S9.6 mutant antibody for improving specificity of r-loop and rna-dna hybrid chain detection and application thereof

Abstract

The application discloses application of S9.6 mutant antibody for improving specificity of R-Loop detection, wherein the threonine at the 6th position in the CDR1 sequence of the heavy chain variable region of the S9.6 antibody is mutated into tryptophan (VH-CDR1-T6W) to reduce the non-specific binding of the antibody to double-stranded RNA (dsRNA), so that the accuracy of R-Loop and RNA / DNA hybrid chain detection is improved. The application uses molecular and cellular biology means to express and purify the S9.6 VH-CDR1-T6W mutant antibody through 293F cells, uses different nucleic acid probes such as R-Loop, RNA / DNA hybrid chain and dsRNA, studies the nucleic acid binding of the VH-CDR1-T6W in flow cytometry (Flow Cytometry) and electrophoretic mobility shift assay (EMSA), and finds that the mutant antibody can maintain the binding capacity to R-Loop and RNA / DNA hybrid chain, and greatly weakens the binding to dsRNA, so that the specificity of R-Loop detection is improved.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and cell biology, and more particularly to an S9.6 mutant antibody capable of improving the detection specificity of R-Loop and RNA-DNA hybrid chains and applications thereof. Background Art

[0002] An R-loop is a triple-stranded nucleic acid structure composed of an RNA / DNA hybrid and single-stranded DNA. It is typically formed when a nascent transcript hybridizes with a DNA template strand, leaving behind a single strand of non-template DNA. R-loops are a dynamic, reversible structure ubiquitous in bacterial, eukaryotic, and viral genomes. Because R-loop accumulation is closely associated with DNA damage, gene transcriptional regulation, and various diseases such as cancer, accurate detection of R-loops has become a crucial technique for studying gene expression, DNA repair, and epigenetics.

[0003] Research on R-loops relies heavily on detection technologies that can accurately locate and quantify R-loop structures. The S9.6 antibody is currently one of the most widely used R-loop detection tools. S9.6 was originally discovered in mice by immunizing them with RNA / DNA hybrids synthesized on φΧ174 single-stranded DNA. It specifically recognizes RNA / DNA hybrids, enabling R-loop detection. This antibody can be used to observe R-loop levels and localization in single cells using immunofluorescence staining, immunoprecipitate proteins with proteins or genomic fragments to identify R-loop interacting proteins, or perform whole-genome sequencing analysis.

[0004] S9.6 binds to RNA / DNA hybrids through the aromatic and basic residues of the heavy chain, recognizing two consecutive 2'-hydroxyl groups on the RNA chain and six consecutive phosphate backbones on the DNA chain in the hybrid. However, this recognition specificity is low. Scientists used surface plasmon resonance technology to find that the binding affinity of S9.6 for RNA / DNA hybrids is only about five times that of dsRNA. Therefore, the ability of this antibody to accurately locate and quantify R-Loop has been questioned. Scientists have now confirmed in experiments such as Dot Blot, IF, IP, and DRIPc-seq that the non-specific binding of S9.6 to dsRNA will result in a lot of background and false-positive signals. Therefore, the presence of dsRNA will interfere with the accuracy of R-Loop detection. In addition, the method of introducing RNase III treatment to remove dsRNA in the experiment is not perfect. Incomplete digestion and non-specific cleavage may occur. To a certain extent, it cannot solve the problem of S9.6 non-specific binding and will introduce additional steps and variables that affect the final results.

[0005] Therefore, the present invention aims to provide an improved S9.6 antibody to fundamentally reduce the binding to dsRNA, thereby enhancing the sensitivity and specificity of R-Loop detection. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide an S9.6 antibody improved by gene mutation, which has higher specificity and sensitivity for R-Loop detection.

[0007] We used cell display and flow cytometry techniques to screen antibody mutation libraries. We isolated highly specific cell populations based on their binding to RNA / DNA hybrids and dsRNA. High-throughput sequencing then obtained monoclonal heavy and light chain variable region DNA sequences. Recombinant vectors were then constructed for expression, purification, and biological detection. We identified a specific S9.6 mutant antibody, VH-CDR1-T6W, which effectively reduced nonspecific binding to dsRNA without compromising its R-Loop binding ability, thereby improving the accuracy of R-Loop and RNA / DNA hybrid detection.

[0008] The above technical objectives of the present invention are achieved through the following technical solutions: an S9.6 mutant antibody that improves the specificity of R-Loop and RNA-DNA hybrid chain detection, wherein the mutation is achieved by replacing the threonine at position 6 in the CDR1 sequence of the heavy chain variable region of the S9.6 antibody with tryptophan (VH-CDR1-T6W) to reduce nonspecific binding to dsRNA.

[0009] Furthermore, the antibody has a heavy chain variable region and / or a light chain variable region;

[0010] Wherein, the heavy chain variable region includes the following complementarity determining regions CDR:

[0011] VH CDR1 shown in SEQ ID NO: 1,

[0012] VH CDR2 shown in SEQ ID NO: 2,

[0013] VH CDR3 shown in SEQ ID NO: 3;

[0014] The light chain variable region includes the following complementarity determining regions (CDRs):

[0015] VL CDR1 shown in SEQ ID NO:4,

[0016] VL CDR2 shown in SEQ ID NO:5,

[0017] VL CDR3 shown in SEQ ID NO: 6;

[0018] The amino acid sequence of the antibody heavy chain variable region is SEQ ID NO: 7; the amino acid sequence of the antibody light chain variable region is SEQ ID NO: 8.

[0019] Furthermore, the S9.6 mutant antibody VH-CDR1-T6W comprises an antibody heavy chain constant region CH and a light chain constant region CL. The antibody heavy chain constant region CH is conventional in the art. In certain embodiments, the antibody heavy chain constant region may comprise a constant region derived from human IgG1 or a constant region derived from mouse IgG2α; the antibody light chain constant region CL is conventional in the art. In certain embodiments, the antibody light chain constant region may be derived from human or mouse Kappa constant region.

[0020] Furthermore, heavy chain and light chain plasmids containing the above variable region sequences were constructed, expressed in 293F cells, and the resulting antibodies were isolated and purified.

[0021] Furthermore, the antibody can specifically recognize RNA / DNA hybrid chains and R-Loop structures by reducing binding to dsRNA, and does not significantly bind to ssRNA, ssDNA and dsRNA molecules.

[0022] The present invention provides an S9.6 mutant antibody VH-CDR1-T6W capable of improving the detection specificity of R-Loop and RNA / DNA hybrid chains, and is a monoclonal antibody against RNA / DNA hybrid chains.

[0023] Furthermore, the S9.6 mutant antibody VH-CDR1-T6W is a monoclonal antibody capable of reducing non-specific binding to dsRNA, which is obtained by cell display of a mutant library, flow cytometry screening, and sequencing analysis.

[0024] Furthermore, the present invention provides two nucleic acid molecules, encoding the heavy chain and light chain variable regions of the antibody of the present invention, respectively.

[0025] Furthermore, the present invention also provides a recombinant expression vector comprising the nucleic acid molecule of the present invention. In certain embodiments, the recombinant expression vector can be obtained by conventional methods in the art.

[0026] Furthermore, the present invention also provides a mammalian host cell comprising the recombinant expression vector of the present invention. In certain embodiments, the host cell is a conventional host cell in the art, as long as it can satisfy the requirements of the recombinant expression vector of the present invention for stable self-replication and effective expression of the nucleic acid molecules carried by it. For example, the host cell can include 293T or 293F cells.

[0027] Furthermore, the present invention relates to a method for preparing the antibody in large quantities, comprising co-transfecting 293F host cells with recombinant expression vectors of the heavy chain and the light chain, culturing the host cells, and recovering and purifying the antibody.

[0028] In summary, the present invention has the following beneficial effects:

[0029] The present invention utilizes cell biology and molecular biology methods to provide an improved S9.6 antibody VH-CDR1-T6W. The nucleic acid binding of the antibody in flow cytometry and EMSA experiments was mainly studied. It was found that the antibody can maintain its binding ability to R-Loop and RNA / DNA hybrid chains, and has good affinity for R-Loop and RNA / DNA hybrid chains. At the same time, it can greatly reduce the binding to dsRNA, and its specificity and sensitivity are improved. It provides a powerful tool for research in related fields. Specific applications include but are not limited to Dot Blot, IF, DRIP and RNA-based pathogen detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 represents the change in the proportion of some mutants in the library after two rounds of screening;

[0031] Figure 2 Flow cytometry was used to verify that the VH-CDR1-T6W mutant antibody had increased binding specificity for RNA / DNA hybrid chains;

[0032] Figure 3 The images show Coomassie blue staining of purified S9.6 WT and VH-CDR1-T6W antibodies;

[0033] Figure 4 Images are shown for EMSA experiments showing that VH-CDR1-T6W attenuates dsRNA binding;

[0034] Figure 5 Images from EMSA experiments show that VH-CDR1-T6W can maintain binding to RNA / DNA hybrid chains;

[0035] Figure 6 Images are shown for EMSA experiments showing that VH-CDR1-T6W can maintain binding to the R-Loop. DETAILED DESCRIPTION

[0036] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0037] It should be noted that the term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. Therefore, the term "antibody" encompasses not only intact or full-length antibodies, but also fragments and variants (including derivatives) of such antibodies and antibody fragments. Examples include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), disulfide-linked Fv (sdFv), di-scFv, chimeric antibodies, HCAbs, and / or dAbs.

[0038] Example 1: Construction and screening of cell display library.

[0039] The sequence of the heavy chain variable region of the murine S9.6 wild-type antibody was selected, and amino acids in the three CDR regions were saturatedly mutagenized. The resulting construct was then transfected into the pBOBI lentiviral vector containing the human heavy chain constant region. A membrane-localizing domain was fused to the N-terminus of the heavy chain to confer displayability. Different fluorescently labeled RNA / DNA hybrids and dsRNA probes were prepared (sequences are shown in Table 1). The synthesis method was to mix two single-stranded oligonucleotides in annealing buffer (10 mM Tris pH 7.5, 50 mM NaCl, 1 mM EDTA), heat denature at 95°C, and gradually cool to anneal.

[0040] Recombinant heavy chain plasmids containing mutant fragments and wild-type light chain plasmids were co-infected with lentiviral packaging in 293T cells, generating a cell-based display library containing 780 different heavy chain mutants. This antibody library was screened using two rounds of flow cytometry against fluorescently labeled RNA / DNA hybrids and dsRNA probes. Positives were enriched based on specific binding. After each round of screening, the target gene was amplified from the cell genome for high-throughput sequencing and amino acid sequence analysis. The proportion of each mutant in the total sequenced was calculated and ranked. The proportion of WT sequences decreased gradually, decreasing by 0.62-fold after the second round of screening compared to the first. Mutants were enriched overall, with the CDR1-T6W mutant increasing by 3.89-fold after the second round of screening compared to the first. Its proportion exceeded that of the WT sequence by 1.51-fold after the second round of screening. Data processing: Percentage = Number of mutant occurrences / Total number of sequencing attempts × 100%. Enrichment factor = Second round of screening / First round of screening. GraphPad Prism software v 9.0 was used for plotting. The specific results are shown in Table 2 and Figure 1 .

[0041] Table 1 RNA / DNA hybrid, dsRNA and R-Loop sequences

[0042]

[0043] Table 2 Proportion and enrichment fold changes of some mutants after two rounds of screening

[0044]

[0045] We analyzed that the optimal mutant sequence was VH-CDR1-T6W, and constructed a plasmid of its variable region sequence for subsequent expression and identification. We combined the following cellular and molecular functional experiments to evaluate the specificity of VH-CDR1-T6W for the R-Loop and RNA / DNA hybrid chain.

[0046] Example 2: Flow cytometry verified that the VH-CDR1-T6W mutant antibody had increased binding specificity to RNA / DNA hybrid chains.

[0047] Based on the sequencing results, the detailed sequences of the heavy and light chain variable regions of the VH-CDR1-T6W antibody obtained above are shown in Table 3. The mutation site is located in the heavy chain variable region CDR1. Using the original wild-type heavy chain plasmid as a template, CDR1-T6W point mutations were performed. Primers were designed for polymerase chain reaction (PCR) amplification of the VH-CDR1-T6W sequence. The PCR-amplified fragment was recovered and reconstructed into the pBOBI vector. Together with the light chain plasmid, it was used to transfect 293T cells, thereby achieving surface display of the single VH-CDR1-T6W antibody.

[0048] Table 3 Variable region sequences of S9.6 mutants

[0049]

[0050] The VH-CDR1-T6W antibody cells cultured above were trypsinized, neutralized with DMEM (10% FBS), and centrifuged at 1000 rpm for 2-3 minutes to remove the supernatant. The cell pellet was resuspended in FACS buffer (2% FBS in PBS) and centrifuged at 1000 rpm for 2-3 minutes to remove the supernatant. FITC-labeled RNA / DNA hybrids, Alexa fluor 647-labeled Tetra dsRNA probes (tetrameric dsRNA prepared based on the interaction between streptavidin and biotin, used to amplify the fluorescent signal), and Pacific Blue-labeled human Ig light chain kappa antibodies were added to the cells and incubated at 4°C in the dark for 1 hour. After incubation, the cells were washed again with FACS buffer. Finally, the cells were resuspended in FACS buffer and filtered into dedicated flow cytometer tubes for flow cytometric analysis.

[0051] The higher the Pacific Blue mean fluorescence signal intensity (MFI), the higher the expression level of VH-CDR1-T6W; the higher the Alexa fluor 647 mean fluorescence signal intensity (MFI), the stronger the binding of VH-CDR1-T6W to dsRNA; the higher the FITC mean fluorescence signal intensity (MFI), the stronger the binding of VH-CDR1-T6W to RNA / DNA hybrid chains. Kappa antibodies were used to eliminate the influence of inconsistent expression levels on the results. The results showed that the VH-CDR1-T6W mutant was more inclined to bind to RNA / DNA hybrid chains than the WT, indicating that its specificity was increased. Moreover, when the antibody expression level was constant, the ability of VH-CDR1-T6W to bind to RNA / DNA hybrid chains did not change; but when the expression level was constant, the ability of VH-CDR1-T6W to bind to dsRNA was significantly reduced, indicating that the increased specificity of the mutant was presented by reducing binding to dsRNA. Result data processing: Graphs were drawn using FlowJo v10.8.1 software. See for specific results. Figure 2 .

[0052] Example 3: Expression and purification of antibodies.

[0053] The heavy and light chain variable region sequences of the VH-CDR1-T6W antibody were constructed into the NAC expression vector, and the 293F suspension cells were subcultured and inoculated in Union 293 culture medium (1:100 with GlutaMAX and 1:100 with D-+-Glucose before use) at a cell density of 0.4-0.6×10 6 cells / mL, 37°C, 150 rpm, 8% CO2 shaker culture. Count the cell suspension and count the cells when the cell density reaches 4×10 6 When the cell density is about 3×10 cells / mL, dilute the cells with Union 293 culture medium to a cell density of 3×10 6 cells / mL for transfection.

[0054] 20 mL of 3 × 10 6For example, using a cell / mL concentration, add 2 mL of Opti-MEM to a centrifuge tube, then add 11.4 μg of the wild-type light chain plasmid and 4.6 μg of the VH-CDR1-T6W heavy chain mutant plasmid in the appropriate ratio. Vortex to mix thoroughly. Then, add the transfection reagent PEI in the appropriate ratio and immediately vortex to mix thoroughly to avoid localized high PEI concentrations. Mix thoroughly and incubate at room temperature for 15 minutes. After incubation, add the transfection system to the diluted cell suspension, shake well, and incubate at 37°C, 135 rpm, and 8% CO2 in a shaker. 24 hours after transfection, add 5 mL of Union 293 culture medium. ProFeed (OPM-293 ProFeed, F081918-001) and VPA (SIGMA, 1069-66-5) are added at a 1:100 ratio (i.e., 250 μl) based on the volume of Union 293 culture medium added.

[0055] On the fifth day of transfection, the cell suspension was centrifuged at 4000 rpm for 10 min, and the supernatant was collected into a 50 mL centrifuge tube. Protein G beads (Genscript) washed with PBS were added and incubated at 4°C for 2 h. After incubation, the beads were washed with Washbuffer (25 mM Tris, 150 mM NaCl, pH=7.2). Finally, the beads mixture was transferred to the chromatography column and the filtrate was allowed to flow down. Cover the column cap at the bottom of the chromatography column, add 300 μL Elution buffer (0.1 M citric acid, pH=3) to the chromatography column and let it stand for 2 min. Remove the column cap and let the liquid flow down the column into a 1.5 mL centrifuge tube. Quickly add 1.5 MTris (pH=11) to neutralize the solution to pH 7. Repeat elution twice. The purified antibodies were used for SDS-PAGE and Coomassie Brilliant Blue staining. See the specific results for details. Figure 3 .

[0056] Example 4: EMSA experiments demonstrated that the specificity of VH-CDR1-T6W was significantly increased.

[0057] 1. The binding ability of VH-CDR1-T6W to dsRNA is significantly weakened:

[0058] 50 mM fluorescently labeled Tetra dsRNA was incubated with a concentration gradient (0 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, and 1000 nM) of WT or VH-CDR1-T6W antibodies in a buffer (10 mM Tris pH 7.5, 1 mM EDTA, 100 mM KCl, 1.5 mM MgCl2, and 200 ng / μL BSA) at room temperature for 30 minutes. After completion of the reaction, samples were electrophoretically migrated on a 6% native polyacrylamide gel using 0.5× Tris-borate buffer (TBE) at 150 V for 1.5 hours at 4°C in the dark. Nucleic acids were visualized by fluorescence scanning using an Azure Biosystems c400 instrument. The results showed that as the antibody concentration increased, the WT antibody bound to more dsRNA substrates, while the VH-CDR1-T6W mutant significantly reduced dsRNA binding. Data processing: Image J and GraphPad Prism software v 9.0 were used for statistical analysis. The percentage of dsRNA substrate bound to the antibody = (grey value of dsRNA bound to the antibody - grey value of background) / (grey value of total dsRNA - grey value of background) × 100%. Detailed results are shown in Tables 4 and Figure 4 .

[0059] Table 4 The proportion of dsRNA substrates that bind to WT and VH-CDR1-T6W

[0060]

[0061] 2. The binding ability of VH-CDR1-T6W to RNA / DNA hybrid chains remains unchanged:

[0062] 50 mM fluorescently labeled RNA / DNA hybrids were incubated with a concentration gradient of WT or VH-CDR1-T6W antibodies (0 nM, 15.625 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, and 500 nM) in a buffer (10 mM Tris pH 7.5, 1 mM EDTA, 100 mM KCl, 1.5 mM MgCl2, and 200 ng / μL BSA) at room temperature for 30 minutes. After completion of the reaction, samples were electrophoretically migrated on a 6% native polyacrylamide gel using 0.5× TBE buffer. Electrophoresis was performed at 150 V for 1 hour at 4°C in the dark. Nucleic acids were visualized by fluorescence scanning using an Azure Biosystems c400 instrument. The results showed that as the antibody concentration increased, the WT and VH-CDR1-T6W antibodies bound to increasing amounts of RNA / DNA hybrids. VH-CDR1-T6W maintained binding to the RNA / DNA hybrids, comparable to the WT, with binding affinities ranging from 62.5 to 125 nM. Data were analyzed using Image J and GraphPad Prism software v 9.0. The percentage of RNA / DNA hybrid substrate bound to the antibody was calculated as (antibody-bound RNA / DNA hybrid grayscale value - background grayscale value) / (total RNA / DNA hybrid grayscale value - background grayscale value) × 100%. Detailed results are shown in Tables 5 and 6. Figure 5 .

[0063] Table 5 The proportion of RNA / DNA hybrid chains binding to WT and VH-CDR1-T6W

[0064]

[0065] 3. The binding ability of VH-CDR1-T6W to R-Loop remains unchanged:

[0066] 50 mM fluorescently labeled R-Loop (sequences shown in Table 1) was incubated with a concentration gradient (0 nM, 15.625 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, and 500 nM) of either WT or VH-CDR1-T6W antibody in a buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 100 mM KCl, 1.5 mM MgCl2, and 200 ng / μL BSA) at room temperature for 30 minutes. After completion of the reaction, the samples were electrophoretically migrated on a 6% native polyacrylamide gel using 0.5× TBE buffer. Electrophoresis was performed at 150 V for 1.5 hours at 4°C in the dark. Nucleic acids were visualized by fluorescence scanning using an Azure Biosystems C400 instrument. The results showed that as the antibody concentration increased, the R-Loop bound by the WT and VH-CDR1-T6W antibodies gradually increased. VH-CDR1-T6W was able to maintain its R-Loop binding, with binding affinities ranging from 62.5 to 125 nM, comparable to the WT. Data were processed using Image J and GraphPad Prism software v 9.0. The percentage of R-Loop substrate bound to the antibody was calculated as (R-Loop grayscale value bound to the antibody - background grayscale value) / (total R-Loop grayscale value - background grayscale value) × 100%. Detailed results are shown in Tables 6 and Figure 6 .

[0067] Table 6 R-loop proportions of WT and VH-CDR1-T6W binding

[0068]

[0069] Example 5: Dot Blot verification that VH-CDR1-T6W specifically binds to the R-Loop and RNA / DNA hybrid chain in the genome and reduces binding to dsRNA.

[0070] Centrifuge 293T cells at 3500 rpm for 2 minutes, discard the supernatant, and centrifuge again at 3500 rpm for 2 minutes. Aspirate the supernatant. Resuspend the cells in 800 μL of TE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA). Add 25 μL of 20% SDS and 7-8 μL of proteinase K solution (10 mg / mL). Gently invert the tube 5-6 times until the solution becomes viscous. Incubate at 37°C overnight (12-14 hours).

[0071] Centrifuge a 2 mL low-density liquid phase separation gel tube (TIANGEN, WM5-2302820) at 12,000-16,000 × g for 30 seconds to pellet the gel. Add 800 μL of DNA extraction solution (EX0108) and pour the overnight incubation solution into the tube. Invert and mix thoroughly to mix the organic and aqueous phases. Centrifuge at 12,000-16,000 × g for 5 minutes to separate the layers.

[0072] Add 1 / 10 volume of 3 M NaOAc, pH 5.2, and 2.5 volumes of anhydrous ethanol to a new 15 mL centrifuge tube. Pour or carefully transfer the upper aqueous phase containing nucleic acids from the gel tube to the 15 mL centrifuge tube. Invert and mix thoroughly. Let stand for 10 minutes until a white DNA precipitate is visible. Wash the DNA pellet with 1.5 mL of 80% ethanol and let stand for 10 minutes. Carefully discard the supernatant and repeat this step three times. After the final wash, carefully remove as much ethanol as possible by pipetting and allow the DNA pellet to air dry. Finally, add 50-100 μL of TE buffer to dissolve the DNA. Use an ultrasonic disruptor (Diagenode Bioruptor Pico) to fragment the genomic DNA to approximately 500 bp. Determine the DNA concentration using a Spark® multi-function microplate reader.

[0073] The samples and related controls were treated with various nucleases, including RNase H (NEB), RNase III (NEB), and RNase A (thermo), at a loading volume of 500 ng. The samples were incubated at 37°C. The samples were transferred to a NC membrane (Millipore, HATF00010) using a Bio-Dot Apparatus (Bio-Rad) in 20× SSC buffer (Sangon Biotech, B548110-0200). The nucleic acids were then crosslinked to the membrane using an SGLinker™ UV crosslinker (SINSAGE). The membrane was blocked with 5% milk in TBST and incubated with primary antibodies (1 µg / mL) prepared from S9.6WT and VH-CDR1-T6W, respectively, overnight at 4°C. An internal control was also incubated with an anti-dsDNA antibody (Abcam, ab27156). The primary antibody was washed with TBST, and the membrane was then incubated with a secondary antibody (anti-mouse HRP, 1:10,000) for 1 hour at room temperature. Finally, the membrane was imaged using an ECL chemiluminescence kit (SUDGEN, 31060) and a MiniChemi chemical imager (SINSAGE).

[0074] Example 6: DRIP and high-throughput sequencing were used to verify that VH-CDR1-T6W specifically detected the R-Loop and RNA / DNA hybrid chain signals in the genome.

[0075] Genomic DNA (with R-Loop) was extracted using phenol / chloroform / isoamyl alcohol in a low-density liquid phase separation gel tube. DNA was precipitated with ethanol / sodium acetate, washed with 80% ethanol, and resuspended in TE buffer. The DNA was digested with restriction endonucleases BsrG1, EcoR1, HindIII, SspI, and XbaI (NEB) overnight at 37°C. Additionally, 8 µg of digested DNA was treated with RNase H (NEB) at 37°C as a control and then subjected to the S9.6 immunoprecipitation step. Four µg of digested DNA was incubated with 10 µg of S9.6 WT or VH-CDR1-T6W antibody in DRIP binding buffer (10 mM NaPO₄ pH 7.0, 140 mM NaCl, 0.05% Triton X-100) with rotation overnight at 4°C. The mixed sample was then incubated with 50 µL of protein G magnetic beads at 4°C with rotation for 4 hours. After the reaction, wash the beads three times with binding buffer by spinning for 10 minutes each time, then centrifuge at 300xg for 2 minutes at 4°C and discard the supernatant. Finally, elute the beads with elution buffer (50 mM Tris pH 8, 10 mM EDTA, 0.5% SDS, Proteinase K) at 55°C with rotation for 45 minutes. The eluted DNA can be directly used for library construction and high-throughput sequencing, or digested with DNase I and recovered for reverse transcription, followed by cDNA library construction and high-throughput sequencing.

[0076] Example VII: IF Verification of VH-CDR1-T6W Specificity in Detecting the Levels and Localization of R-Loop and RNA / DNA Hybrid Chains in Cells.

[0077] HeLa cells were plated in six-well plates containing slides (Citoglas) 1-2 days before the experiment and cultured to a confluence of 40-60%. Cells were fixed with ice-cold methanol for 15 minutes on ice, then washed three times with cold PBS and shaken at 20 rpm for 5 minutes. Cells were permeabilized with cold 0.5% Triton X-100 in PBS for 15 minutes on ice, then washed three times with cold PBS and shaken at 20 rpm for 5 minutes. A dark box was lined with damp paper towels and a slide placed on it. The slide was placed face-up on the slide. The slide border was marked with an immunohistochemistry pen. 3% BSA in PBS was carefully dropped onto the edge of the slide and gently shaken to ensure complete coverage. The slide was blocked overnight at 4°C. The blocking solution was discarded, and the primary antibody (S9.6WT or VH-CDR1-T6W antibody diluted 1:400 in 1% BSA + PBST) was added dropwise and incubated at room temperature for 1 hour. The slides were then washed three times with PBST, each time for 5 minutes. Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) secondary antibody (Thermo, A11029) diluted 1:500 in 1% BSA + PBST was then added dropwise and incubated at room temperature for 1 hour in the dark. The slides were washed three times with PBST, each for 5 minutes; washed once with PBS, each for 5 minutes; and finally washed once with ddH2O, each for 5 minutes. Two to three drops of DAPI (Thermo, 2416488) were added to a new slide, and the slide was placed upside down on a glass cover. The slides were then mounted with neutral resin (Yisheng Bio). Staining was observed using a CKX53 inverted fluorescence microscope (Olympus), and images were processed using Image J.

[0078] Example 8: Pathogen detection based on the binding properties of RNA / DNA hybrid chains by S9.6.

[0079] Accurately identifying pathogens that cause infections is crucial for effective treatment and control. Detecting pathogen RNA using the binding properties of monoclonal antibody S9.6 to RNA / DNA hybrids is a rapid and effective method. The VH-CDR1-T6W antibody provided by the present invention can also be used for pathogen detection, reducing nonspecific binding of dsRNA to a certain extent and improving detection accuracy.

[0080] For example, in the detection of Bacillus anthracis, the pagA gene (encoding the protective antigen of anthrax toxin) is used as a target for detection of Bacillus anthracis. First, the target gene is amplified using PCR. During PCR, the probe is biotinylated using either a 5'-biotinylated primer or biotinylated dATP. Exonuclease digests the 5'-phosphorylated forward strand, leaving a biotinylated single-stranded DNA probe. The single-stranded DNA is then separated and purified by agarose gel electrophoresis.

[0081] In practical applications, total RNA is extracted directly from pathogen cultures or infected host cells and tissues using Trizol reagent. A biotinylated single-stranded DNA probe is then hybridized to the target RNA. The hybrid strand is then captured with streptavidin. The hybrid strand is recognized by the S9.6VH-CDR1-T6W motif, allowing for the detection of pathogen-specific RNA. This method allows for the specific capture and detection of pathogen RNA transcripts without the need for sequence amplification. This method can be adapted to detect any pathogen (including other bacteria, parasites, and viruses), demonstrating its broad applicability.

[0082] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. An S9.6 mutant antibody that improves the detection specificity of R-Loop and RNA / DNA hybrid chains, characterized by: The mutation is achieved by replacing the threonine at position 6 in the CDR1 sequence of the heavy chain variable region of the S9.6 antibody with tryptophan (VH-CDR1-T6W) to reduce nonspecific binding to dsRNA; The antibody has a heavy chain variable region and / or a light chain variable region; Wherein, the heavy chain variable region includes the following complementarity determining regions CDR: VH CDR1 shown in SEQ ID NO: 1, VH CDR2 shown in SEQ ID NO: 2, VH CDR3 shown in SEQ ID NO: 3; The light chain variable region includes the following complementarity determining regions (CDRs): VL CDR1 shown in SEQ ID NO:4, VL CDR2 shown in SEQ ID NO:5, VL CDR3 shown in SEQ ID NO:

6.

2. The antibody according to claim 1, characterized in that: The amino acid sequence of the antibody heavy chain variable region is SEQ ID NO: 7; the amino acid sequence of the antibody light chain variable region is SEQ ID NO:

8.

3. The antibody according to claim 2, characterized in that: The heavy chain and light chain plasmids containing the above variable region sequences were constructed and expressed in 293F cells and the resulting antibodies were isolated and purified.

4. The antibody according to claim 1, wherein: The antibody can specifically recognize RNA / DNA hybrid chains and R-Loop structures by reducing binding to dsRNA, and does not significantly bind to ssRNA, ssDNA and dsRNA molecules.

5. An application of an S9.6 mutant antibody to improve R-Loop detection specificity, the application method comprising: The S9.6 VH-CDR1-T6W antibody according to any one of claims 1 to 3 has increased specificity in recognizing and binding to R-Loop and RNA / DNA hybrid chain probes in flow cytometry and EMSA experiments.

Images