A 6mA modified single-stranded DNA sequence specifically binding with myh9 protein and application thereof in sepsis treatment

Abstract

The application relates to the technical field of biological medicine, and discloses a 6mA modified single-stranded DNA sequence specifically combined with MYH9 protein and application of the 6mA modified single-stranded DNA sequence in sepsis treatment; the single-stranded DNA sequence is a 6mA modified single-stranded DNA sequence specifically combined with MYH9 protein N 6 a methylated adenine (6mA) modified single-stranded DNA sequence, and the nucleotide sequence is shown in SEQ ID NO:1; after 6mA methylation modification of adenine (A) at a specific position, the 6mA modified single-stranded DNA sequence can be specifically recognized and combined with MYH9 protein, and the combination specificity is verified in vitro / in vivo through EMSA, Biotin DNA pulldown WB experiments and MYH9-DNA IP experiments. The 6mA modified single-stranded DNA is combined with MYH9 protein, activates a downstream JAK1-STAT6 signal path, promotes M2 phenotype polarization of macrophages, inhibits excessive inflammatory reactions of the organism, improves survival rate, reduces pathological damage of spleen tissues, improves pathological phenotypes such as weight loss and body temperature drop of sepsis mice, and then plays an anti-sepsis role. The 6mA modified single-stranded DNA sequence effectively solves the clinical pain points of existing non-specific anti-inflammatory treatment, and has good drug development potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a 6mA modified single-stranded DNA sequence that specifically binds to the MYH9 protein and its application in the treatment of sepsis. Background Technology

[0002] The statements in this section provide only background information relevant to the disclosure of this application and may not constitute prior art.

[0003] Sepsis, caused by infection triggering a dysregulation of the host response, leads to life-threatening organ dysfunction. It is a disease with high morbidity and mortality rates in clinical critical care medicine, placing a heavy burden on the healthcare system. Currently, clinical treatment of sepsis mainly relies on symptomatic treatments such as antibiotics to control infection, fluid resuscitation, and anti-inflammatory drugs to suppress the inflammatory response. However, there are currently no FDA-approved sepsis-specific drugs, and antibiotics face the risk of resistance. Existing treatments are insufficient to fundamentally regulate the body's inflammatory imbalance, failing to effectively intervene in sepsis and resulting in limited overall treatment efficacy. Therefore, developing novel anti-sepsis therapeutic molecules has significant clinical importance and application value.

[0004] Macrophage polarization is closely related to the occurrence and development of sepsis. Macrophages are classified into M1 and M2 types. M1 macrophages mainly secrete pro-inflammatory factors, inducing excessive inflammatory responses in the body and exacerbating organ damage in sepsis. M2 macrophages mainly secrete anti-inflammatory factors, inhibiting inflammatory responses, promoting tissue repair, and thus playing a protective role against sepsis. Therefore, regulating the immunophenotype of macrophages and promoting their polarization towards the M2 phenotype, thereby improving the body's inflammatory imbalance, can serve as an important strategy for sepsis intervention and an effective approach to treating sepsis.

[0005] DNA aptamers are short, single-stranded DNA sequences that can specifically bind to target proteins and can be used as functional nucleic acid molecules to regulate biological processes. The MYH9 protein is a protein that specifically recognizes and binds to 6mA-modified single-stranded DNA sequences. While it is not directly related to sepsis, our research found that when a specific 6mA-modified DNA sequence specifically binds to the MYH9 protein, it can initiate the regulation of the JAK1-STAT6 downstream signaling pathway, thereby affecting the expression of inflammatory factors and the polarization state of macrophages.

[0006] Currently, no drugs modified with 6mA are marketed or in clinical trials globally. Related research primarily focuses on RNA regulation, and drug development using 6mA-modified DNA remains a blank. Existing technologies have not disclosed 6mA-modified DNA sequences that can be specifically recognized by the MYH9 protein, nor have they disclosed the physiological pathways activated upon such recognition. Therefore, developing 6mA-modified DNA sequences that can be specifically recognized by the MYH9 protein and exert antiseptic effects is of great significance in overcoming the deficiencies in current technologies. Summary of the Invention

[0007] The purpose of this invention is to address the problem that current technologies struggle to fundamentally regulate inflammatory imbalances in the body, resulting in limited therapeutic effects. This invention provides a 6mA-modified single-stranded DNA sequence that specifically binds to the MYH9 protein and its application in the treatment of sepsis, filling the technological gap of lacking functional nucleic acid molecules that can regulate inflammation and intervene in the sepsis process through MYH9 protein recognition and binding to 6mA-modified nucleic acids. The 6mA-modified single-stranded DNA (ssDNA) sequence provided by this invention can specifically bind to the MYH9 protein, improving macrophage polarization and inflammatory imbalance by regulating the JAK1 / STAT6 signaling pathway. Animal experiments have verified its promising potential for anti-septic applications, making it a novel nucleic acid candidate molecule that can provide new molecular tools and technological foundations for the research and development of sepsis intervention and treatment.

[0008] The technical solution of the present invention is as follows: In one aspect, the present invention provides a 6mA-modified single-stranded DNA molecule that specifically binds to the MYH9 protein. Specifically, it provides a DNA aptamer, which is an ssDNA molecule with the sequence shown in SEQ ID NO:1, wherein the first adenine (A) residue in the “GAATTGCCCT” part of the sequence is modified with 6mA methylation.

[0009] 6mA is N 6 -Methyladenine; that is, adenine residues N 6 Methylation modification occurs at the position.

[0010] The sequence shown in SEQ ID NO:1 (6mA) 1st ) is: 5'-GCCACCACTTACTACCTTCCTCTAAG6mAATTGCCCTTTAACTCATCACGCTAACATCGCCG-3'.

[0011] The 6mA-modified single-stranded DNA sequence specifically binds to the MYH9 protein, initiating the downstream anti-inflammatory signaling pathway JAK1-STAT6 regulation, promoting macrophage polarization towards the M2 type, inhibiting sepsis-related excessive inflammatory response, and alleviating sepsis-related organ damage. The binding specificity of the 6mA-modified single-stranded DNA sequence to the MYH9 protein was verified in vitro and in vivo using electrophoresis (EMSA), biotinylated DNA pulldown Western blotting (BIB), and MYH9-DNA immunoprecipitation (MYH9-DNA IP) assays. The results showed that the 6mA-modified single-stranded DNA sequence can bind to the MYH9 protein and treat sepsis.

[0012] Preferably, the antiseptic effect specifically refers to a decrease in the level of pro-inflammatory factors and an increase in the level of anti-inflammatory factors.

[0013] In another aspect, the present invention provides the use of a 6mA-modified single-stranded DNA molecule that specifically binds to the MYH9 protein as described above in the preparation of an antiseptic drug.

[0014] In another aspect, the present invention provides a pharmaceutical composition comprising a 6mA-modified single-stranded DNA molecule that specifically binds to the MYH9 protein as described above.

[0015] Preferably, it also includes a delivery carrier or a pharmaceutically acceptable excipient.

[0016] Preferably, it also includes a pharmaceutically acceptable carrier.

[0017] Preferably, the delivery carrier is, for example, lipid nanoparticles (LNPs); LNPs, as the core delivery carrier, can efficiently deliver 6mA modified single-stranded DNA sequences to target cells (such as macrophages), avoiding sequence degradation by nucleases in vivo, and further improving the bioavailability and targeting of the sequence; while pharmaceutically acceptable carriers, including but not limited to physiological saline, phosphate buffer, etc., can be selected according to the administration method (intravenous injection, intraperitoneal injection, etc.) to assist LNP delivery and reduce toxic side effects.

[0018] The mechanism of action of the aforementioned pharmaceutical composition as an antiseptic drug is as follows: LNP delivers a 6mA-modified single-stranded DNA sequence to target cells. This sequence specifically binds to the MYH9 protein, regulating the inflammatory response and promoting macrophage polarization towards the M2 type, thereby exerting an antiseptic effect. The binding specificity of the sequence to the MYH9 protein was verified in vitro and in vivo using EMSA, Biotin DNA pulldown WB, and DNA IP methods. The verification results showed that the 6mA-modified single-stranded DNA sequence can bind to the MYH9 protein and treat sepsis.

[0019] Preferably, the delivery carrier also includes extracellular vesicles, polymer carriers, or inorganic nanocarriers.

[0020] Preferably, pharmaceutically acceptable excipients include microcrystalline cellulose, lactose, or starch.

[0021] Compared with existing technologies, the advantages of this invention are: 1. High binding specificity: Through in vitro and in vivo verification using EMSA, DNA pulldown, and DNA IP experiments, the 6mA-modified ssDNA sequence of this invention can be specifically recognized and bound by the MYH9 protein. This specific binding initiates downstream anti-inflammatory signals and regulates macrophage polarization, acting on sepsis-related inflammatory pathways without the risk of drug resistance. This targeted drug therapy is fundamentally different from existing non-specific anti-inflammatory treatments. 2. Excellent stability: Combined with LNP delivery, 6mA modification can enhance the recognition and binding ability of ssDNA sequence to MYH9 protein, effectively resisting in vivo nuclease degradation, improving the cellular uptake efficiency and in vivo stability of DNA sequence, overcoming the defects of ordinary DNA being easily degraded and having low cellular uptake rate, thereby enhancing the in vivo anti-septic effect. 3. Clear Efficacy: Animal experiments have demonstrated that the 6mA-modified ssDNA sequence of this invention, after being delivered into the body via LNP and recognized and bound by the MYH9 protein, promotes macrophage polarization towards the M2 type. In a sepsis mouse model, it can significantly improve the survival rate of mice, reduce spleen organ damage, upregulate the expression level of serum anti-inflammatory factor IL-10, inhibit the expression level of serum pro-inflammatory factor IL-6, and effectively improve physiological phenotypes such as weight loss and abnormal body temperature in sepsis mice. It has significant potential for the treatment of sepsis and provides a new candidate molecule for the treatment of sepsis. 4. High practicality: The DNA sequence synthesis process of this invention is mature, simple, and can be prepared on a large scale. It can be combined with LNPs and pharmaceutically acceptable carriers to form drug compositions, which are easy to prepare into drug formulations such as injections and lyophilized powders. They are easy to store and administer, meet clinical drug administration needs, and are suitable for clinical translation and application. Attached Figure Description

[0022] Figure 1 This is a diagram verifying the specific binding of the 6mA-modified ssDNA sequence of this invention to the MYH9 protein; p <0.0001; Figure 2 This invention relates to the effect of a 6mA-modified ssDNA sequence on macrophage polarization in vitro. Figure 3 The effects of the 6mA-modified ssDNA sequence of this invention on peritoneal macrophage polarization in vivo and the expression level of the anti-inflammatory factor IL-10 in mouse serum were investigated. Figure 4 The present invention enables the 6mA-modified ssDNA sequence to be specifically recognized by the MYH9 protein, thereby regulating macrophage polarization by activating the downstream signaling pathway mediated by the MYH9 protein (JAK1-STAT6 pathway); Figure 5 The effect of the 6mA modified ssDNA sequence of this invention on spleen organ damage in septic mice (HE-stained pathological section, ×200, scale bar = 50μm). Figure 6 This invention relates to the effect of 6mA-modified ssDNA on the levels of pro-inflammatory / anti-inflammatory factors in the serum of septic mice. Figure 7 The present invention relates to the effects of 6mA modified ssDNA sequence on the survival rate and physiological phenotype (body temperature / body weight) of septic mice. Detailed Implementation

[0023] The specific embodiments listed in this invention are merely examples, and the invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of this invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of this invention should be covered within its scope. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments whose manufacturers are not specified are commercially available conventional products. To better illustrate this invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this invention can be practiced even without certain specific details. In other embodiments, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of this invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0025] The experimental animals involved in the following examples are specifically as follows: SPF grade 6-8 week old male C57BL / 6N mice, weighing 20-25g, purchased from Sichuan Vital River Laboratory Animal Technology Co., Ltd., Experimental Animal Production License No.: SCXK (Chuan) 2023-0040. The feeding conditions are: 5 mice are housed per cage, the temperature of the feeding environment is controlled at 22±2℃, the humidity is controlled at 50%±15%, a 12 h / 12 h circadian rhythm is adopted, and the mice can freely access food and water. All experiments involving mice were approved by the Experimental Animal Management Ethics Committee of the Institute of Blood Transfusion, Chinese Academy of Medical Sciences, and the handling of mice and all experimental operations were carried out in strict accordance with the specifications of the ethical review requirements for experimental animals.

[0026] The features and performances of the present invention are further described in detail below with reference to the examples.

[0027] Example 1: Synthesis and modification of 6mA-modified DNA sequences Sequence synthesis: According to the sequence shown in SEQ ID NO: 1 (6mA 1st ): 5'-GCCACCACTTACTACCTTCCTCTAAG6mAATTGCCCTTTAACTCATCACGCTAACATCGCCG-3'; the sequence shown in SEQ ID NO: 2 (6mA 2nd ): 5'-GCCACCACTTACTACCTTCCTCTAAGA6mATTGCCCTTTAACTCATCACGCTAACATCGCCG-3'; the 6mA-modified DNA sequences and unmodified DNA sequences used in this patent are all entrusted to a professional biotechnology company (Sangon Biotech (Shanghai) Co., Ltd.) for synthesis. The synthesis process is strictly carried out in accordance with the preset nucleotide sequences and 6mA modification site requirements. The company performs quality control on the synthesized sequences through mass spectrometry detection, verifying that the synthesized sequences and modifications are completely consistent with the design, with a purity of ≥99.95%. The sequences that have passed the quality inspection are dissolved in Ultra-ddH2O and stored at -80℃ for later use.

[0028] Example 2: Verification of specific recognition and binding between 6mA-modified ssDNA and MYH9 protein To verify the specific binding of the sequence shown in SEQ ID NO: 2 of the present invention to the MYH9 protein, the present invention adopts EMSA, Biotin DNA pulldown WB and MYH9-DNA IP for multi-dimensional verification respectively.

[0029] 1. Experimental methods (1) Prokaryotic expression and purification of recombinant fusion protein GST-MYH9-His6 The pGEX-6p-1 recombinant vector containing the mouse MYH9 coding sequence (NM_022410.4), which was verified by sequencing, was transformed into Transetta (DE3) competent cells; single colonies were picked and inoculated into LB medium containing ampicillin for activation overnight; 10 mL of the activated bacterial culture was expanded into 1 L of LB medium containing ampicillin and cultured at 37°C with shaking until OD. 600 =0.6, cooled to 16℃, then IPTG was added to a final concentration of 1 mM, and the mixture was cultured at 16℃ with shaking for 16 hours to induce protein expression.

[0030] The bacterial culture was collected by centrifugation. After washing the precipitate with PBS, it was resuspended in buffer T (20 mM Tris-HCl, pH 8.0, 50 mM NaCl), sonicated on ice, and the supernatant was collected by centrifugation. The supernatant was incubated with Glutathione Sepharose 4B beads (GE17-0756-01, Cytiva) at 4°C for 3 hours. The beads were washed sequentially with high-salt buffer T, buffer T, and gradient L-glutathione pre-elution buffer, and then eluted four times with 20 mM L-glutathione elution buffer. The eluent was collected and dialyzed to remove L-glutathione.

[0031] After dialysis, the sample was incubated with Ni-NTA His-bind beads at 4°C for 3 hours, washed sequentially with PBS and gradient imidazole pre-elution buffer, and eluted 4 times with 250 mM imidazole elution buffer. The eluent was collected and dialyzed to remove imidazole. All buffers were mixed with protease inhibitors before use.

[0032] (2) EMSA detection of the binding specificity of 6mA-modified ssDNA to recombinant GST-MYH9-His6 fusion protein 50 nM biotin-labeled 6mA modified ssDNA probes and dsDNA probes (negative control) were taken separately, and recombinant MYH9 protein at concentration gradients of 0, 50, and 150 nM was added. The mixture was then topped up to 20 μL with EMSA binding buffer (100 mM Tris, 500 mM KCl, 10 mM DTT, 2.5% glycerol, 5 mM MgCl2, pH 7.5) and incubated at room temperature for 30 minutes to form a complex. The sample was loaded onto an 8% TBE non-denaturing gel and electrophoresed at 100 V for 60 minutes. The gel was then transferred to a membrane (380 mA, 30 minutes), cross-linked twice under 1500 mJ / cm² UV light, and observed for migration arrest bands after Western blotting.

[0033] (3) Biotin DNA pulldown assay to detect the binding specificity of the 6mA-modified ssDNA sequence to endogenous MYH9 protein in cells. Take 5 μg of control ssDNA (Ctrl) containing 5'-Biotin labeling and ssDNA sequence modified with 6mA (6mA), respectively. 1st The virus was transfected into iBMDM cells cultured in 150 mm cell culture dishes. Two hours after transfection, the cells were washed three times with pre-chilled PBS buffer containing 1 mM PMSF (product model: ST505, manufacturer: Beyotime), followed by two UV crosslinking treatments at an intensity of 1500 mJ / cm². The treated cells were collected and lysed, and the protein concentration of the lysate was adjusted to 2.5 mg / mL for later use.

[0034] The DNA pulldown procedure is as follows: Take 10 μL of streptavidin magnetic beads (product model: 65002, manufacturer: Invitrogen), and block them with IP blocking solution (composed of TE buffer containing 0.2 mg / mL sonicated salmon sperm DNA and 0.5 mg / mL BSA) at 4°C for 1 hour; add the blocked magnetic beads to the prepared cell lysis buffer and gently rotate and incubate at 4°C for 30 minutes.

[0035] After incubation, wash the magnetic beads six times with RIPA buffer and once with TE buffer. Add 40 μL of 1.3× NuPAGE™ LDS buffer to the washed magnetic beads and boil at 95°C for 10 minutes to obtain the supernatant. Use the obtained supernatant for immunoblotting experiments. Note that a protease inhibitor mixture must be added to the RIPA buffer before use.

[0036] (4) Detection of the binding specificity of MYH9-DNA IP to 6mA-modified ssDNA sequence by real-time quantitative PCR (RT-qPCR). Take 5 μg of unmodified control DNA (Ctrl) and 6mA modified DNA (6mA) respectively. 1st The virus was transfected into iBMDM cells cultured in 150 mm cell culture dishes. Two hours after transfection, the cells were washed three times with pre-cooled PBS buffer containing 1 mM PMSF.

[0037] After washing, cells were fixed with 1.1% formaldehyde (product model: F8775, manufacturer: Sigma-Aldrich) and crosslinked at 37°C for 15 minutes. Then, 2.5M glycine (2 / 5 volume ratio) was added to terminate the crosslinking, and the cells were incubated at room temperature for 5 minutes. After fixation, the cells were washed three times with pre-chilled PBS buffer containing 1 mM PMSF. Cells were then collected and lysed using lysis buffer (containing 1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1, a mixture of protease inhibitors, and 1 mM DTT).

[0038] Take an equal volume of protein sample and dilute it with dilution buffer (containing 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl) to a protein concentration of 2.5 mg / mL for later use.

[0039] Take 20 μL of protein A agarose beads (product model: AS046, manufacturer: Abclonal), wash and centrifuge them, then mix them with the prepared samples above and incubate at 4°C for 1 hour. After incubation, centrifuge the mixture and transfer the supernatant to a new centrifuge tube; take 1 / 100 of the supernatant volume as the input sample, add 2.5 μg of anti-MYH9 antibody (product model: 81204-1-RR, manufacturer: Proteintech) to the remaining supernatant, and incubate overnight at 4°C.

[0040] Take 5 μL of protein G magnetic beads (product model: 10004D, manufacturer: ThermoFisherScientific), wash and centrifuge them, and add them to the sample that has been incubated overnight. Incubate at 4°C for 2 hours by rotation.

[0041] After incubation, the oligonucleotide complex bound to the magnetic beads was washed with 1 mL of the corresponding buffer for 5 minutes at 4°C each time. The washing sequence was as follows: wash once with low-salt washing buffer (containing 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl); wash once with high-salt washing buffer (containing 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl); wash once with LiCl washing buffer (containing 0.25 M LiCl, 1% IGEPAL-CA630, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris, pH 8.1); and wash twice with TE washing buffer.

[0042] After washing, add 100 μL of elution buffer (containing 0.1 M NaHCO3 and 1% SDS), incubate by rotation for 20 minutes, repeat elution twice, and collect the supernatant. Add 20 μL of 5 M NaCl to the supernatant and incubate at 50 °C for 8 hours; then add 1 μL of proteinase K (concentration 10 mg / mL, product model: EO0492, manufacturer: ThermoFisher Scientific), 4 μL of 0.5 M EDTA, and 8 μL of 1 M Tris-HCl (pH 6.8), and incubate at 45 °C for 2 hours.

[0043] The DNA was purified using a ChIP DNA purification and concentration kit (product model: D5025, manufacturer: ZYMO RESEARCH) according to the manufacturer's instructions. The DNA was eluted with 20 μL of elution buffer, followed by qPCR detection. In the MYH9-DNA-IP qPCR experiment, the calculation method differed from the previous one; a percentage-based method was used, as shown in the following formula: %Input = 2^[Ct(Input) - Ct(IP) - log2(dilution factor)] × 100% In the formula, %Input represents the proportion of target DNA fragments enriched after immunoprecipitation with the target antibody to the initial total chromatin Input; Ct(Input) and Ct(IP) are the Ct values ​​of the Input sample and the antibody IP-precipitated sample, respectively, and the Ct values ​​are the technical mean of repeated samples. The result is converted into a percentage relative to Input. The higher the %Input value, the stronger the binding and enrichment ability of the MYH9 protein for 6mA modified DNA sequences.

[0044] 2. Experimental Results The purified recombinant fusion protein GST-MYH9-His6 was determined to have a protein concentration of 50 ng / μL using a qubit3 assay (manufacturer: Thermo Fisher Scientific). Figure 1 As shown in Figure A, the purified GST-MYH9-His6 fusion protein was identified by SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining, and the purity met the experimental requirements.

[0045] If the incubation group of MYH9 fusion protein with 6mA-modified ssDNA labeled with the Biotin probe shows a migration arrest band that increases with increasing protein concentration, while the negative control and blank control groups show no band, this indicates that the two proteins specifically bind. Figure 1 EMSA results in lane B showed that lanes 1-3 contained probe-labeled 6mA-modified single-stranded DNA (6mA). 1st ssDNA (50 nM) was incubated with MYH9 fusion protein at different concentration gradients (0, 50, 150 nM) (the migration retardation bands were significantly enhanced with increasing MYH9 protein concentration). Lanes 4-6 contained probe-labeled 6mA modified double-stranded DNA (6mA). 1st The 6mA modified ssDNA sequence of the present invention was incubated with the MYH9 fusion protein at the above concentration gradient (no migration arrest band). This demonstrates that the 6mA modified ssDNA sequence of the present invention can directly and specifically bind to the MYH9 fusion protein.

[0046] like Figure 1 The DNA pulldown Western blotting results in C showed that the 6mA-modified ss DNA labeled with biotin could effectively enrich MYH9 protein from cell lysates, while the unmodified control DNA could only enrich weak or almost undetectable MYH9 protein bands; Figure 1 The MYH9-DNA IP results in D showed that the MYH9 protein antibody significantly enriched 6mA-modified ssDNA, while the enrichment level for unmodified DNA was extremely low, and no obvious enrichment signal was observed in the negative control group. Both forward and reverse affinity enrichment experiments demonstrated that the MYH9 protein can still specifically recognize and bind to specific 6mA-modified ssDNA sequences in the intracellular environment.

[0047] The above experiments confirm from both in vitro and in vivo perspectives that the 6mA-modified ss DNA sequence described in this invention can be specifically recognized and bound by the MYH9 protein, and that this binding depends on a specific 6mA modification on the DNA sequence.

[0048] Example 3: The regulatory effect of 6mA-modified ssDNA on the expression of inflammatory factors and macrophage polarization phenotype after being recognized and bound by the MYH9 protein. To verify that our designed 6mA-modified ssDNA sequence affects macrophage immune phenotype after being specifically recognized by MYH9, we conducted in vitro and in vivo cell experiments.

[0049] 1. Experimental Methods (1) The human HEK293T cell line and the mouse iBMDM cell line were purchased from a reputable cell bank. The cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution.

[0050] The isolation and culture steps for mouse peritoneal macrophages (PMs) are as follows: Mice were euthanized by cervical dislocation, and mouse peritoneal macrophages (PMs) were isolated from their peritoneal cavity using 3 mL of PBS buffer. After 8 hours of culture, non-adherent cells were removed; adherent cells were reseeded with growth medium and cultured overnight for subsequent transfection and other treatments.

[0051] (2) Cell transfection: One day before transfection, seed cells in 12-well plates and culture until confluence reaches 70%-80%; following the instructions of the Lipofectamine RNAiMAX transfection reagent (catalog number 13778-150, Invitrogen), the transfection conditions are as follows: For iBMDM cells, transfect 50 ng of 6mA-modified (6mA) 1st / 6mA 2nd ) or unmodified ssDNA sequence (Ctrl), after 4 hours of transfection, cells were collected to extract RNA, and qPCR was used to detect M2 type markers in cells. Arg1 mRNA expression levels; for primary peritoneal macrophages (PMs), transfection with 50 ng 6mA-modified (6mA) mRNA... 1st The cells were transfected with either ssDNA sequence (Ctrl) or unmodified ssDNA sequence. After 48 hours, the cell pellet was collected, and the expression ratio of CD206 positive cells, an M2 marker, on PMs was detected by flow cytometry.

[0052] (3) In vivo delivery: LNP-only treatment group (LNP), LNP-wrapped unmodified DNA treatment group (LNP+Ctrl), LNP-wrapped 6mA modified DNA treatment group (LNP+6mA) 1st The final concentration in the packaging was adjusted to 80 μg / mL. The product was administered intraperitoneally at a dose of 0.5 mg / kg. Anesthesia was performed 24 hours later, and blood was collected from the tail vein to obtain serum. Simultaneously, peritoneal fluid was collected and centrifuged (400g, 5min) to collect PMs. Flow cytometry was used to detect M2 type (CD11b) peritoneal macrophages. + F4 / 80 + CD206 + The expression ratio of IL-10 was determined by ELISA, and the expression level of IL-10 in serum was detected.

[0053] (4) qPCR detection Arg1 Expression level In step (2) of this embodiment, after the iBMDM cell transfection treatment was completed, Ctrl and 6mA were collected respectively. 1st and 6mA 2nd RNA was extracted from samples in three treatment groups, and cDNA was synthesized by reverse transcription to serve as a template for detection by qPCR. Arg1 The transcriptional level was determined, and the detection primers used are shown in Table 1. Two methods were employed. Δ ΔCt Method calculation Arg1 The relative expression level, in Gapdh Normalize the internal reference gene.

[0054] Table 1. qPCR primers for Example 3

[0055] (5) Flow cytometry detection of CD206 expression level The PMs transfected with DNA sequences collected in step (2) and the PMs obtained after in vivo LNP delivery of DNA sequences collected by centrifugation of ascites fluid in step (3) were processed separately: washed once with PBS, then blocked with purified anti-mouse CD16 / 32 antibody (catalog number 101302, BioLegend) and stained with M2 type (CD11b). + F4 / 80 + CD206 + Antibody; data were acquired using a Beckman DxFlex flow cytometer and analyzed using FlowJo software (v10.8.1).

[0056] (6) Serum IL-10 level detection Serum samples were collected from the four groups of mice in this embodiment 24 hours after DNA sequence delivery via LNP in vivo, and IL-10 levels were detected by ELISA.

[0057] 2. Experimental Results like Figure 2 As shown, after in vitro transfection with iBMDM, compared with transfection with unmodified ssDNA sequence (Ctrl group), only 6mA was transfected. 1st The experimental group, Arg1 The mRNA expression level was significantly increased ( p <0.0001, Figure 2 A) After in vitro transfection of PMs, 6 mA was transfected.1st Experimental group M2 (CD11b) + F4 / 80 + CD206 + The proportion of positive cells increased significantly. p <0.01, Figure 2 B).

[0058] like Figure 3 As shown, in step (3), mice were intraperitoneally injected with physiological saline, empty LNP vector, LNP+Ctrl, or LNP+6mA. 1st Subsequent PMs were collected, and flow cytometry results showed that LNP+6mA was transfected. 1st The experimental group, CD11b + F4 / 80 + CD206 + The positive rate increased more significantly, while the above indicators in the negative control group were not significantly different from those in the LNP control group. p >0.05); ELISA detection showed that the serum anti-inflammatory factor IL-10 level in this group was significantly higher than that in other groups ( p <0.001).

[0059] The above experimental results demonstrate that after 6mA-modified ssDNA is bound by MYH9 protein, it can effectively promote macrophage polarization to the M2 type and inhibit inflammatory response. Moreover, this regulatory effect depends on the binding of MYH9 protein, clarifying that this regulatory effect is the core mechanism for intervening in sepsis. At the same time, LNP delivery can effectively improve the cellular uptake efficiency of DNA, ensuring that it can play a regulatory role.

[0060] Example 4: 6mA-modified ssDNA activates the downstream signaling pathway JAK1-STAT6 after being recognized by the MYH9 protein, thus affecting macrophage polarization phenotype. To verify that our designed 6mA-modified ssDNA sequence is specifically recognized by MYH9 and affects macrophage immune phenotype through the JAK1-STAT6 signaling pathway.

[0061] 1. Experimental Methods (1) Construction Myh9 Knockdown iBMDM cell lines: Highly efficient and experimentally validated cell lines screened from the Sigma website. Myh9The short hairpin RNA (shRNA) sequence was obtained; this sequence was constructed into the pLKO.1-Puro vector using T4 ligase to obtain a recombinant plasmid, which was then transformed into Trans1-T1 competent cells (product model: CD501, manufacturer: Transgen). The recombinant plasmid and lentiviral helper plasmids (psPAX2, pMD2G) were co-transfected into 293T cells using polyethyleneimine (PEI); 48 hours after transfection, the cell supernatant containing lentivirus was collected and filtered. The filtered lentiviral supernatant was co-cultured with iBMDM cells, followed by selection with 3 μg / mL puromycin for 1 week to obtain... Myh9 The iBMDM cell line with stable gene knockdown was used. The gene knockdown efficiency of this cell line was detected by RT-qPCR and Western blotting.

[0062] (2) Western blot analysis of the effect of 6mA-modified ssDNA transfection on JAK1 and STAT6 expression in iBMDM cell lines Proteins were extracted from cell lysates using RIPA lysis buffer (ab156034, Abcam) containing a mixture of 1% protease inhibitors (product model: B14001, manufacturer: Selleck), a mixture of phosphatase inhibitors (product model: P003, manufacturer: NCM Biotech), and 1 mM DTT (product model: R0861, manufacturer: ThermoFisher Scientific). The extracted proteins were separated by electrophoresis on a 4-20% SDS-PAGE gel and transferred to a PVDF membrane; subsequently, Western blotting was performed on the membrane for protein immunoblotting. The antibodies used for the Western blotting experiment are as follows: MYH9 (81204-1-RR, Proteintech, 1:10000), STAT6 (51073-1-AP, Proteintech, 1:1000), pSTAT6(Tyr641) (AP0456, Abclonal, 1:500), JAK1 (A11963, Abclonal, 1:1000), pJAK1(Tyr1034 / 1035) (74129, Cell Signaling Technology, 1:500), were modified with 6mA of ssDNA (6mA). 1st ) and control unmodified ssDNA (Ctrl) transfection of wild-type iBMDM and Myh9Gene knockdown cells were used, with a blank control group included. Total protein was extracted from the cells after the above treatment, and Western blotting was used to detect the phosphorylation levels of JAK1 protein at Y1034 / 1035 and STAT6 protein at Y641. Myh9 The effect of gene knockdown on the phosphorylation of both.

[0063] 2 Experimental Results The JAK1-STAT6 signaling pathway is a crucial pathway for M2 macrophage polarization, primarily activated through protein phosphorylation, ultimately regulating the transcription of M2 macrophage-related genes. For example... Figure 4 As shown, 6mA 1st In the treatment group, the phosphorylation level of JAK1 protein at sites Y1034 / 1035 was significantly upregulated, while the Ctrl treatment group showed no significant response; JAK1 protein activation can activate downstream STAT6 protein, which, after 6mA... 1st Following DNA treatment, the phosphorylation level at the Y641 site of the STAT6 protein also significantly increased, suggesting that 6mA... 1st DNA can regulate the phosphorylation of JAK1 and its downstream protein STAT6 through MYH9; the verification results show that... Myh9 Gene knockdown completely eliminated 6mA-modified ssDNA-induced phosphorylation of JAK1 and STAT6 proteins. These experimental results indicate that MYH9 is an indispensable key factor in the 6mA-modified ssDNA-induced phosphorylation of JAK1 and STAT6 proteins.

[0064] Example 5: In vivo antiseptic effect of 6mA-modified ssDNA sequence 1. Experimental Methods (1) Grouping of experimental animals Eight-week-old male C57BL / 6 mice (weighing 20 kg) were used. 25g), divided into the non-modeling group (saline injection group), the modeling group (LPS+LNP injection), and the model treatment group (LNP+Ctrl, LNP+6mA). 1st Mice were randomly divided into groups of 10.

[0065] (2) Construction of sepsis model and drug administration After 7 days of acclimatization to the four groups of mice in this embodiment, three groups were randomly selected to induce a mouse sepsis model by intraperitoneal injection of lipopolysaccharide (LPS) combined with LNP. The LPS dose used was 10 mg / kg. One hour after modeling, two groups of modeling mice were randomly selected and intraperitoneally injected with LNP+Ctrl and LNP+6mA, respectively. 1stTreatment was administered; according to the LNP packaging DNA sequence final concentration of 80 μg / mL as described in Example 3, the injection dose was 0.5 mg / kg, and the other group was injected with an equal volume of physiological saline.

[0066] (3) Serum IL 6. IL-10 level detection Serum samples were collected from the four groups of mice in this example 24 hours after infection, and IL-1 levels were detected by ELISA. 6. IL-10 level.

[0067] (4) Statistics on changes in body weight and body temperature and survival curves During the LPS infection treatment period, the weight, body temperature and mortality of mice in each group of this example were recorded daily, and the changes in weight, body temperature and survival score of mice in each group were statistically analyzed.

[0068] (5) Pathological examination After the experiment, spleen tissues from mice in each group of this example were collected and fixed in 4% paraformaldehyde (PFA) solution. After removing the PFA fixative, the samples were placed in 30% sucrose solution and incubated overnight at 4°C. The mouse spleen tissues were frozen and embedded, and cut into 10-micrometer-thick sections. The sections were stained with HE according to the instructions of the hematoxylin-eosin (HE) staining kit (catalog number G1120, Solarbio).

[0069] 2. Experimental Results A mouse sepsis model was constructed using lipopolysaccharide (LPS) induction. The model included a saline control group (Saline), an LPS model control group (LPS+LNP), a control DNA (unmodified) treatment group (LPS+LNP+Ctrl), and a 6mA modified DNA treatment group (LPS+LNP+6mA¹). st The results of the comprehensive evaluation of the mouse spleen histopathological morphology, serum inflammatory factor levels, survival status, and changes in body temperature / body weight are as follows: (1) Histopathological analysis of spleen tissue like Figure 5 As shown: Saline group: The spleen tissue structure was intact, the boundary between the white pulp and the red pulp was clear, the lymphocytes were densely and regularly arranged, and there was no inflammatory infiltration or structural damage. The white pulp (lymphoid follicles) is shown in the figure: a dark purple-blue, round / oval area of ​​densely packed lymphocytes; the red pulp is shown in the figure: a light pink area between the follicles.

[0070] LPS+LNP group: The original intact round lymphoid follicles atrophied and fused on a large scale, and the white pulp boundary became blurred and disappeared; the whole tissue was congested with deep red blood on a large scale, the red pulp was severely congested and expanded, and the red and white pulp structure was disordered.

[0071] LPS+LNP+Ctrl group: atrophy and reduced number of white pulp lymphoid follicles, large areas of red pulp congestion and infiltration, complete blurring of the boundary between red and white pulp, and destruction of the spleen's immune structure.

[0072] LPS+LNP+6mA¹ st Group: Splenic damage was significantly less than that in the first two LPS control groups. Multiple well-formed, deep purple white pulp (lymphoid follicles) with clear outlines were observed. The extent and degree of congestion and infiltration of the red pulp were significantly reduced, the boundary between the red and white pulp was restored, and the splenic structure was protected.

[0073] (2) Serum inflammatory factor level analysis The levels of pro-inflammatory cytokine IL-6 and anti-inflammatory cytokine IL-10 in mouse serum were detected using ELISA. IL-6 is a marker pro-inflammatory cytokine routinely used in clinical practice for early diagnosis, disease stratification, and efficacy assessment of sepsis. After the onset of sepsis, IL-6 is rapidly and massively released into the bloodstream, and its serum concentration is positively correlated with systemic inflammatory storm, organ damage, and mortality risk, making it a core objective indicator for evaluating the severity of sepsis. IL-10 is a key endogenous anti-inflammatory factor used to evaluate the body's inflammatory compensation and anti-inflammatory response capabilities.

[0074] The test results are as follows: Serum pro-inflammatory factor IL-6 test results (e.g.) Figure 6 As shown in A): Compared with the saline control group, the IL-6 concentration in the LPS+LNP group was significantly increased (approximately 2000–3500 pg / mL); the IL-6 level in the LPS+LNP+Ctrl group was significantly higher than that in the LPS+LNP group. p <0.01), suggesting that control DNA could further aggravate the inflammatory response; while LPS+LNP+6mA¹ st The IL-6 concentration in the group was significantly reduced to near baseline levels, and significantly lower than that in the LPS+LNP group and the LPS+LNP+Ctrl group. p The difference was <0.0001), which was not significantly different from the saline control group, indicating that the 6mA-modified ssDNA of the present invention can effectively inhibit the excessive pro-inflammatory response induced by sepsis.

[0075] Serum anti-inflammatory factor IL-10 test results (e.g.) Figure 6As shown in B): Compared with the saline group, the serum IL-10 concentration in the LPS+LNP group and the LPS+LNP + Ctrl control group was significantly increased (approximately 3000–4000 pg / mL). p <0.0001); Although both groups showed compensatory increases in IL-10, the intensity of the inflammatory stimulation far exceeded the body's own anti-inflammatory compensatory capacity. The mice still showed severe systemic inflammation, destruction of spleen immune tissue, and rapid death. Simple low-amplitude increases in IL-10 could not reverse sepsis damage.

[0076] LPS+LNP +6mA¹ st The serum IL-10 concentration in the experimental group was significantly increased to approximately 5000 pg / mL, which was significantly higher than that in the LPS+LNP group and the LPS+LNP+Ctrl control group. p <0.0001). Compared with the compensatory increase in IL-10 in the model control group, the 6mA modified DNA of this invention can induce the body to produce higher levels of the anti-inflammatory factor IL-10. Sufficient IL-10 can effectively antagonize excessive inflammatory response, correct the imbalance of pro-inflammatory / anti-inflammatory factors in sepsis, and ultimately alleviate organ damage and improve the survival rate of mice.

[0077] (3) Survival Analysis Figure 7 As shown in A: LPS + LNP group: All mice died within 2 days of model establishment; In the LPS+LNP+Ctrl group, almost all mice died within one day, indicating that unmodified ssDNA (Ctrl) produces a synergistic pro-inflammatory effect, further exacerbating the fatal damage caused by sepsis.

[0078] LPS+LNP+6mA¹ st Group: Under the same N / P=12 LNP delivery system, the number of mouse deaths decreased significantly after the introduction of 6mA methylated ssDNA: only a small number of individuals died within 2–4 days, and the 8-day endpoint survival rate was approximately 60%; the difference was significant compared with the LPS+LNP group and the LPS+LNP+Ctrl group. p <0.01).

[0079] (4) Analysis of changes in body temperature and weight Figure 7 As shown in B: LPS+LNP group: Body temperature dropped sharply, with the lowest value being only about 32.8℃, which was the lowest body temperature among the four groups, indicating that LPS induced systemic inflammatory shock; all mice in this group died within 2 days, which corresponds to the phenotype of severe hypothermia.

[0080] The LPS+LNP+Ctrl group exhibited severe hypothermia and extremely strong inflammatory damage, leading to the rapid death of all mice. This demonstrates that the combined use of unmodified DNA synergistically exacerbates septic shock.

[0081] LPS+LNP+6mA¹ st The body temperature drop in the group was significantly smaller than that in the LPS+LNP group and the LPS+LNP+Ctrl group, and the lowest body temperature was maintained above 34℃. One day after modeling, the body temperature continued to rise steadily, and no severe hypothermia of shock level occurred throughout the process, indicating that 6mA modified nucleic acid can significantly inhibit excessive inflammation and alleviate hypothermic injury caused by sepsis.

[0082] Figure 7 As shown in C: LPS+LNP group and LPS+LNP+Ctrl group: All mice died within a short period of time, and weight data were only available for the first 2 days.

[0083] LPS+LNP+6mA¹ st Group: After modeling, the body weight decreased in stages, but after hitting bottom on the 3rd to 4th day, it continued to rise, indicating that the body's inflammation and consumption were relieved and that it had the ability to repair damage.

[0084] The results, based on survival curves, body temperature, and body weight, confirm that in the N / P=12 LNP delivery system, only 6mA methylated ssDNA effectively alleviates LPS-induced severe sepsis damage. Unmodified nucleic acids combined with LNP offer no protective effect and instead exacerbate inflammatory shock and death in animals. This example uses an N / P=12 LNP system for comparative validation. Under this N / P condition, both blank LNP and LNP loaded with unmodified ssDNA exhibit significant pro-inflammatory and lethal effects, maximizing the efficacy difference brought about by 6mA modification and more directly highlighting the decisive role of 6mA modification in the anti-inflammatory activity of nucleic acids.

[0085] The above results indicate that 6mA-modified ssDNA significantly improves the survival outcome of septic mice, reduces body temperature fluctuations and weight loss, alleviates pathological damage to spleen tissue, and effectively reduces the level of pro-inflammatory cytokine IL-6 and increases the level of anti-inflammatory cytokine IL-10, thus correcting the inflammatory imbalance. These results fully demonstrate that the 6mA-modified ssDNA sequence described in this patent (essentially a DNA aptamer-like nucleic acid molecule) has a significant protective effect in the intervention and treatment of sepsis and possesses promising clinical application prospects.

[0086] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. A 6mA-modified single-stranded DNA molecule that specifically binds to the MYH9 protein, characterized in that, The ssDNA molecule with the sequence shown in SEQ ID NO:1 has a 6mA methylation modification on the first adenine residue in the "GAATTGCCCT" part of the sequence.

2. The application of the 6mA-modified single-stranded DNA molecule that specifically binds to MYH9 protein as described in claim 1 in the preparation of antiseptic drugs.

3. A pharmaceutical composition, characterized in that, This includes a 6mA-modified single-stranded DNA molecule that specifically binds to the MYH9 protein, as described in claim 1.

4. The pharmaceutical composition according to claim 3, characterized in that, It also includes delivery carriers or pharmaceutically acceptable excipients.

5. The pharmaceutical composition according to claim 4, characterized in that, The delivery carrier is a lipid nanoparticle, extracellular vesicle, polymer carrier, or inorganic nanocarrier.

6. The pharmaceutical composition according to claim 5, characterized in that, It also includes pharmaceutically acceptable carriers.

7. The pharmaceutical composition according to claim 6, characterized in that, Pharmaceutically acceptable carriers include physiological saline or phosphate buffer.

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