive control.

[0141] Using frozen sections combined with SSB-SG affinity imaging, observation of the root tip meristem revealed a significant increase in the number of cells undergoing DNA damage repair in the high-temperature treatment group compared to the control, with signal characteristics similar to those in the bleomycin-treated group. Figure 8 This result indicates that this method can be used to accurately detect DNA damage induced by high-temperature stress in Arabidopsis root tip meristem cells.

[0142] It should be noted that the difference between "frozen section combined with SSB-SG affinity imaging technology" and "frozen section combined with P5SG affinity imaging technology" in Example 3 is only that the fusion protein is replaced by SSB-SG instead of P5SG.

[0143] Example 9: Detection of DNA damage in soybean root tip induced by DNA damaging agents and high temperature stress using SSB-SG DNA damage affinity imaging technology.

[0144] The applicant subjected soybean seedlings aged 3-5 days to a 35°C high-temperature stress treatment for 6 hours. Frozen sections were prepared from the root tip meristem using SSB-SG affinity imaging technology, followed by SSB-SG staining. The results showed a significant increase in the number of cells undergoing DNA damage repair under high-temperature treatment. Figure 9 ).

[0145] This study confirms that SSB-SG affinity imaging technology can be effectively applied to the detection of DNA damage in crops such as soybean. Combined with SSB-SG affinity imaging technology, this result demonstrates that single-stranded binding proteins from various organisms can be fused with fluorescent proteins for precise detection of DNA damage.

[0146] Example 10: Comparison of Imaging Effects of Different Fluorescence Microscopy Devices

[0147] Samples treated with fluorescent labeling were imaged and observed using both laser confocal microscopy and conventional fluorescence microscopy. Confocal microscopy (the applicant used a Zeiss 980 laser confocal microscope) allowed for clear acquisition of fluorescence localization images of the samples. Figure 3 , Figure 4 , Figure 5 , Figure 7 , Figure 8 and Figure 9 );

[0148] Clear fluorescence imaging results can also be obtained using a conventional fluorescence microscope (the applicant used a Zeiss Axio imager. M2 upright fluorescence microscope). Figure 10 Both methods can effectively observe and record the target fluorescence signal. Figure 10 This image shows the DNA damage repair detection results of soybean W82 root tip after high-temperature treatment.

[0149] This embodiment demonstrates that the technical solution does not have strict limitations on fluorescence microscopy imaging equipment. The fluorescence imaging effect that can be achieved by a confocal microscope can also be achieved by a regular fluorescence microscope, demonstrating good equipment applicability and versatility.

[0150] Example 11: Detection of DNA damage in Hi5 insect cells treated with DNA damaging agents using P5SG DNA damage affinity imaging technology and SSB-SG DNA damage affinity imaging technology respectively.

[0151] Hi5 cells (also known as HighFive cells) are the ovarian cell line of the white armyworm (BTI-TN-5B1-4). Hi5 cells were transferred to culture medium containing 0.17 µM bleomycin for 22 hours, then transferred to glass-bottomed culture dishes and cultured for 2 hours. After cell attachment, the culture medium was aspirated, and the cells were washed twice with 1×PBS for 5 min each time. The cells were then fixed with 4% PFA (containing 0.1% Triton-100 to a final concentration) for 1 h, followed by P5SG and SSB-SG staining.

[0152] The purified P5SG or SSB-SG fusion protein was added to the treated Hi5 cell culture medium, ensuring that the working concentration of the fusion protein was 5 μM. The cells were incubated overnight at 4°C, washed three times with 1×PBS for 5 min each time, and then observed under a microscope after adding anti-quenching oil containing DAPI.

[0153] The only difference between SSB-SG staining and P5SG staining is that the fluorescent protein P5 in SSB-SG staining is replaced with the fluorescent protein SSB.

[0154] The results showed that bleomycin treatment significantly increased the ongoing DNA damage repair process. Figure 11 This study confirms that P5SG affinity imaging and SSB-SG affinity imaging technologies can be effectively applied to the detection of DNA damage in animal cells such as Hi5.

[0155] Example 12: Detection of DNA damage in soybean pollen tubes induced by high-temperature stress using P5SG DNA damage affinity imaging technology and SSB-SG DNA damage affinity imaging technology respectively.

[0156] Pollen tubes are extremely sensitive to high temperatures: high temperatures easily lead to DNA breakage, ROS accumulation, pollen tube growth stagnation, and sterility, directly affecting seed set rate and yield. The applicant collected fresh, open flowers between 8 and 10 AM, removed the anthers, and dispersed pollen into sterile, dry petri dishes. Soybean germination medium was added; the control group was placed at 25°C and germinated in the dark for 2 hours, while the high-temperature group was placed at 35°C and germinated in the dark for 2 hours.

[0157] Pollen tubes after germination were fixed using cryosection combined with P5SG affinity imaging and cryosection combined with SSB-SG affinity imaging. After immunofluorescence incubation, the samples were observed under a confocal microscope. The results showed that the fluorescence signals of single-chain binding proteins P5SG and SSB-SG in pollen tubes treated with high temperature were significantly higher than those in the control group. Figure 14 ).

[0158] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0159] (1) This method uses fluorescent fusion proteins to achieve in situ labeling of single-stranded DNA sites. The detection process does not require antibodies, lysis, or complex pretreatment, and can truly reflect the in situ state of DNA damage repair in cells. By using the number of fluorescent cells and fluorescence intensity as detection indicators, the degree of DNA damage repair can be quantitatively detected. The detection results are accurate and quantifiable, avoiding errors caused by subjective judgment.

[0160] (2) The detection operation process is simple. The observation can be completed with the help of a laser confocal microscope. The detection efficiency is high and it is suitable for the detection and analysis of large-scale samples.

[0161] (3) Since this method is not limited by species and is non-toxic, it has potential applications in different scenarios such as animal biology research, plant germplasm evaluation, drug genotoxicity screening, and plant stress tolerance research.

[0162] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of fluorescent fusion protein in DNA damage repair detection, characterized in that, The applications include: The fluorescent fusion protein and the sample to be tested were incubated and then subjected to fluorescence imaging to obtain the damage repair status of the DNA to be tested in the sample to be tested. The sample to be tested contains the DNA to be tested; Before incubating the fluorescent fusion protein and the test sample, the application further includes fixing and permeabilizing the test sample. The fluorescent fusion protein is formed by fusing a single-stranded DNA-binding protein and a fluorescent protein. The fluorescence imaging was performed using a laser confocal microscope or a fluorescence microscope. The fluorescent fusion protein was obtained through heterologous expression and isolation / purification. The fluorescent fusion protein can label DNA single-strand sites exposed in situ during DNA damage. Based on the intensity of the fluorescence signal, it can quantitatively detect the degree of DNA damage repair in cell or tissue samples and reflect the presence and abundance of single-stranded DNA.

2. The application according to claim 1, characterized in that, The sample to be tested is selected from any one of the following: cell sample or tissue sample.

3. The application according to claim 1, characterized in that, When the sample to be tested is a tissue sample, after the permeabilization process, the application further includes slicing the sample to be tested.

4. The application according to claim 3, characterized in that, The sectioning process can be performed using any of the following methods: frozen sectioning or paraffin sectioning.

5. The application according to claim 4, characterized in that, The sample to be tested originates from any of the following organisms: plants, animals, bacteria, fungi, archaea, or protists.

6. The application according to any one of claims 1-5, characterized in that, The single-stranded DNA-binding protein is derived from prokaryotes or viruses.

7. The application according to claim 6, characterized in that, The prokaryotes are selected from any one of the following: Escherichia coli, Actinomycetes, or Cyanobacteria.

8. The application according to claim 6, characterized in that, The virus is selected from any one of the following: bacteriophage, tobacco mosaic virus, or tulip mosaic virus.

9. The application according to claim 6, characterized in that, The fluorescent protein is selected from any one of the following: StayGold, EGFP, mNeonGreen, sfGFP, mClover3, mScarlet-I, or mWasabi.

10. The application according to claim 1, characterized in that, The fluorescent fusion protein has any one of the following amino acid sequences: SEQ ID NO: 1 or SEQ ID NO: 3.

Images