Controllable DNA self-assembly immunostimulatory system and application thereof

Abstract

The present application relates to a controllable DNA self-assembly immune stimulation system and application thereof, the DNA self-assembly immune stimulation system comprises: DNA self-assembly immune stimulation sequence and inhibitory element;The DNA self-assembly immune stimulation sequence includes domain P1 and domain P2, the domain P1 and domain P2 are palindromic sequence, the base number of the domain P1 and domain P2 is between 12-45nt;The inhibitory element includes B domain or CR-ODN 2P chain;The DNA self-assembly immune stimulation sequence and domain B are connected by at least one enzyme cutting site, modifiable site or chemical bond to realize inhibitory effect;The base sequence of the CR-ODN 2P chain is completely complementary to the DNA self-assembly immune stimulation sequence and forms DNA and RNA hybridization complex to realize inhibitory effect.The system of the present application can accurately control the activation of cGAS-STING pathway.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and medicine, and more particularly to a controllable DNA self-assembly immunostimulation system and its applications, specifically a DNA self-assembly immunostimulation system. Background Technology

[0002] The cyclic GMP-AMP synthase (cGAS)–interferon gene stimulating factor (STING) pathway is an important innate immune pathway that plays a crucial role in regulating the immune response. cGAS is the core component of the cGAS–STING pathway, recognizing dsDNA in the cytoplasm and catalyzing the synthesis of the second messenger 2',3'-cGAMP. cGAMP binds to the STING protein, activating the STING signaling pathway, inducing phosphorylation of STING and TANK-binding kinase 1 (TBK1), ultimately leading to the activation of transcription factors IRF3 and NF-κB, promoting the production of type I interferon and other inflammatory factors. The cGAS–STING immune pathway plays a vital role in maintaining immune homeostasis, resisting pathogen invasion, inhibiting tumor growth, and preventing autoimmune diseases.

[0003] The strict regulation of the cGAS-STING pathway is crucial in the immune system. Moderate activation of this pathway helps clear pathogen infections, recognize and suppress tumor cells, and initiate adaptive immune responses. However, excessive or persistent activation of the cGAS-STING pathway can lead to an overreaction of the immune system, thereby triggering autoimmune diseases or chronic inflammatory diseases and promoting tumor proliferation and invasion.

[0004] Currently, the activation of cGAS-STING by widely studied and used small molecule agonists is irreversible, making it difficult to precisely control its site and duration of action according to treatment needs, and easily triggering a series of adverse reactions in the body.

[0005] Current technologies cannot effectively control the activation of cGAS-STING. Therefore, how to achieve precise regulation of the cGAS-STING pathway has become an urgent problem to be solved. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a controllable DNA self-assembly immunostimulation system and its application. The DNA self-assembly immunostimulation system comprises a DNA self-assembly immunostimulatory sequence and an inhibitory element. The inhibitory element suppresses the self-assembly process of the DNA self-assembly immunostimulatory sequence. Subsequently, the activation of the cGAS-STING pathway is further precisely controlled by controlling the linkage of domain B with the DNA self-assembly immunostimulatory sequence through restriction enzyme sites or chemical bonds, or by controlling the complementary pairing of the CR-ODN 2P chain with the DNA self-assembly immunostimulatory sequence.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a DNA self-assembly immunostimulation system, the DNA self-assembly immunostimulation system comprising: a DNA self-assembly immunostimulation sequence and an inhibitory element;

[0009] The DNA self-assembly immunostimulatory sequence includes domain P1 and domain P2, both of which are palindromic sequences, and the number of bases in domains P1 and P2 is between 12 and 45 nt.

[0010] The suppression element includes a B-domain or a CR-ODN 2P chain;

[0011] The DNA self-assembly immunostimulatory sequence and domain B are linked through at least one restriction site, a modifiable site, or a chemical bond to achieve the inhibitory effect.

[0012] The base sequence of the CR-ODN 2P chain is completely complementary to the DNA self-assembly immunostimulatory sequence to form a DNA-RNA hybrid complex, thereby achieving the inhibitory effect.

[0013] The present invention provides a controllable DNA self-assembly immunostimulation system, comprising a DNA self-assembly immunostimulation sequence and an inhibitory element. When the inhibitory element is a B domain, it is linked to the DNA self-assembly immunostimulation sequence (hereinafter referred to as ODN-2P) through an enzyme cleavage site, a modifiable site, or a chemical bond to construct the AP-H system. Controlling this linkage structure inhibits its self-assembly ability. When the inhibitory element is a CR-ODN 2P chain, the D / R system is constructed. By blocking the hybridization between the RNA chain CR-ODN 2P and ODN-2P, the self-assembly behavior of ODN-2P is inhibited.

[0014] Preferably, the DNA self-assembled immunostimulatory sequence and domain B are linked together through at least one restriction site, a modifiable site, or a chemical bond to form an immunostimulatory monomer.

[0015] Preferably, at least two groups of immunostimulatory monomers are self-assembled by nucleases, oxidoreductases, or oxidoreductases to obtain a controllable DNA self-assembly immunostimulatory system.

[0016] Preferably, the nuclease used to cleave the restriction site includes depurinyl / depyrimidine endonuclease 1.

[0017] In this invention, the degree of control over the DNA self-assembly immunostimulatory system varies in different cells. For example, in cancer cells, the expression level of depurinyl / depyrimidine endonuclease 1 in the cytoplasm is higher than in normal cells. This endonuclease is used as the enzyme cleavage site to activate the immune system, resulting in higher stimulation of the immune system in disease cells and lower activation in normal cells. Therefore, the DNA self-assembly immunostimulatory system can exert a cell-selective effect.

[0018] Preferably, the modifiable site includes DNA thiomodification.

[0019] Preferably, the modifiable site is interrupted by hypochlorous acid or myeloid peroxidase, etc.

[0020] Preferably, the chemical bond includes a disulfide bond.

[0021] Preferably, the chemical bond is broken by either dithiothreitol or glutathione as a reducing agent.

[0022] Preferably, the immunostimulatory monomer removes domain B by nuclease or reducing agent, and after removal, domains P1 and P2 can complement each other to form a double-stranded DNA self-assembly immunostimulatory system.

[0023] The nuclease and reducing reagent provided by this invention can act at the enzyme cleavage site or cleave chemical bonds, causing the ODN-2P and B domains in the AP-H system to be separated. Due to the reduced hybridization stability, the ODN-2P and B domains separate. The released ODN-2P can undergo intermolecular base pairing and then self-assemble in solution to form a long double-stranded DNA molecule, which can activate the cGAS protein, thereby stimulating the cGAS-STING immune pathway.

[0024] Preferably, the domain B is complementary to either domain P1 or domain P2 in the DNA self-assembly immunostimulatory sequence.

[0025] The controllable DNA self-assembly immunostimulation system AP-H provided by the present invention forms a stable hairpin structure through complementary pairing of domain B with domain P1 or domain P2, which prevents intermolecular base pairing and thus loses its self-assembly ability.

[0026] Preferably, the number of complementary base pairs is 5-11 bases. For example, it can be 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, or 11 bases, etc.

[0027] Preferably, the connection between domain P1 and domain P2 in the DNA self-assembly immunostimulatory sequence further includes a photolytic linker.

[0028] Preferably, the photolyzable linker is cleaved under light to achieve photocontrolled inactivation of the DNA self-assembly immunostimulation system.

[0029] The photolytic linker provided by this invention is connected to domains P1 and P2. When light is applied to it, the photolytic linker is cleaved, and a break occurs between P1 and P2. The assembled long double-stranded DNA molecule breaks into short DNA fragments and loses its ability to stimulate the cGAS-STING pathway.

[0030] Preferably, the light includes either ultraviolet light or visible light.

[0031] Preferably, the photolytic linker is selected from any one of photolytic groups or chemical bonds such as o-nitrobenzyl and its derivatives, coumarin, and azo bonds.

[0032] Preferably, the DNA self-assembly immunostimulatory sequence and the CR-ODN 2P chain form a DNA-RNA hybrid complex through complete complementary pairing.

[0033] Preferably, after the complex is cleaved by ribonuclease to break the CR-ODN 2P chain, the P1 and P2 domains in the DNA self-assembly immunostimulatory sequence can complementarily pair to form a double-stranded DNA self-assembly immunostimulatory sequence.

[0034] In this invention, ribonuclease H can cleave the RNA sequence in the DNA / RNA hybrid double strand. Therefore, under the cleavage of ribonuclease H, CR-ODN 2P is degraded, releasing ODN-2P, which then self-assembles to form a long double-stranded DNA molecule, which is used to activate the cGAS-STING pathway.

[0035] In a second aspect, the present invention provides the application of the DNA self-assembly immunostimulation sequence in the controllable DNA self-assembly immunostimulation system described in the first aspect in the preparation of products that activate the cGAS-STING immune pathway.

[0036] Thirdly, the present invention provides the application of the controllable DNA self-assembly immunostimulation system described in the first aspect in the preparation of products that precisely regulate the cGAS-STING immune pathway.

[0037] Compared with the prior art, the present invention has at least the following beneficial effects:

[0038] (1) This invention utilizes endogenous enzymes and exogenous light as control elements to construct a controllable DNA self-assembly immunostimulation system for fine-tuning the cGAS–STING immune pathway. Compared to small molecule drug agonists, this DNA self-assembly immunostimulation system exhibits excellent spatiotemporal controllability in regulating the cGAS–STING pathway. It can precisely regulate the timing, location, and degree of cGAS–STING pathway activation according to specific needs, thereby effectively avoiding the adverse effects of over-activation of this pathway (including inflammatory responses and tissue damage). This provides a reliable solution for medical research on diseases related to this pathway, including the development of tumor immunotherapy and antiviral and antitumor vaccines, as well as the prevention and treatment of immune system diseases.

[0039] (2) The controllable DNA self-assembly immunostimulation system provided by this invention is designed based on the high programmability and modifiability of DNA. Without changing the nucleotide sequence, only different modifying groups need to be introduced. This system can respond to different endogenous and exogenous control factors, thereby achieving controllable regulation of the cGAS-STING pathway and providing a feasible approach for the regulation of the organism's immune system and basic biological pathways. Compared with the traditional screening and development of immunotherapeutic drugs and agonists, this method has the advantages of simplicity, convenience, and versatility. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the design and self-assembly principle of ODN-2P.

[0041] Figure 2 This is a diagram showing the activation results of ODN-2P self-assembly.

[0042] Figure 3 This is a schematic diagram illustrating the activation principle of the controlled DNA self-assembly immune stimulation system (AP-H).

[0043] Figure 4 This is a diagram showing the activation results of the controlled DNA self-assembly immune stimulation system (AP-H).

[0044] Figure 5 This is a diagram showing the activation results of the controlled DNA self-assembly immune stimulation system (AP-H) in cells.

[0045] Figure 6 This is a diagram showing the activation results of the controlled DNA self-assembly immune stimulation system (AP-H) in different cells.

[0046] Figure 7 This is a schematic diagram of the activation principle and activation effect of the controllable DNA self-assembly immunostimulation system (D / R).

[0047] Figure 8 This is a schematic diagram illustrating the activation principle of the controlled DNA self-assembly immune stimulation system (AP-H-PC).

[0048] Figure 9 This is a diagram showing the activation results of the controlled DNA self-assembly immune stimulation system (AP-H-PC).

[0049] Figure 10 This is a diagram showing the activation results of the controlled DNA self-assembly immunostimulation system (AP-H-PC) in cells. Detailed Implementation

[0050] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0051] The various instruments, consumables, and reagents not specifically described in the embodiments of this invention are all conventional commercially available products in the art and can be obtained through commercial means. The specific experimental materials and their sources listed in the following embodiments are merely exemplary and are not intended to limit the invention. Materials that are the same as or similar in type, model, quality, properties, or functions to the following tissues, cells, reagents, and instruments can be used to implement this invention.

[0052] The reagents used in the following examples were sourced from the following sources:

[0053] lipo 3000: Purchased from Thermo Fisher Scientific Inc., USA, part number: L3000015.

[0054] Recombinant human cGAS protein: provided by MedChemExpress, Inc., USA, catalog number: HY-P72337.

[0055] APE1: Purchased from New England Biotechnology Co., Ltd., USA, item number: M0282S.

[0056] Human monocytic leukemia cells: purchased from ATCC, catalog number: CL-0233.

[0057] ELISA kit: purchased from Beijing Solarbio Technology Co., Ltd., catalog number: SEKH-0410-96T.

[0058] RNA extraction kit: purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP451.

[0059] Human skin fibroblasts: from Professor Chen Qi's research group at Fujian Normal University.

[0060] Cytoplasmic protein extraction kit: purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number: P0027.

[0061] RNase H: Purchased from New England Biotechnology Co., Ltd., USA, product number: M0297S.

[0062] Example 1

[0063] This embodiment describes the design and preparation of a controllable DNA self-assembly immunostimulation system.

[0064] Design and preparation of controllable DNA self-assembly immunostimulation systems ODN-2P, AP-H, D / R, and AP-H-PC: ODN-2P was designed as an oligonucleotide containing two palindromic sequences (P1 and P2). Figure 1 This diagram illustrates the design and self-assembly principle of ODN-2P. AP-H, D / R, and AP-H-PC were all modified, extended, and improved based on ODN-2P, with the relevant nucleic acid sequences synthesized by Sangon Biotech (Shanghai) Co., Ltd. and Huzhou Hippo Biotechnology Co., Ltd. In AP-H, the ODN-2P and B domains are connected by a base-free site (AP site), which can be cleaved by apurinyl / pyrimidine endonuclease 1 (APE1), as shown in Table 1. All DNA and RNA powders were first dissolved in sterile, enzyme-free water, and then the absorbance was measured using a UV spectrophotometer to calculate the accurate concentration. All reactions were carried out in Tris-HCl buffer (40 mM Tris-HCl, 100 mM NaCl, and 5 mM MgCl2, pH 7.4).

[0065] Table 1

[0066]

[0067]

[0068] The AP-H sequence is “P1-P2-idSp-B”, specifically TTGAGCGCTCAATC ACTGCAGTGA / idSp / TGAGCGCT.

[0069] The AP-H-PC sequence is “P1-iPCLink-P2-idSp-B”, specifically TTGAGCGCTCAA / iPCLink / TCACTGCAGTGA / idSp / TGAGCGCT.

[0070] Example 2

[0071] This embodiment is used to verify the activation effect of DNA self-assembly immunostimulatory sequences of different lengths on the cGAS-STING pathway.

[0072] (1) Self-assembly detection

[0073] First, the self-assembly ability of ODN-2P was verified. ODN-2P was dispersed in 4 μM Tris-HCl buffer and incubated at 37°C for 1 h. Denaturing and non-denaturing polyacrylamide gel electrophoresis were then used for analysis. (Details follow...) Figure 2 As shown in Figures a and b, ODN-2P is a single oligonucleotide chain that can self-assemble in buffer to generate a series of long double-stranded DNA assemblies. Next, the P1 and P2 domains of ODN-2P were designed with different sequences or lengths to verify their self-assembly ability; the specific sequences are shown in Table 2. Agarose gel electrophoresis was used to verify the self-assembly ability of these ODN-2P sequences or lengths. The results showed that changes in the P1 and P2 domain sequences or lengths of ODN-2P did not affect its self-assembly ability. ODN-2P with altered sequences or lengths could effectively self-assemble into long double-stranded DNA, laying the foundation for its cGAS-stimulated ability.

[0074] Table 2

[0075]

[0076] (2) Activation effect detection

[0077] 4 μM ODN-2P was thoroughly mixed with 200 μM ATP, 200 μM GTP, and 1 μM recombinant human cGAS protein, and heated in a metal bath at 37 °C for 5 h, followed by heating at 95 °C for 10 min to terminate the reaction. No DNA was added to the control group. The products obtained from the reaction were analyzed using high-performance liquid chromatography (HPLC).

[0078] Specific results are as follows Figure 2 As shown in c and d, ODN-2P molecules of different sizes can self-assemble in the buffer solution, effectively activating the cGAS protein and stimulating it to convert the reaction substrates ATP and GTP into the product cGAMP. These results indicate that ODN-2P possesses self-assembly capability, and its self-assembly product can effectively stimulate the cGAS protein to produce small-molecule cGAMP.

[0079] Example 3

[0080] This embodiment is used to verify the activation effect of the DNA self-assembly immune stimulation system (AP-H).

[0081] This embodiment designs a controllable DNA self-assembly immunostimulation system, AP-H. An 8-base domain B, connected by an AP site, extends from ODN-2P. Domain B can complementaryly pair with some bases in the P1 sequence, causing AP-H to form a stable hairpin structure and lose its self-assembly ability. Through the recognition and cleavage of the AP site by APE1, a break occurs between domain B and ODN-2P, releasing ODN-2P, which then self-assembles to produce a long-chain DNA product, thereby activating the cGAS–STING pathway. The specific principle is as follows: Figure 3 As shown.

[0082] (1) Self-assembly detection

[0083] In Tris-HCl buffer, 4 μM of nAP-H and AP-H chains were thoroughly mixed with 0 or 400 U / mL APE1, respectively, and incubated at 37°C for 1 h for digestion. Agarose gel electrophoresis analysis was then performed, and the results are as follows: Figure 4 In the a-type, the AP-H chain containing only the AP site can undergo efficient self-assembly after APE1 cleavage, producing long DNA fragments.

[0084] (2) Activation effect detection

[0085] 4 μM of uncleaved nAP-H and AP-H chains were thoroughly mixed with 200 μM ATP, 200 μM GTP, and 1 μM recombinant human cGAS protein. The mixture was heated in a metal bath at 37 °C for 5 h, followed by heating at 95 °C for 10 min to terminate the reaction. The products were analyzed by high-performance liquid chromatography (HPLC). The results are as follows: Figure 4 As shown in b and c, after the addition of APE1, the AP-H sequence containing the AP site can effectively activate the cGAS protein and promote its catalytic synthesis of cGAMP. Conversely, the H sequence without the AP site cannot activate the cGAS protein.

[0086] Example 4

[0087] This embodiment is used to verify the activation effect of the controlled DNA self-assembly immunostimulation system (AP-H) in cells.

[0088] (1) Cell transfection

[0089] Human monocytic leukemia cells (THP-1 cells) were cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. THP-1 cells were seeded into wells of a plate, and cell adhesion was induced using PMA. After replacing the medium with fresh medium, the cells were cultured for another 24 hours. Then, the cells were transfected with the lipo 3000 transfection reagent to obtain the nAP-H and AP-H nucleic acid sequences, respectively.

[0090] The specific steps are as follows: Dissolve the DNA strand in Optimum Antibody (OPT) serum-free medium, and dissolve Lipo 3000 in another tube of OPT medium. Add the OPT solution containing 400 nM DNA to the solution containing Lipo 3000, with a DNA to Lipo 3000 ratio of 1 μg DNA: 1.5 μL Lipo 3000. Mix thoroughly and incubate at room temperature for 15 min, then add it to THP-1 cells. After incubation for 4 h, discard the cell culture supernatant, replace with 1640 medium, and continue culturing the cells at 37°C for 6 h.

[0091] (2) Activation effect detection

[0092] The cultured cells were lysed using cell lysis buffer, and cGAMP was extracted and detected using an ELISA kit. The results are as follows: Figure 5 In section a, RNA was extracted from cells using an RNA extraction kit and used for RT-qPCR quantitative analysis of the RNA expression levels of specific immune factors (CXCL10 and IFN-β). The results are as follows: Figure 5 b and c in the example.

[0093] Cells were cultured at 37°C for another 16 hours. The cell culture supernatant was collected, and the IFN-β immune factor secreted by the cells was detected using an ELISA kit. The results are as follows: Figure 5 As shown in figure d, AP-H successfully activated the cGAS–STING pathway intracellularly, inducing cGAMP production and an increase in the expression of a series of immune factors. Conversely, the H sequence without the AP site failed to successfully induce activation of the cGAS–STING pathway intracellularly.

[0094] (3) Effect of enzyme content on activation effect

[0095] THP-1 cells were seeded in 6-well plates and induced to adhere using PMA. After replacing the medium with fresh medium, the cells were cultured for another 24 hours. Then, siRNAs knocking down APE1 expression (siRNA1 and siRNA2) were transfected into the cells using the Lipo 3000 transfection reagent at a concentration of 100 nM. After 48 hours, the cells were lysed using a cytoplasmic protein extraction kit, and the cytoplasmic proteins were collected for Western blotting analysis to detect the APE1 expression level in the cytoplasm. The results are as follows: Figure 5 The figure shows that siRNA effectively knocked down APE1 in the cytoplasm of THP-1 cells.

[0096] 48 hours after siRNA transfection, the supernatant was removed, and the cells were re-transfected with ODN-2P and AP-H sequences. After 4 hours of culture, the medium was replaced with fresh medium, and the cells were cultured at 37°C for another 16 hours. The cell culture supernatant was then collected, and the secretion of IFN-β immune factor by the cells was detected using an ELISA kit. The results are as follows: Figure 5 As shown in the figure, transfection with AP-H into cells with APE1 knockdown failed to successfully activate the cGAS–STING pathway, while the positive sequence ODN-2P, which does not require APE1 triggering, was unaffected by APE1 knockdown.

[0097] The above results indicate that the activation of the intracellular cGAS–STING pathway by AP-H is strictly controlled by the expression level of APE1 in the cytoplasm, verifying the feasibility of the APE1-triggered DNA self-assembly immune stimulation system regulating the intracellular cGAS–STING pathway.

[0098] Example 5

[0099] This embodiment is used to verify the activation effect of the controlled DNA self-assembly immunostimulation system (AP-H) in different cells.

[0100] (1) The effect of different cells on activation effect

[0101] THP-1 cells with high APE1 expression and human skin fibroblasts (BJ cells) with low APE1 expression were selected and cultured at 37°C and 5% CO2. THP-1 cells were cultured in 1640 medium containing 10% fetal bovine serum, and BJ cells were cultured in DMEM medium containing 10% fetal bovine serum. THP-1 and BJ cells were seeded into well plates. THP-1 cells were induced to adhere using PMA, and after replacing the medium with fresh medium, they were cultured for another 24 hours. BJ cells did not require induction to adhere. When the cells reached a suitable density, they were transfected with nucleic acid sequences (ODN-2P, H, and AP-H). Four hours after transfection, the cell culture supernatant was discarded, and the medium was replaced with fresh medium. The cells were cultured at 37°C for another 16 hours. The cell culture supernatant was then collected, and the cGAMP produced and IFN-β secreted by the cells were detected using an ELISA kit. The results are shown below. Figure 6 As shown in the figure, AP-H effectively activates the cGAS–STING pathway only in THP-1 cells with high APE1 expression in the cytoplasm, while it fails to successfully stimulate the cGAS pathway in THP-1 cells with low APE1 expression in the cytoplasm. This fully verifies that AP-H can achieve cell-selective activation of the cGAS–STING pathway.

[0102] Example 6

[0103] This embodiment is used to verify the activation effect of the DNA self-assembly immunostimulation system (D / R).

[0104] A DNA self-assembly immunostimulatory system, D / R, triggered by RNase H, was designed. This system inhibits the self-assembly ability of ODN-2P by hybridizing a DNA / RNA hybrid chain (CR-ODN 2P) with ODN-2P, which is completely complementary to ODN-2P. RNase H cleaves the RNA sequence in the DNA / RNA hybrid double strand. Therefore, under RNase H cleavage, CR-ODN 2P is degraded, releasing ODN-2P, which then self-assembles to produce a long double-stranded DNA molecule, activating the cGAS–STING pathway. The specific mechanism is as follows: Figure 7 As shown in Figure a.

[0105] The same concentration of ODN-2P was thoroughly mixed with CR-ODN 2P or CD-ODN 2P chains (the sequence of which is completely identical to CR-ODN 2P, except that the nucleotides are replaced with deoxyribonucleotides instead of ribonucleotides) and annealed in a 95°C metal bath for 5 min, then slowly cooled to room temperature to obtain D / R and D / D complexes for further experiments. 4 μM of D / R and D / D chains were thoroughly mixed with 0 or 100 U / mL RNase H in Tris-HCl buffer, incubated at 37°C for 1 h, and analyzed by agarose gel electrophoresis. The results are shown below. Figure 7 Figure b shows that only D / R can undergo effective self-assembly after RNase H cleavage to produce long DNA fragments, while double-stranded DNA fragments D / D show no significant changes after RNase H treatment.

[0106] 4 μM of RNase H-pre-cleaved or unpre-cleaved D / R and D / D chains were thoroughly mixed with 200 μM ATP, 200 μM GTP, and 1 μM recombinant human cGAS protein. The mixture was heated in a metal bath at 37 °C for 5 h, followed by heating at 95 °C for 10 min to terminate the reaction. The products were analyzed by high-performance liquid chromatography (HPLC). The results are as follows: Figure 7 As shown in Figure c, only DNA / RNA hybrid double-stranded D / R can significantly activate cGAS protein after RNase H triggering. D / R without RNase H triggering and D / D (regardless of whether they are triggered by RNase H) cannot significantly stimulate cGAS protein.

[0107] THP-1 cells were seeded in well plates, and cell adhesion was induced using PMA. After replacing the medium with fresh medium, the cells were cultured for another 24 hours before nucleic acid transfection (D / R and D / D) and the secretion of IFN-β by the cells were measured. Results are as follows: Figure 7As shown in Figure d, D / R successfully activated the cGAS–STING pathway intracellularly upon RNase H triggering, inducing an increase in IFN-β expression levels. In contrast, the control group D / D, unable to be triggered by RNase H, could not effectively activate the cGAS–STING pathway and therefore could not lead to increased IFN-β secretion. These results demonstrate that D / R can successfully activate the intracellular cGAS–STING pathway upon RNase H triggering, validating the feasibility of this RNase H-triggered DNA self-assembly immunostimulatory system regulating the intracellular cGAS–STING pathway.

[0108] Example 7

[0109] This embodiment is used to verify the activation effect of an enzyme-activated, light-controlled inactivation orthogonally regulated DNA self-assembly immunostimulatory system (AP-H-PC).

[0110] A photolytic linker containing an o-nitrobenzyl group is introduced between the P1 and P2 domains of AP-H. Under the catalytic cleavage of APE1, AP-H-PC undergoes the same self-assembly behavior as AP-H, activating the cGAS-STING pathway. Applying UV light causes the photolytic linker to cleave, resulting in a break between P1 and P2. The assembled long double-stranded DNA molecule breaks into short DNA fragments, thus losing its ability to stimulate the cGAS-STING pathway. By coordinating endogenous APE1 stimulation or exogenous light stimulation, the activation time of the cGAS-STING pathway can be well controlled, avoiding the adverse effects of overactivation. The specific principle is as follows... Figure 8 As shown.

[0111] (1) Illumination detection

[0112] 4 μM AP-H and AP-H-PC chains were thoroughly mixed with 0 or 400 U / mL APE1 in Tris-HCl buffer and incubated at 37 °C for 1 h. The cleaved AP-H and AP-H-PC chains were then irradiated under 365 nm UV light for 0 or 10 min (power 5 mW / cm²). 2 Finally, the obtained product was analyzed by agarose gel electrophoresis, and the results are as follows: Figure 9 As shown in Figure a, the long DNA fragments generated by the self-assembly of the AP-H-PC chain containing photolytic linkers after activation by APE1 are broken down into shorter DNA fragments under light. In contrast, the self-assembly products of the AP-H chain without photolytic linkers are unaffected by light.

[0113] (2) Activation effect detection

[0114] 4 μM of AP-H and AP-H-PC chains pre-cleaved with APE1 were irradiated under 365 nm UV light for 0 or 10 min (power 5 mW / cm²). 2 The mixture was then thoroughly mixed with 200 μM ATP, 200 μM MTP, and 1 μM recombinant human cGAS protein, and incubated in a metal bath at 37°C for 5 h, followed by heating at 95°C for 10 min to terminate the reaction. The product was analyzed by high performance liquid chromatography.

[0115] The results are as follows Figure 9 As shown in Figures b and c, compared to the unilluminated group, the cGAMP content of AP-H-PC stimulated by light exposure decreased significantly after light exposure, while the ability of AP-H to stimulate cGAS protein was not significantly affected by UV light exposure.

[0116] Example 9

[0117] This embodiment is used to verify the activation effect of the DNA self-assembly immunostimulatory system (AP-H-PC) in cells.

[0118] THP-1 cells were seeded into well plates, and cell adhesion was induced using PMA. After replacing the medium with fresh medium, the cells were cultured for another 24 hours before transfection with nucleic acids (AP-H and AP-H-PC). The nucleic acids were pre-treated at a wavelength of 365 nm and a power of 5 mW / cm². 2 The cells were irradiated with UV light for 10 min, and 4 h after transfection, the cell culture supernatant was discarded and replaced with fresh culture medium. The cells were then cultured at 37°C for another 6 h. cGAMP was then extracted from the cells and detected using an ELISA kit. The results are as follows: Figure 10 Alternatively, continue culturing cells at 37°C for 16 hours, then collect the cell culture supernatant and use an ELISA kit to detect the IFN-β secreted by the cells. The results are as follows: Figure 10 b.

[0119] As shown in the figure, UV light irradiation significantly reduced the production of cGAMP and IFN-β pro-inflammatory factors in AP-H-PC-stimulated cells. However, the production of cGAMP and IFN-β induced by AP-H within cells was not significantly affected by UV light irradiation. These results indicate that the stimulation of the cGAS–STING pathway by AP-H-PC can be effectively terminated by exogenous light irradiation.

[0120] THP-1 cells were seeded into well plates, and cell adhesion was induced using PMA. After replacing the culture medium with fresh medium, the cells were cultured for another 24 hours before transfection with nucleic acids (AP-H and AP-H-PC). At different time points after transfection (0, 2, 4, 6, and 8 hours), the cells were placed at a wavelength of 365 nm with a power of 5 mW / cm².2 The cells were irradiated with UV light for 10 min (twice, 5 min each time, with a 2 min interval). Four hours after transfection, the culture medium was replaced with fresh medium, and the cells were cultured at 37°C for another 6 h. Then, cGAMP was extracted from the cells and detected using an ELISA kit. The results are as follows: Figure 10 c. Alternatively, continue culturing cells at 37°C for 16 hours, then collect the cell culture supernatant and use an ELISA kit to detect the IFN-β secreted by the cells. The results are as follows: Figure 10 The results indicate that the earlier the light exposure is terminated, the worse the effect of this system on activating the cGAS-STING pathway in cells. This verifies that the APE1-activated, light-controlled inactivation DNA self-assembly immunostimulation system can achieve on-demand orthogonal regulation of the intracellular cGAS-STING immune pathway and precisely regulate the spatial, temporal, and intensity of cGAS-STING pathway activation.

[0121] In summary, this application achieves precise control of the cGAS-STING immune pathway by combining DNA self-assembly immune stimulation sequences with inhibitory elements. Compared with the traditional screening and development of immunotherapeutic drugs and agonists, this method has the advantages of being simple, convenient, and universal.

[0122] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A controllable DNA self-assembly immune stimulation system, characterized in that, The DNA self-assembly immunostimulation system includes: a DNA self-assembly immunostimulation sequence and an inhibitory element; The DNA self-assembly immunostimulatory sequence consists of domain P1 and domain P2, both of which are palindromic sequences. The nucleotide sequence of domain P1 is SEQ ID NO:1, and the nucleotide sequence of domain P2 is SEQ ID NO:

2. The suppression element is a structural domain B or a CR-ODN 2P chain; The DNA self-assembly immunostimulatory sequence and domain B are linked via a base-free site. The domain B is complementary to either domain P1 or domain P2 in the DNA self-assembly immunostimulatory sequence. The number of complementary base pairs is 5-11 bases; The CR-ODN 2P chain is an RNA chain that is completely complementary to the bases of the DNA self-assembly immunostimulatory sequence, and hybridizes with the DNA self-assembly immunostimulatory sequence to form a DNA / RNA hybrid chain.

2. The controllable DNA self-assembly immunostimulation system according to claim 1, characterized in that, The DNA self-assembly immunostimulatory sequence and domain B are linked through a base-free site to form an immunostimulatory monomer. At least two groups of immunostimulatory monomers self-assembled under the action of nucleases to obtain double-stranded DNA self-assembled immunostimulatory sequences.

3. The controllable DNA self-assembly immunostimulation system according to claim 1 or 2, characterized in that, Nucleases used to cleave the baseless sites include depurinase / depyrimidine endonuclease 1.

4. The controllable DNA self-assembly immunostimulation system according to claim 2, characterized in that, The immunostimulatory monomer removes domain B via a nuclease, and after removal, domains P1 and P2 complement each other to form a double-stranded DNA self-assembled immunostimulatory sequence.

5. The controllable DNA self-assembly immunostimulation system according to claim 1, characterized in that, The DNA self-assembly immunostimulatory sequence also includes a photolyzable linker between domains P1 and P2.

6. The controllable DNA self-assembly immunostimulation system according to claim 5, characterized in that, The photolyzable linker is cleaved under light, thereby achieving photocontrolled inactivation of the DNA self-assembly immunostimulation system.

7. The controllable DNA self-assembly immunostimulation system according to claim 6, characterized in that, The light includes either ultraviolet light or visible light; The photolytically degradable linker is selected from any one of o-nitrobenzyl, coumarin, and azo bond.

8. The controllable DNA self-assembly immunostimulation system according to claim 1, characterized in that, The DNA self-assembly immunostimulatory sequence and the CR-ODN 2P chain form a DNA-RNA hybrid chain through complete complementary pairing.

9. The controllable DNA self-assembly immunostimulation system according to claim 8, characterized in that, After the hybrid strand is cleaved by ribonuclease, the P1 and P2 domains in the DNA self-assembly immunostimulatory sequence complement each other to form a double-stranded DNA self-assembly immunostimulatory sequence.

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