S9.6 mutant antibody for improving detection specificity of R-Loop and RNA-DNA hybrid chain and application of S9.6 mutant antibody

Abstract

The invention discloses application of an S9.6 mutant antibody for improving R-Loop detection specificity, threonine at the sixth site on a CDR1 sequence of a heavy chain variable region of the S9.6 antibody is mutated into tryptophan (VH-CDR1-T6W) to reduce non-specific binding of the S9.6 antibody to double-stranded RNA (dsRNA), so that the accuracy of R-Loop and RNA / DNA hybrid chain detection is improved. According to the present invention, molecular and cell biology means are adopted, the S9.6 VH-CDR1-T6W mutant antibody is expressed and purified through 293F cells, different nucleic acid probes such as R-Loop, RNA / DNA hybrid chains and dsRNA are adopted, the nucleic acid binding conditions of the VH-CDR1-T6W in flow cytometry and a gel migration experiment (EMSA) are researched, it is found that the mutant antibody can maintain the binding capacity to the R-Loop and the RNA / DNA hybrid chains, and the mutant antibody can be used for detecting the nucleic acid binding conditions of the VH-CDR1-T6W. And the combination of dsRNA is greatly weakened, 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 specifically, to an S9.6 mutant antibody capable of improving the detection specificity of R-Loop and RNA-DNA hybrid chains and its application. Background Art

[0002] R-Loop is a three-stranded nucleic acid structure composed of RNA / DNA hybrid strands and single-stranded DNA. It is usually formed by the hybridization of a nascent transcript with a DNA template strand and leaving a non-template DNA single strand. R-Loop is commonly found in bacteria, eukaryotes, and viral genomes and is a dynamic, reversible structure. Since the accumulation of R-Loop is closely related to DNA damage, gene transcription regulation, and cancer and other diseases, the accurate detection of R-Loop has become an important technical means to study gene expression, DNA repair, epigenetics, and other fields.

[0003] The study of R-Loop is highly dependent on detection technology that can accurately locate and quantify the structure of R-Loop. S9.6 antibody is one of the most widely used R-Loop detection tools. S9.6 was originally discovered in mice by immunizing mice with RNA / DNA hybrid chains synthesized on φΧ174 single-stranded DNA as antigens. It can detect R-Loop by specifically recognizing RNA / DNA hybrid chains. This antibody can be used for immunofluorescence staining to observe the level and location of R-Loop in single cells, and can also be used for immunoprecipitation with proteins or fragmented genomes to find R-Loop interacting proteins or whole genome sequencing analysis, etc.

[0004] The binding of S9.6 to RNA / DNA hybrid chains is mediated by the aromatic residues and basic residues of the heavy chain, and it recognizes two consecutive 2'-hydroxyl groups on the RNA chain and six consecutive phosphate backbones on the DNA chain in the hybrid chain. However, this recognition specificity is low. Scientists used surface plasmon resonance technology to find that the binding affinity of S9.6 to recognize RNA / DNA hybrid chains is only about 5 times that of dsRNA, so the antibody has been questioned in accurately locating and quantifying R-Loop. At present, scientists have confirmed in experiments such as Dot Blot, IF, IP, and DRIPc-seq that the non-specific binding of S9.6 to dsRNA will cause a lot of background and false positive signals, so 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. There will be incomplete digestion and non-specific cutting. To a certain extent, it cannot solve the problem of non-specific binding of S9.6, and it will introduce additional steps and variables to 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 prior art, the purpose of the present invention is to provide an S9.6 antibody improved by gene mutation, which has higher specificity and sensitivity for the detection of R-Loop.

[0007] Cell display and flow sorting techniques were used to screen antibody mutation libraries. Highly specific cell populations were sorted based on the binding of RNA / DNA hybrid chains and dsRNA. Monoclonal heavy chain variable region and light chain variable region DNA sequences were obtained by high-throughput sequencing. Recombinant vectors were then constructed for expression, purification and biological detection. We identified a specific S9.6 mutant antibody VH-CDR1-T6W, which can effectively reduce nonspecific binding to dsRNA without reducing its binding ability to R-Loop, thereby improving the accuracy of R-Loop and RNA / DNA hybrid chain detection.

[0008] The above technical purpose of the present invention is achieved through the following technical scheme: a S9.6 mutant antibody that improves the detection specificity of R-Loop and RNA-DNA hybrid chains, wherein the mutation is achieved by replacing the threonine at position 6 on 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] Further, 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 region 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 CDR: VL CDR1 shown in SEQ ID NO:4, VL CDR2 shown in SEQ ID NO:5, VL CDR3 shown in SEQ ID NO:6; 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.

[0010] Furthermore, the S9.6 mutant antibody VH-CDR1-T6W comprises an antibody heavy chain constant region CH and a light chain constant region CL, wherein the antibody heavy chain constant region CH is conventional in the art, and 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, and in certain embodiments, the antibody light chain constant region may be derived from human or mouse Kappa constant region.

[0011] Furthermore, heavy chain and light chain plasmids having the above variable region sequences were constructed, expressed in 293F cells, and the obtained antibodies were isolated and purified.

[0012] 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.

[0013] The invention provides an S9.6 mutant antibody VH-CDR1-T6W which can improve the detection specificity of R-Loop and RNA / DNA hybrid chains, and is a monoclonal antibody against RNA / DNA hybrid chains.

[0014] Furthermore, the S9.6 mutant antibody VH-CDR1-T6W is a monoclonal antibody capable of reducing non-specific binding to dsRNA obtained by cell display of a mutant library, flow cytometry screening, and sequencing analysis.

[0015] 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.

[0016] 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.

[0017] 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 requirement that the recombinant expression vector of the present invention can stably replicate itself and the carried nucleic acid molecule can be effectively expressed. For example, the host cell can include 293T or 293F cells.

[0018] 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 heavy chains and light chains, culturing the host cells, and recovering and purifying the antibody.

[0019] In summary, the present invention has the following beneficial effects: The present invention provides an improved S9.6 antibody VH-CDR1-T6W by using cell biology and molecular biology methods, and mainly studies the nucleic acid binding of the antibody in flow cytometry and EMSA experiments. It is found that the antibody can maintain the binding ability to R-Loop and RNA / DNA hybrid chains, has good affinity to R-Loop and RNA / DNA hybrid chains, and can greatly weaken the binding to dsRNA. The specificity and sensitivity are improved, providing 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

[0020] Figure 1 The change in the proportion of some mutants in the library after two rounds of screening; Figure 2 Flow cytometry was used to verify that the VH-CDR1-T6W mutant antibody had increased binding specificity to RNA / DNA hybrid chains; Figure 3 The images show the Coomassie blue staining of S9.6 WT and VH-CDR1-T6W antibodies after purification; Figure 4 The picture shows the EMSA experiment of VH-CDR1-T6W attenuating the binding to dsRNA; Figure 5 The picture shows the EMSA experiment that VH-CDR1-T6W can maintain binding to RNA / DNA hybrid chains; Figure 6 The images show an EMSA experiment showing that VH-CDR1-T6W is able to maintain binding to the R-Loop. DETAILED DESCRIPTION

[0021] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0022] It should be noted that the "antibody" described herein refers to immunoglobulin molecules and immunologically active parts of immunoglobulin molecules. Therefore, the term "antibody" not only covers complete antibodies or full-length antibodies, but also includes fragments of the antibodies and variants (including derivatives) of the antibodies and antibody fragments. For example, it includes but is 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.

[0023] Example 1: Construction and screening of cell display library.

[0024] The sequence of the heavy chain variable region of the mouse S9.6 wild-type antibody was selected, and the amino acids in the three CDR regions were saturated mutated and constructed into the pBOBI lentiviral vector containing the human heavy chain constant region, in which the heavy chain N-terminal fusion membrane localization domain was conferred with displayability of the antibody. Prepare RNA / DNA hybrid chains and dsRNA probes with different fluorescent labels (specific sequences are shown in Table 1). The synthesis method is 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 and anneal.

[0025] The heavy chain recombinant plasmid containing the mutant fragment and the wild-type light chain plasmid were co-infected with 293T cells by lentiviral packaging, and a cell display library containing 780 different heavy chain mutants was constructed. The antibody library was used for two rounds of flow cytometry screening against fluorescently labeled RNA / DNA hybrid chains and dsRNA probes, and positive enrichment was obtained according to the 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, and the proportion of each mutant in the total sequence obtained by sequencing was calculated and sorted. It can be found that the proportion of WT sequences is gradually decreasing, and the proportion after the second round of screening is 0.62 times lower than that after the first round of screening. The mutants are enriched as a whole, among which the proportion of CDR1-T6W mutants after the second round of screening increased by 3.89 times compared with the first round of screening. After the second round of screening, its proportion exceeded the WT sequence, which is 1.51 times that of WT. Data processing: Percentage = number of mutant appearances / total number of sequencing times × 100%. Enrichment multiple = second round of screening / first round of screening. GraphPad Prism software v 9.0 was used for drawing. The specific results are shown in Table 2 and Figure 1 .

[0026] Table 1 RNA / DNA hybrid chain, dsRNA and R-Loop sequences Table 2 Proportion and enrichment fold changes of some mutants after two rounds of screening We analyzed that the optimal mutant sequence is VH-CDR1-T6W, and constructed a plasmid of its variable region sequence for subsequent expression and identification. The specificity of VH-CDR1-T6W to R-Loop and RNA / DNA hybrid chain was evaluated in combination with the following cellular and molecular functional experiments.

[0027] Example 2: Flow cytometry verified that the VH-CDR1-T6W mutant antibody had increased binding specificity to RNA / DNA hybrid chains.

[0028] According to the sequencing results, the specific sequences of the heavy chain and light chain variable regions of the VH-CDR1-T6W antibody obtained above are shown in Table 3, where the mutation site is located in the heavy chain variable region CDR1. The original wild-type heavy chain plasmid was used as a template for CDR1-T6W point mutation, and 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, and used together with the light chain plasmid for 293T cell transfection, thereby realizing the display of a single VH-CDR1-T6W antibody on the cell membrane surface.

[0029] Table 3 Variable region sequences of S9.6 mutants The cultured VH-CDR1-T6W antibody cells were digested with trypsin, neutralized with DMEM medium (containing 10% FBS), and centrifuged at 1000 rpm for 2-3 min to remove the supernatant. Resuspend the cell pellet with FACS buffer (2% FBS in PBS), centrifuge at 1000 rpm for 2-3 min to remove the supernatant. Add FITC fluorescently labeled RNA / DNA hybrid chain, Alexa fluor 647 fluorescently labeled Tetra dsRNA probe (tetrameric dsRNA prepared based on the interaction between streptavidin and biotin to amplify the fluorescent signal) and Pacific Blue fluorescently labeled human Ig light chain Kappa antibody to incubate the cells, and react at 4°C in the dark for 1 hour. After incubation, add FACS buffer again to wash the cells. Finally, resuspend the cells with FACS buffer and filter into a dedicated flow tube for flow analysis.

[0030] 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 force between VH-CDR1-T6W and dsRNA; the higher the FITC mean fluorescence signal intensity (MFI), the stronger the binding force between VH-CDR1-T6W and RNA / DNA hybrid chains. Kappa antibody was 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 WT, indicating that its specificity was increased. And 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 the binding with dsRNA. Results Data processing: FlowJo v10.8.1 software was used for drawing. For specific results, see Figure 2 .

[0031] Example 3: Expression and purification of antibodies.

[0032] The heavy chain 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 before use; 1:100 with D-+-Glucose) 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.

[0033] Take 20 mL of 3 × 10 6cells / mL concentration as an example, add 2 mL Opti-MEM to the centrifuge tube, add 11.4 μg wild-type light chain plasmid and 4.6 μg VH-CDR1-T6W heavy chain mutant plasmid in proportion, vortex mix, then add transfection reagent PEI in proportion, vortex mix immediately to avoid excessive local PEI concentration, mix thoroughly and incubate at room temperature for 15 minutes. After the transfection system is incubated, add it to the diluted cell suspension, shake evenly and place it in a shaker at 37°C, 135rpm, 8% CO2 for culture. 24 hours after transfection, add 5 mL Union 293 culture medium, and add ProFeed (OPM-293 ProFeed, F081918-001) and VPA (SIGMA, 1069-66-5) at a ratio of 1:100 (i.e. 250μl) according to the volume of Union 293 culture medium added.

[0034] On the fifth day of transfection, the cell suspension was centrifuged at 4000 rpm for 10 min, the supernatant was collected into a 50 mL centrifuge tube, Protein G beads (Genscript) washed with PBS were added, and the mixture was rotated 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, all 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. 1.5 MTris (pH=11) was quickly added to neutralize the solution to pH 7. Repeat elution twice. The purified antibodies were used for SDS-PAGE and Coomassie Brilliant Blue staining. For specific results, see Figure 3 .

[0035] Example 4: EMSA experiments demonstrated that the specificity of VH-CDR1-T6W was significantly increased.

[0036] 1. The binding ability of VH-CDR1-T6W to dsRNA is significantly weakened: 50 mM fluorescently labeled Tetra dsRNA and a series of concentration gradients (0 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, 1000 nM) of WT or VH-CDR1-T6W antibody were mixed in a buffer (10 mM Tris pH 7.5, 1 mM EDTA, 100 mM KCl, 1.5 mM MgCl 2, 200 ng / μL BSA) and incubated at room temperature for 30 minutes. After the reaction was completed, the samples were electrophoretically migrated in a 6% non-denaturing polyacrylamide gel using 0.5×Tris-borate buffer (TBE buffer), and electrophoresed at 150V constant voltage at 4°C in the dark for 1.5 hours. Finally, the nucleic acids were displayed by fluorescence scanning using an Azure biosystems c400 instrument. The results showed that as the antibody concentration increased, the dsRNA substrate bound by the WT antibody gradually increased, while the VH-CDR1-T6W mutant was able to significantly reduce the binding to dsRNA. Data processing: Image J and GraphPad Prism software v 9.0 were used for statistics. The percentage of dsRNA substrates bound to the antibody = (gray value of dsRNA bound to the antibody - background gray value) / (total dsRNA gray value - background gray value) × 100%. The specific results are shown in Tables 4 and Figure 4 .

[0037] Table 4 The proportion of dsRNA substrates binding to WT and VH-CDR1-T6W 2. The binding ability of VH-CDR1-T6W to RNA / DNA hybrid chains remains unchanged: 50 mM fluorescently labeled RNA / DNA hybrid strand and a series of concentration gradients (0 nM, 15.625 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM) of WT or VH-CDR1-T6W antibody were mixed in a buffer (10 mM Tris pH 7.5, 1 mM EDTA, 100 mM KCl, 1.5 mM MgCl 2, 200 ng / μL BSA) and incubated at room temperature for 30 minutes. After the reaction was completed, the samples were electrophoretically migrated in a 6% non-denaturing polyacrylamide gel with 0.5×TBE buffer, and electrophoresed at 150V constant voltage for 1 hour at 4°C in the dark. Finally, the nucleic acid was displayed by fluorescence scanning using the Azure biosystems c400 instrument. The results showed that with the increase of antibody concentration, the RNA / DNA hybrid chains bound by WT antibody and VH-CDR1-T6W antibody gradually increased, and VH-CDR1-T6W was able to maintain the binding to RNA / DNA hybrid chains, which was not much different from WT, and the binding affinity was between 62.5 and 125 nM. Data processing: Image J and GraphPad Prism software v 9.0 were used for statistics. The percentage of RNA / DNA hybrid chain substrates bound to antibodies = (gray value of RNA / DNA hybrid chains bound to antibodies-background gray value) / (gray value of total RNA / DNA hybrid chains-background gray value) × 100%. The specific results are shown in Tables 5 and Figure 5 .

[0038] Table 5 The proportion of RNA / DNA hybrid chains binding to WT and VH-CDR1-T6W 3. The binding ability of VH-CDR1-T6W to R-Loop remains unchanged: 50 mM fluorescently labeled R-Loop (specific sequence see Table 1) and a series of concentration gradients (0 nM, 15.625 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM) of WT or VH-CDR1-T6W antibody were incubated in a buffer (10 mMTris pH 7.5, 1 mM EDTA, 100 mM KCl, 1.5 mM MgCl 2, 200 ng / μL BSA) and incubated at room temperature for 30 minutes. After the reaction was completed, the samples were electrophoretically migrated in a 6% non-denaturing polyacrylamide gel using 0.5×TBE buffer, and electrophoresed at 150V constant voltage at 4°C in the dark for 1.5 hours. Finally, the nucleic acids were displayed by fluorescence scanning using the Azure biosystems c400 instrument. The results showed that with the increase in antibody concentration, the R-Loop bound by the WT antibody and the VH-CDR1-T6W antibody gradually increased, and VH-CDR1-T6W was able to maintain binding to the R-Loop, which was not much different from WT, and the binding affinity was between 62.5 and 125 nM. Data processing: Image J and GraphPad Prism software v 9.0 were used for statistics. The percentage of R-Loop substrate bound to the antibody = (R-Loop gray value of the bound antibody - background gray value) / (total R-Loop gray value - background gray value) × 100%. The specific results are shown in Tables 6 and Figure 6 .

[0039] Table 6 R-loop proportions of WT and VH-CDR1-T6W binding Example 5: Dot Blot verified that VH-CDR1-T6W specifically binds to the R-Loop and RNA / DNA hybrid chain in the genome and reduces the binding to dsRNA.

[0040] 293T cells were centrifuged at 3500 rpm for 2 min, the supernatant was discarded, and then centrifuged at 3500 rpm for 2 min, and the supernatant was aspirated. The cells were resuspended in 800 μL TE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA), 25 μL 20% SDS and 7-8 μL proteinase K solution (10 mg / mL) were added, and the tube was gently inverted 5-6 times until the solution became viscous, and incubated at 37°C overnight (12-14 hours).

[0041] Centrifuge a 2 mL low-density liquid phase separation gel tube (TIANGEN, WM5-2302820) at 12,000-16,000×g for 30 s to pellet the gel. Add 800 μL DNA extraction solution (EX0108), pour the overnight incubation solution into the tube, invert and mix thoroughly to mix the organic phase and aqueous phase. Centrifuge at 12,000-16,000×g for 5 min to separate the layers.

[0042] Add 1 / 10 volume of 3 M NaOAc pH 5.2 and 2.5 volumes of absolute ethanol to a new 15 mL centrifuge tube. Directly pour or pipette carefully transfer the upper aqueous phase containing nucleic acids in the gel tube to the above 15 mL centrifuge tube, invert and mix, and let stand for 10 min until a white DNA precipitate is visible. Wash the DNA precipitate 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 last wash, carefully remove as much ethanol as possible by pipetting and let the DNA precipitate dry. Finally, add 50-100 μL of TE buffer to dissolve. Use an ultrasonic disruptor (Diagenode Bioruptor Pico) to lyse the genomic DNA to about 500 bp. Measure the DNA concentration using a Spark® multi-function microplate reader.

[0043] The above samples and related controls were treated with different nucleases, including RNase H (NEB), RNase III (NEB) and RNase A (thermo) at a loading amount of 500 ng, and the samples were incubated at 37°C. The samples were transferred to NC membrane (Millipore, HATF00010) in 20×SSC buffer (Sangon Biotech, B548110-0200) by Bio-Dot Apparatus (Bio-Rad). Then the nucleic acid was cross-linked to the membrane by SGLinkerTM UV cross-linker (SINSAGE). The membrane was blocked with 5% milk (in TBST), and then the primary antibody incubation solution (1µg / mL) was prepared with S9.6WT and VH-CDR1-T6W, respectively, and incubated at 4°C overnight. At the same time, the internal control was incubated with Anti-dsDNA antibody (Abcam, ab27156). The primary antibody was washed with TBST, and then incubated with secondary antibody (anti-mouse HRP, 1:10,000) at room temperature for 1 hour. Finally, the membrane was imaged using an ECL chemiluminescence kit (SUDGEN, 31060) and a MiniChemi chemical imager (SINSAGE).

[0044] 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.

[0045] Genomic DNA (with R-Loop) was extracted by phenol / chloroform / isoamyl alcohol extraction in a low-density liquid phase separation gel tube, and DNA was precipitated with ethanol / sodium acetate, washed with 80% ethanol, and resuspended in TE buffer. Restriction endonucleases BsrG1, EcoR1, HindIII, SspI, XbaI (NEB) were digested overnight at 37°C. In addition, 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. 4 µg of digested DNA was incubated with 10 µg of S9.6WT or VH-CDR1-T6W antibody in DRIP binding buffer (10 mM NaPO4 pH7.0, 140 mM NaCl, 0.05% Triton X-100) with rotation at 4°C overnight. The mixed sample was incubated with 50 µL proteinG magnetic beads at 4°C with rotation for 4 hours. After the reaction, wash the beads three times with binding buffer for 10 minutes each time, then centrifuge at 300xg at 4℃ for 2 min, and discard the supernatant. Finally, incubate and elute with Elution buffer (50 mM Tris pH 8, 10 mMEDTA, 0.5% SDS, Proteinase K) at 55℃ for 45 min. The eluted DNA can be directly used for library construction and high-throughput sequencing detection, or digested with DNase I and recovered RNA for reverse transcription, and then tested by cDNA library construction and high-throughput sequencing.

[0046] Example VII: IF verification of VH-CDR1-T6W specific detection of the level and location of R-Loop and RNA / DNA hybrid chains in cells.

[0047] HeLa cells were plated in six-well plates containing glass slides (Citoglas) 1-2 days before the experiment and cultured to a density of 40-60%. Cells were fixed with ice-cold methanol on ice for 15 min, then washed three times with cold PBS and shaken on a shaker at 20 rpm for 5 min. Cells were permeabilized with cold 0.5% Triton X-100 (in PBS) on ice for 15 min, then washed three times with cold PBS and shaken on a shaker at 20 rpm for 5 min. A wet paper towel was placed in a dark box, and a glass slide was placed on it. The front of the glass slide was placed on the glass slide, and the border of the glass slide was marked with an immunohistochemistry pen. 3% BSA (in PBS) was carefully dropped on the edge of the glass slide, and then gently shaken to ensure that the glass slide was completely covered, and blocked overnight at 4°C. Discard the blocking solution, add the primary antibody (S9.6WT or VH-CDR1-T6W antibody diluted with 1% BSA+PBST1:400) and incubate at room temperature for 1 hour. Wash the slides three times with PBST, and let stand for 5 minutes each time. Then add the Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) secondary antibody (Thermo, A11029) diluted with 1% BSA+PBST 1:500, and incubate at room temperature in the dark for 1 hour. Wash the slides three times with PBST, let stand for 5 minutes each time; wash once with PBS, let stand for 5 minutes; finally wash once with ddH2O, let stand for 5 minutes. Take a new slide, drop 2-3 drops of DAPI (Thermo, 2416488), cover the slide upside down on the slide, and seal the slide with neutral resin (Yisheng Bio). Observe the staining with a CKX53 inverted fluorescence microscope (Olympus), and the result map is processed with Image J.

[0048] Example 8: Detect pathogens based on the binding properties of RNA / DNA hybrid chains by S9.6.

[0049] Accurately identifying pathogens that cause infection is essential for effective treatment and control of infection. Using the binding properties of monoclonal antibody S9.6 to RNA / DNA hybrid chains to detect the RNA of pathogens is a rapid and effective method. The VH-CDR1-T6W antibody provided by the present invention can also be used for pathogen detection, and can reduce the non-specific binding of dsRNA to a certain extent, thereby improving the accuracy of detection.

[0050] Taking the detection of Bacillus anthracis as an example, the pagA gene (encoding the protective antigen of anthrax toxin) is used as the target for detecting Bacillus anthracis. First, the target gene is amplified by PCR. During the PCR process, the biotin labeling of the probe is achieved by directly using a 5'-biotin-labeled primer or biotin-labeled dATP. The 5'-phosphorylated forward strand is digested with an exonuclease, leaving behind a biotin-labeled single-stranded DNA probe. The single-stranded DNA is separated by agarose gel electrophoresis and purified by gel recovery.

[0051] In actual application, Trizol reagent is used to directly extract total RNA from pathogen culture or infected host cells and tissues, biotinylated single-stranded DNA probe is hybridized with target RNA, and then streptavidin is used to capture the hybrid chain, and the pathogen-specific RNA is detected by S9.6VH-CDR1-T6W recognizing the hybrid chain. In the absence of sequence amplification, the pathogen RNA transcript is specifically captured and detected. This method can be adjusted to detect any pathogen (other bacteria, parasites, viruses, etc.) and has wide applicability.

[0052] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A S9.6 mutant antibody that improves the detection specificity of R-Loop and RNA / DNA hybrid chains, characterized in that: 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 non-specific binding to dsRNA.

2. An antibody according to claim 1, characterized in that: 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 region 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 CDR: VL CDR1 shown in SEQ ID NO:4, VL CDR2 shown in SEQ ID NO:5, VL CDR3 shown in SEQ ID NO:

6.

3. An antibody according to claim 2, 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.

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

5. An antibody according to claim 1, characterized in that: 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.

6. An application of an S9.6 mutant antibody for improving the specificity of R-Loop detection, the application method comprising: The S9.6 VH-CDR1-T6W antibody according to claims 1 to 4 has increased specificity in recognizing and binding to R-Loop and RNA / DNA hybridization chain probes in flow cytometry and EMSA experiments, and includes but is not limited to applications in Dot Blot, IF (Immunofluorescence), DRIP (DNA-RNA immunoprecipitation), and RNA-based pathogen detection.

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