Novel immunostimulation regulation and control method based on nucleic acid topological structure conversion

Abstract

The invention discloses a novel immunostimulation regulation and control method based on nucleic acid topological structure conversion. The novel immunostimulatory regulation method comprises the following steps: eliminating immunogenicity by controlling a topological structure of an immunostimulatory DNA molecule, mixing a linear nucleic acid molecule, splint DNA and DNA ligase, carrying out a ligation reaction, carrying out separation and purification to obtain a circular nucleic acid molecule, and carrying out an in-vitro enzyme activity experiment and a cell experiment by using the circular nucleic acid molecule. An immunostimulatory nucleic acid molecule is inhibited to activate a cGAS-STING pathway; a functional element is modified at a specific position of nucleic acid, so that the functional element is subjected to structural transformation under the stimulation of a corresponding trigger element, and the stimulation capability of cGAS is recovered. According to the method disclosed by the invention, the time-space controllable activation of the cGAS-STING signal path can be realized.

Description

Technical Field

[0001] The present invention belongs to the fields of chemistry and biotechnology, and relates to a new method for regulating immune stimulation based on nucleic acid topological structure conversion. Background Art

[0002] Identifying exogenous DNA is one of the basic defense mechanisms in the body's immune system and is crucial for maintaining the host-microbe balance. Under normal physiological conditions, DNA exists in the nucleus and mitochondria of eukaryotic cells. If DNA appears in the cytoplasm, it usually means that the cell is in an abnormal state. The main sources of cytoplasmic DNA are divided into two aspects. On the one hand, it is exogenous DNA such as viruses and bacteria, and on the other hand, it is self-damaged DNA. In bacteria, bacteria use mechanisms such as restriction endonucleases and CRISPR to protect the host and resist invading exogenous DNA and phages. In mammals, foreign invading pathogen DNA can be quickly recognized and cleared through powerful DNA receptors. In 2013, the Chen Zhijian research group discovered a broad-spectrum cytoplasmic DNA receptor, cyclic GMP-AMP synthase (cGAS). Activated cGAS converts one molecule of ATP and one molecule of GTP into one molecule of cyclic GMP-AMP (2’3’-cGAMP) and two molecules of diphosphate.

[0003] As an important intracellular DNA receptor, cGAS plays a very important role in resisting the invasion of exogenous pathogens. It has currently been found that cGAS can resist the infection of a variety of viruses, including adenovirus, human cytomegalovirus, and human immunodeficiency virus. In addition to exogenous DNA that can activate cGAS, host DNA can also activate cGAS under specific conditions. Under intracellular stress, mitochondrial DNA is released from the mitochondria into the cytoplasm, thereby activating cGAS. In addition, when DNA damage occurs in the cell, resulting in the release of nuclear DNA into the cytoplasm, cGAS can also be activated.

[0004] The cGAS-STING pathway plays an important role in many aspects. By sensing exogenous DNA in the cytoplasm, it can quickly recognize the invasion of pathogens such as bacteria and viruses and initiate the body's defense program to timely clear pathogen infections. At the same time, the cGAS protein can also recognize endogenous nucleic acids accumulated in the cytoplasm of senescent, damaged or tumor cells and initiate downstream signaling pathways to timely clear these abnormal cells. Therefore, the cGAS / STING pathway also plays an important role in tumor immune surveillance and spontaneous anti-tumor immunity. In addition, the cGAS-STING pathway is also involved in the regulation of various physiological processes such as cell senescence, autophagy and apoptosis.

[0005] Given the important role of the cGAS-STING pathway in numerous biological processes, a large number of studies have been dedicated in recent years to modulating this pathway for potential therapeutic applications. Current regulatory methods mainly fall into three categories. The first is to deliver exogenous DNA for activation, the second is to utilize endogenous damaged DNA for activation, and the third is to deliver STING agonists, such as cGAMP analogs or some small molecules to directly activate the STING protein. However, it is worth noting that these existing methods mentioned are not specific, and non-specific or excessive activation of the immune signaling pathway may induce an overabundance of cytokines, leading to autoimmune disorders. Therefore, there is a need to develop spatiotemporally controllable methods to achieve activation of the cGAS-STING signaling pathway "on demand".

[0006] Intramolecular cyclization reaction is a reaction that causes a molecular chain to form a cyclic structure. DNA cyclization refers to the head-to-tail connection of a single-stranded DNA molecule into a ring. The usual method of forming a ring is to ligate it into a ring with the assistance of a DNA splint strand using DNA ligase, that is, linear DNA (the ring-forming strand) hybridizes with a DNA splint strand that is partially complementary to its two ends to form a nicked structure, and the two ends of the ring-forming strand are closed by DNA ligase ligation. Compared with linear DNA, circular DNA has better anti-digestion properties and biological stability.

[0007] Therefore, there is an urgent need to develop a spatiotemporally controllable method to achieve activation and precise regulation of the cGAS-STING signaling pathway in living cells "on demand". Summary of the Invention

[0008] Aiming at the deficiencies of the prior art and the actual needs, the present invention provides a new method for immunostimulation regulation based on nucleic acid topological structure conversion, aiming to cyclize immunostimulatory nucleic acid molecules modified with functional elements by T4 DNA ligase for spatiotemporal selective regulation of the cGAS-STING pathway. The technical solution adopted is: cyclize the immunostimulatory nucleic acid molecule intramolecularly by T4 DNA ligase to achieve good anti-digestion effect and biological stability; modify the immunostimulatory nucleic acid molecule with functional elements to achieve conformational conversion under specific conditions (such as light, reducing agent, etc.) and restore its linear structure, thereby restoring its ability to stimulate the cGAS protein to catalyze the synthesis of cGAMP, so as to achieve "spatiotemporal" controllable activation of the cGAS-STING signaling pathway.

[0009] In a first aspect, the present invention provides a new method for regulating immune stimulation based on nucleic acid topological structure conversion. The new method for regulating immune stimulation includes: eliminating the immunogenicity of an immunostimulatory DNA molecule by controlling its topological structure, mixing a linear nucleic acid molecule, a splint DNA, and a DNA ligase for a ligation reaction, separating and purifying to obtain a circular nucleic acid molecule, performing in vitro enzyme activity experiments and cell experiments with the circular nucleic acid molecule to inhibit the activation of the cGAS-STING pathway by the immunostimulatory nucleic acid molecule; modifying a functional element at a specific position of the nucleic acid to cause a structural conversion under the stimulation of a corresponding triggering factor and restoring the stimulating ability of cGAS.

[0010] The method of the present invention can achieve "time-space" controllable activation of the cGAS-STING signaling pathway.

[0011] Preferably, the sequence of the linear nucleic acid molecule is as shown in SEQ ID NO.1, and the nucleic acid sequence of the splint DNA includes the sequence shown in SEQ ID NO.2.

[0012] SEQ ID No.1: TATATGGGGGGATATATATACGTATA.

[0013] SEQ ID NO.2: CTAGTTTTCTAGCATATATATACGCGTGTTTTCACG.

[0014] Preferably, the molar ratio of the linear nucleic acid molecule to the splint DNA is 1:(1 - 5), such as 1:1, 1:2, 1:3, 1:4, 1:5.

[0015] Preferably, the DNA ligase includes T4 DNA ligase.

[0016] Preferably, the temperature of the ligation reaction is 14°C - 20°C (such as 14°C, 15°C, 17°C, 19°C or 20°C), and the time is 10 - 20 h (such as 10 h, 14 h, 15 h, 17 h, 19 h or 20 h).

[0017] Preferably, the functional element includes a photosensitive group and / or a disulfide bond.

[0018] Preferably, the triggering factor stimulation includes light stimulation or chemical stimulation.

[0019] As a preferred technical solution, the new method for regulating immune stimulation based on nucleic acid topological structure conversion of the present invention includes the following steps:

[0020] (1) Mix the linear nucleic acid molecule, the splint DNA, and T4 DNA ligase to carry out a ligation reaction at 14°C - 20°C. The molar ratio of the linear nucleic acid molecule to the splint DNA is 1:(1 - 5). React for 10 - 20 h to obtain a circular nucleic acid molecule, separate and purify it, and perform exonuclease digestion characterization on the purified product.

[0021] (2) Stimulate the circular nucleic acid molecule purified in step (1) in vitro or after transfection into living cells. Among them, the circular nucleic acid molecule modified with a functional group is subjected to light stimulation or chemical stimulation to obtain a corresponding linear immunostimulatory nucleic acid molecule.

[0022] (3) All circular nucleic acid molecules eliminate the immunogenicity of the original linear nucleic acid molecule and inhibit its activation of the cGAS - STING pathway. However, the circular nucleic acid molecule modified with a functional group is transformed into a linear nucleic acid molecule under light stimulation or chemical stimulation, stimulating the cGAS protein to activate the cGAS - STING pathway.

[0023] Preferably, the transfection time in step (2) is 4 - 6 h, such as 4 h, 5 h, or 6 h.

[0024] Preferably, the molar ratio of the linear nucleic acid molecule to the cGAS protein in step (3) is 1:(0.3 - 1), such as 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.8, 1:1.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The method of the present invention can achieve "time - space" controllable activation of the cGAS - STING signaling pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the gel electrophoresis diagram of the circular nucleic acid molecule prepared in Preparation Example 1 of the present invention;

[0028] Figure 2 It is the gel electrophoresis diagram of the circular nucleic acid molecule prepared in Preparation Example 2 of the present invention;

[0029] Figure 3 It is the test result diagram of the anti - digestion effect of the circular nucleic acid molecules obtained in Preparation Example 1 and Preparation Example 2 of the present invention;

[0030] Figure 4 It is the test result diagram of the structural conversion ability of the linear and circular nucleic acid molecules in Preparation Example 1 and Preparation Example 2 of the present invention;

[0031] Figure 5 It is the test result diagram of the structural conversion ability of the linear and circular nucleic acid molecules in Preparation Example 1 and Preparation Example 2 of the present invention;

[0032] Figure 6 This is the result graph of the in vitro enzyme activity experiment in Example 3 of the present invention;

[0033] Figure 7 This is the result graph of the intracellular IFN-β molecule detection in Example 4 of the present invention;

[0034] Figure 8 This is the result graph of the intracellular cGAMP molecule detection in Example 4 of the present invention. Detailed implementation manners

[0035] To further illustrate the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with examples and drawings. It can be understood that the specific implementation manners described herein are only used to explain the present invention, rather than limiting the present invention.

[0036] For those not specifying specific techniques or conditions in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For those reagents or instruments not specifying the manufacturer, they are all conventional products that can be obtained through regular channels.

[0037] Raw materials:

[0038] The DNA is from Sangon Biotech Co., Ltd.;

[0039] T4 DNA ligase, Exonuclease I, and Exonuclease III are purchased from New England Biolabs;

[0040] Lipo 3000 is purchased from Thermo Fisher Scientific;

[0041] RIPA and PMSF are purchased from Solarbio Life Sciences Co., Ltd.;

[0042] THP-1 cells are from the American Type Culture Collection (ATCC).

[0043] Preparation Example 1

[0044] This preparation example provides a product of intramolecular ligation of an immunostimulatory nucleic acid molecule. The ligation product is catalyzed by T4 DNA ligase to form a phosphodiester bond between the 5'-terminal phosphate group of P26 and its own 3'-terminal hydroxyl group with the assistance of a splint DNA strand. The preparation method includes the following steps:

[0045] In this preparation example, the sequences of P26 and the splint DNA are shown in SEQ ID No.1 and SEQ ID No.2 respectively:

[0046] SEQ ID No.1: 5'-p-TATATGGGGGGATATATATACGTATA(P26);

[0047] SEQ ID No.2: 5'-CTAGTTTTCTAGCATATATATACGCGTGTTTTCACG(S36).

[0048] (1) Annealing

[0049] Add 20 μmol / L of the immunostimulatory nucleic acid molecule and 60 μmol / L of the splint DNA to a 200 μL reaction system, react at 80 °C for 5 min, and anneal to room temperature.

[0050] (2) Ligation

[0051] Add 100 U of T4 DNA ligase to every 1 nmol of P26 and react at 16 °C for 16 h.

[0052] (3) Gel extraction and purification

[0053] Add loading buffer to each tube of the obtained product, heat at 80 °C in a metal bath for 5 min to terminate the reaction, immediately cool on ice, recover by running on a 12% dPAGE gel, and load 40 μL per well. Run the gel at 200 V for 30 min, cut the corresponding band, add it to a 1.5 mL centrifuge tube, mash it, add 600 μL of 1× reaction buffer, heat in a metal bath at 55 °C and soak overnight. The next day, centrifuge at 14000 rpm for 10 min, aspirate the supernatant into a new centrifuge tube. Re-add 200 μL of 1× reaction buffer to the residue, heat in a metal bath at 55 °C for 120 min, centrifuge under the same conditions, and aspirate the supernatant into a new centrifuge tube, 300 μL per tube.

[0054] (4) Precipitation with ice-cold ethanol

[0055] Add 30 μL of NaAc (3 M, pH = 5.2) to each tube, shake well and centrifuge. Since the DNA sequences used are short, 2 μL of nucleic acid co-precipitant needs to be added to each tube. Finally, add 900 μL of ice-cold ethanol, shake well and precipitate at -20 °C for 1 h. After precipitation, centrifuge the samples at 4 °C and 14000 rpm for 10 min, carefully pour out the supernatant, trying not to disturb the precipitate. Then wash with 600 μL of 70% ethanol, shake well, centrifuge at 20 °C and 14000 rpm for 5 min, and repeat twice.

[0056] (5) DNA quantification

[0057] The precipitate obtained in the above step (4) was volatilized sufficiently in a metal bath at 80 °C to remove ethanol. An appropriate amount of water was added according to the amount of the precipitate, and it was shaken well. Quantification was performed using Nanodrop to obtain a circular immunostimulatory nucleic acid molecule. The gel result of the preparation process of the obtained circular immunostimulatory nucleic acid molecule C26 is as Figure 1 shown.

[0058] Preparation Example 2

[0059] The difference from Preparation Example 1 is only that P26 described in this preparation example contains a photosensitive group (its sequence is shown in SEQ ID No. 3), and the other raw materials and preparation methods are the same as those in Preparation Example 1.

[0060] SEQ ID No. 3: 5’-p-TATATGGG / iPCLinker / GGGATATATATACGTATA (L26-PC). Wherein iPCLinker is the iPC linker photocleavage site modification.

[0061] The gel result of the preparation process of the obtained circular immunostimulatory nucleic acid molecule C26-PC is as Figure 2 shown.

[0062] Preparation Example 3

[0063] The difference from Preparation Example 1 is only that P26 described in this example contains a disulfide bond (its sequence is shown in SEQ ID No. 4), and the other raw materials and preparation methods are the same as those in Preparation Example 1.

[0064] SEQ ID No. 4: 5’-p-TATATGGG / iSH-HS / GGGATATATATACGTATA (L26-S). Wherein iSH-HS is the disulfide bond modification.

[0065] Example 1

[0066] Anti-digestion effect test.

[0067] Characterize the anti-digestion effect of the circular C26 prepared in Preparation Example 1 and Preparation 2. The steps are as follows:

[0068] Take a part of the obtained product and digest it with exonuclease. The concentration of Exonuclease I is 2 U / μL, the concentration of Exonuclease III is 10 U / μL, the final concentration of DNA is 2 μM, the final volume is 20 μL. After cutting in a metal bath at 37 °C for 1 h, an equal volume of urea glycerol loading buffer was added, and it was heated in a metal bath at 80 °C for 5 min to terminate the reaction. Run gel electrophoresis on 12% dPAGE. The result is as Figure 3As shown, the linear immunostimulatory nucleic acid molecules are completely cleaved by the exonuclease, while the prepared circular immunostimulatory nucleic acid molecules cannot be cleaved by the exonuclease. Figure 3 It can be seen that the cyclic C26 prepared in Preparation Example 1 and the cyclic C26-PC prepared in Preparation 2 have good anti-enzymatic cleavage effects.

[0069] Example 2

[0070] Structural transformation ability test.

[0071] The structural conversion ability of the cyclic C26-PC prepared in Preparation Example 2 and the C26-S prepared in Preparation Example 3 was characterized as follows:

[0072] The circular immunostimulatory nucleic acid molecule with PC-Linker prepared in Preparation Example 2 was irradiated with ultraviolet light at 120 mW for 3 min, and an equal volume of urea and glycerol loading buffer was added. The reaction was terminated by heating in a metal bath at 80°C for 5 min, and the reaction was run on a 12% dPAGE gel. The results are shown in FIG. Figure 4 As shown, only the circular immunostimulatory nucleic acid molecules with photosensitive groups are opened under light conditions and converted into linear DNA molecules.

[0073] As can be seen from the figure, the circular immunostimulatory nucleic acid molecule with PC-Linker prepared in Preparation Example 2 can open the circular immunostimulatory nucleic acid molecule under specific conditions and transform it into a linear structure.

[0074] The cyclic immunostimulatory nucleic acid molecule with disulfide bonds prepared in Preparation Example 3 was cleaved with DTT and TCEP at 37°C for 4 hours. After the reaction, an equal volume of urea and glycerol loading buffer was added and heated in a metal bath at 80°C for 5 minutes to terminate the reaction. The 12% dPAGE gel was run. The results are as follows. Figure 5 As shown, only the circular immunostimulatory nucleic acid molecules with disulfide bonds were opened in the presence of DTT or TCEP to become linear immunostimulatory nucleic acid molecules.

[0075] Example 3

[0076] To evaluate the stimulatory effect of linear and circular immunostimulatory nucleic acid molecules on the catalytic activity of cGAS protein in solution, L26 (Shanghai Sangon Biotechnology Co., Ltd.) was thoroughly mixed with 200 μM ATP, 200 μM GTP, and 1 μM recombinant human cGAS protein to a final concentration of 2 μM in a reaction system (40 mM Tris-HCL, 100 mM NaCl, and 5 mM MgCl 2, pH 7.4). The mixed solution was placed in a metal bath at 37 °C and heated for 5 h, and then immediately placed at 95 °C and heated for 10 min to terminate the reaction. The results are as Figure 6 shown. Only the cyclic immunostimulatory nucleic acid molecule with a photosensitive group can be opened under light conditions and become a linear structure, and has a similar ability to stimulate the production of cGAMP by the cGAS protein as the original P26.

[0077] Example 4

[0078] (1) Intracellular IFN-β detection

[0079] THP-1 cells were seeded in a 24-well plate at a density of 5×10 5 , and the cells were induced to adhere by PMA. After changing to fresh medium, the cells were cultured for another 24 h. The cyclic and linear immunostimulatory nucleic acid molecules prepared in Preparation Example 1 and Preparation Example 2 were delivered to the cells using the transfection reagent lipo 3000, and the cells were incubated in an incubator at 37 °C for 4 h. Subsequently, the supernatant was removed and the cells were washed once with PBS. Then, fresh medium was added to each well of the cells, and the cells were incubated at 37 °C for another 16 h. After that, the cell culture supernatant was collected for enzyme-linked immunosorbent assay (ELISA) to detect the IFN-β molecules therein. The results are as Figure 7 shown. Only the cyclic immunostimulatory nucleic acid molecule with a photosensitive group can be opened under light conditions and become a linear structure, and has a similar ability to produce the cytokine IFN-β as the original P26.

[0080] (2) Intracellular cGAMP detection

[0081] THP-1 cells were seeded in a 24-well plate at a density of 5×10 per well 5 , and the cells were induced to adhere by PMA. After changing to fresh medium, the cells were cultured for another 24 h. When the cell confluence reached 80-90%. The cyclic and linear immunostimulatory nucleic acid molecules prepared in Preparation Example 1 and Preparation Example 2 were delivered to the cells using the transfection reagent lipo 3000, and the cells were incubated in an incubator at 37 °C for 4 h. Subsequently, the supernatant was removed and the cells were washed once with PBS. Then, fresh medium was added to each well of the cells, and the cells were incubated at 37 °C. After 6 h, the cell culture supernatant was discarded, and the cells were washed twice with pre-cooled PBS. M-PER TM mammalian cell protein extraction reagent was added to the cells and placed on ice for 5 min. After the cells were completely lysed, the lysate was collected and centrifuged in a centrifuge. The centrifugation conditions were 14,000 g, 4 °C, and 10 min. Then, the supernatant was taken and the precipitate was discarded for enzyme-linked immunosorbent assay (ELISA) to detect the cGAMP molecules therein. The results are as Figure 8As shown, the results prove that after cyclization, it can inhibit the stimulation of cGAS protein to produce cGAMP, while the cyclic immunostimulatory nucleic acid molecule with a photosensitive group can be opened under light conditions to become a linear structure, and has the ability to stimulate cGAS protein to produce cGAMP similar to that of the original P26.

[0082] In summary, the method of the present invention can accurately and specifically regulate the cGAS-STING signaling pathway in living cells, avoiding the induction of excessive cytokines.

[0083] The applicant declares that the present invention uses the above embodiments to illustrate the detailed method of the present invention, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A novel method for immunostimulation regulation based on nucleic acid topological structure conversion, characterized in that: The novel immunostimulatory regulation method comprises: eliminating the immunogenicity of immunostimulatory DNA molecules by controlling their topological structure, mixing linear nucleic acid molecules, splint DNA and DNA ligase for ligation reaction, separating and purifying to obtain circular nucleic acid molecules, using the circular nucleic acid molecules for in vitro enzyme activity experiments and cell experiments to inhibit the activation of the cGAS-STING pathway by immunostimulatory nucleic acid molecules; and restoring the stimulatory ability of cGAS by modifying functional elements at specific positions of the nucleic acid so that the structure undergoes transformation under the stimulation of corresponding triggering factors.

2. The novel method for regulating and controlling immune stimulation according to claim 1, characterized in that: The linear nucleic acid molecule sequence includes a sequence such as SEQ ID NO.1, and the nucleic acid sequence of the splint DNA includes a sequence such as SEQ ID NO.

2.

3. The novel method for regulating and controlling immune stimulation according to claim 1 or 2, characterized in that: The molar ratio of the linear nucleic acid molecule to the splint DNA is 1:(1-5).

4. The novel method for regulating and controlling immune stimulation according to any one of claims 1 to 3, characterized in that: The temperature of the connection reaction is 14°C-20°C, and the time is 10-20h.

5. The novel method for regulating and controlling immune stimulation according to any one of claims 1 to 4, characterized in that: The DNA ligase includes T4 DNA ligase.

6. The novel method for regulating and controlling immune stimulation according to any one of claims 1 to 5, characterized in that: The functional element includes a photosensitive group and / or a disulfide bond.

7. The novel method for regulating and controlling immune stimulation according to any one of claims 1 to 6, characterized in that: The triggering element stimulation includes light stimulation or chemical stimulation.

8. The novel method for regulating and controlling immune stimulation according to any one of claims 1 to 7, characterized in that: The novel immunostimulation regulation method comprises the following steps: (1) mixing a linear nucleic acid molecule, a splint DNA and a T4 DNA ligase for a 14°C-20°C ligation reaction, wherein the molar ratio of the linear nucleic acid molecule to the splint DNA is 1:(1-5), reacting for 10-20 hours to obtain a circular nucleic acid molecule, separating and purifying it, and characterizing the purified product by exonuclease digestion; (2) stimulating the circular nucleic acid molecules purified in step (1) in vitro or after transfection into living cells, wherein the circular nucleic acid molecules modified with functional groups are subjected to light stimulation or chemical stimulation to obtain corresponding linear immunostimulatory nucleic acid molecules; (3) All circular nucleic acid molecules eliminate the immunogenicity of the original linear nucleic acid molecules and inhibit their activation of the cGAS-STING pathway. Circular nucleic acid molecules modified with functional groups are transformed into linear nucleic acid molecules under light stimulation or chemical stimulation, stimulating cGAS protein to activate the cGAS-STING pathway.

9. The novel method for regulating and controlling immune stimulation according to claim 8, characterized in that: The transfection time in step (2) is 4-6 hours.

10. The novel method for regulating and controlling immune stimulation according to claim 8 or 9, characterized in that: The molar ratio of the linear nucleic acid molecule to the cGAS protein in step (3) is 1:(0.3-1).

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