Liquid-liquid phase separation method for adjusting cGAS and DNA

Abstract

The invention discloses a method for regulating liquid-liquid phase separation of cGAS and DNA (Deoxyribose Nucleic Acid). The present invention provides a method of modulating the liquid-liquid phase separation of cGAS and DNA wherein the method comprises the step of modulating the affinity between cGAS and a helper protein. In some embodiments, the helper protein includes a helper protein that promotes cGAS activity or a helper protein that inhibits cGAS activity. The invention creatively finds that the formation of phase separation and subsequent signal transduction can be regulated by regulating the binding strength of the cGAS and the auxiliary protein of the cGAS. The invention creatively proposes that the protein-dsDNA LLPS in vitro and in living cells and the cGAS-mediated immune response are reversibly regulated by changing the affinity of the auxiliary protein and the cGAS.

Description

Technical Field

[0001] The present invention relates to a method for regulating liquid-liquid phase separation of cGAS and DNA, and in particular to a novel method for manipulating phase separation and signal transduction in vitro and in living cells, and belongs to the fields of cell biology, chemical biology and molecular biology. Background Art

[0002] Cyclic GMP-AMP synthase (cGAS) plays a key role in the cGAS-STING (stimulator of interferon genes) pathway. cGAS acts as a DNA sensor, binding to double-stranded DNA (dsDNA) and subsequently inducing type I interferon expression, thereby significantly promoting innate immune responses.

[0003] cGAS can recognize exogenous DNA that appears in the cytoplasm as an important indicator of pathogen infection, and catalyze GTP and ATP to synthesize cyclic GMP-AMP (cGAMP). When the cGAS protein binds to double-stranded DNA (dsDNA), cGAS undergoes a conformational change, activating its enzymatic activity and producing cGAMP. cGAMP binds to and activates the endoplasmic reticulum protein STING, which then transmits the signal to the cell nucleus through the STING pathway, regulating gene transcription, thereby triggering an effective immune response in the cell. The liquid-liquid phase separation (LLPS) of cGAS and dsDNA is essential for enhancing the enzymatic activity of cGAS. To date, a variety of human and viral proteins have been found to affect the formation of liquid-liquid phase separation between cGAS and dsDNA, thereby regulating the enzymatic activity of cGAS in cells.

[0004] Some proteins, G3BP1 and PCBP1, have been found to accelerate the response of cGAS to dsDNA by promoting the oligomerization and LLPS-induced aggregation phase of cGAS, thereby enhancing the enzymatic activity of cGAS. Other proteins, such as KSHV-ORF52, HSV1-VP22, and VZV-ORF9, inhibit the activity of cGAS. These proteins appear to disrupt the LLPS of cGAS and dsDNA and exclude cGAS from the aggregation phase, strongly inhibiting the activity of cGAS. However, how these proteins enhance or inhibit cGAS-dsDNA LLPS and the enzymatic activity of cGAS at the molecular level is unknown. Summary of the invention

[0005] Problem that the invention aims to solve

[0006] In previous studies, since the molecular mechanism of cGAS phase separation is not very clear, there is no clear method for regulating the phase separation of cGAS. In the present invention, it is found through research that the binding force between cGAS, dsDNA and its auxiliary proteins plays an important role in forming phase separation and signal transduction. The present invention creatively discovered a method for regulating the formation of phase separation and subsequent signal transduction by regulating the binding strength of cGAS and its auxiliary proteins. These methods for regulating binding strength include but are not limited to chemical, light-induced and other methods to change the binding strength of cGAS and its auxiliary proteins, thereby playing a role in regulating activity.

[0007] Solutions for solving problems

[0008] In a first aspect of the present invention, a method for regulating liquid-liquid phase separation of cGAS and DNA is provided, wherein the method comprises the step of regulating the affinity between cGAS and an auxiliary protein.

[0009] In some optional embodiments, the auxiliary protein includes an auxiliary protein that promotes cGAS activity or an auxiliary protein that inhibits cGAS activity.

[0010] In some optional embodiments, the auxiliary protein that promotes cGAS activity is selected from PCBP1 (SEQ ID NO: 2) or G3BP1 (SEQ ID NO: 3).

[0011] In some optional embodiments, the auxiliary protein that inhibits cGAS activity is selected from ORF9 (SEQ ID NO: 4), ORF52 (SEQ ID NO: 5) or VP22 (SEQ ID NO: 6).

[0012] In some embodiments, regulating the liquid-liquid phase separation of cGAS and DNA includes irreversibly or reversibly promoting the formation of the liquid-liquid phase separation of cGAS and DNA.

[0013] In some embodiments, the cGAS and the auxiliary protein are respectively bound to the multimerization domain of the multimer, and the affinity between cGAS and the auxiliary protein is regulated by regulating the multimerization domain to form a multimer.

[0014] In some embodiments, the multimer is an inducible multimer.

[0015] In some embodiments, the cGAS and the accessory protein are fused to the FRB multimerization domain and the FKBP multimerization domain, respectively.

[0016] In some specific embodiments, rapamycin or an analog thereof is used as a multimerizing agent to promote the formation of dimers between the FRB multimerization domain and the FKBP multimerization domain.

[0017] In some embodiments, the cGAS and the accessory protein are fused to the pMag multimerization domain and the nMag multimerization domain, respectively.

[0018] In some specific embodiments, blue light is used as a multimerizing agent to promote the formation of dimers between the pMag multimerization domain and the nMag multimerization domain.

[0019] In the second aspect of the present invention, a composition is provided, wherein the composition comprises a first fusion protein and a second fusion protein, wherein the first fusion protein comprises cGAS and a first multimerization domain; the second fusion protein comprises an auxiliary protein and a second multimerization domain; and the first multimerization domain and the second multimerization domain form a multimer under the induction of a multimerizing agent.

[0020] In some optional embodiments, the auxiliary protein includes an auxiliary protein that promotes cGAS activity or an auxiliary protein that inhibits cGAS activity.

[0021] In some optional embodiments, the auxiliary protein that promotes cGAS activity is selected from PCBP1 (SEQ ID NO: 2) or G3BP1 (SEQ ID NO: 3).

[0022] In some optional embodiments, the auxiliary protein that inhibits cGAS activity is selected from ORF9 (SEQ ID NO: 4), ORF52 (SEQ ID NO: 5) or VP22 (SEQ ID NO: 6).

[0023] In some optional embodiments, the composition further comprises a polymerizing agent.

[0024] In the third aspect of the present invention, any one of the following uses of the composition according to the second aspect of the present invention is provided:

[0025] (a) Use in the preparation of a reagent for regulating liquid-liquid phase separation of cGAS and DNA;

[0026] (b) Use in the preparation of an agent for regulating the cGAS-STING pathway;

[0027] (c) Use in the preparation of drugs for preventing, treating and / or assisting in the treatment of diseases associated with the cGAS-STING pathway.

[0028] Effects of the Invention

[0029] The present invention creatively discovered that the formation of phase separation and subsequent signal transduction can be regulated by regulating the binding strength between cGAS and its auxiliary proteins.

[0030] The present invention creatively proposes to reversibly regulate protein-dsDNA LLPS in vitro and in living cells, as well as cGAS-mediated immune responses, by changing the affinity between accessory proteins and cGAS.

[0031] In some embodiments, the present invention has discovered a method for regulating the interaction between accessory proteins and cGAS by chemical induction or light induction to regulate cGAS phase separation and downstream signal transduction. It is proposed that strengthening the interaction between accessory proteins and cGAS can change their influence on cGAS signal activation from inhibition to promotion, thereby providing a molecular basis for designing chemical and light-induced cGAS immune signaling systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : A schematic diagram of the principle of the method provided by the present invention. Figure 1 A: Schematic diagram of chemical induction method; Figure 1 B in the figure: Schematic diagram of the light-induced method; in the figure, Proteins represents auxiliary proteins.

[0033] Figure 2 : Schematic diagram of the experimental results of the chemical induction method in the embodiment of the present invention. Among them, Figure 2 Figure A shows that rapamycin increases the binding of cGAS to accessory proteins: Figure 2 B in the figure is an in vitro phase separation experiment; Figure 2 C in the figure is the intracellular phase separation experiment; Figure 2 D in the figure shows that the phosphorylation levels of downstream signaling molecules TBK1 and IRF3 in cells are increased.

[0034] Figure 3 : RT-qPCR analysis of IFN-β mRNA expression levels. Figure 3 A to Figure 3 Panel C shows the relative abundance of IFN-β mRNA analyzed by RT-qPCR. Figure 3 The A in it targets protein ORF9; Figure 3 B in the target protein ORF52; Figure 3 C in Figure 1 is directed to protein VP22. Data are presented as mean ± standard error (N≥3). Figure 3 A to Figure 3 In C, WT indicates that cGAS and accessory proteins are not fused with FRB / FKBP; FRB-FKBP indicates that cGAS and accessory proteins are fused with FRB / FKBP.

[0035] Figure 4 : Schematic diagram of the experimental results of the light induction method in the embodiment of the present invention. Among them, Figure 4 A in the figure shows that 5 minutes of blue light irradiation can promote cGAS phase separation in vitro; Figure 4 B in the figure shows that the activity of cGAS in vitro increased 2-5 times under 120 minutes of blue light irradiation; Figure 4 C and Figure 4 D in the figure shows that 5-10 minutes of blue light irradiation can induce intracellular cGAS to enter phase transition aggregation points; Figure 4 Panel E shows that the intracellular light-induced cGAS phase transition is reversible. DETAILED DESCRIPTION

[0036] Various exemplary embodiments, features and aspects of the present invention will be described in detail below. The word "exemplary" used here means "used as an example, embodiment or illustrative". Any embodiment described here as "exemplary" is not necessarily interpreted as being superior or better than other embodiments.

[0037] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0038] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0039] In this specification, the word "may" includes both performing a certain process and not performing a certain process.

[0040] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0041] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0042] <Method for regulating liquid-liquid phase separation of cGAS and DNA>

[0043] In the present invention, a method for regulating the liquid-liquid phase separation of cGAS and DNA is provided, wherein the method comprises the step of regulating the affinity between cGAS and an auxiliary protein.

[0044] As used herein, the term "liquid-liquid phase separation" or "LLPS" refers to the reversible process by which biomacromolecules such as proteins and nucleic acids condense into liquid membraneless organelles driven by weak multivalent interactions.

[0045] In some specific embodiments, regulating the liquid-liquid phase separation of cGAS and DNA includes irreversibly or reversibly promoting the formation of the liquid-liquid phase separation of cGAS and DNA.

[0046] For reversibly promoting the formation of liquid-liquid phase separation of cGAS and DNA, as demonstrated in the examples, cGAS and auxiliary proteins are fused with pMag / nMag, and dimerization is induced by blue light irradiation to increase the affinity of cGAS and auxiliary proteins, thereby promoting the liquid-liquid phase separation of cGAS and DNA. When the blue light irradiation stops, cGAS is separated from the phase transition aggregation point, that is, it returns to a state of inhibiting the formation of liquid-liquid phase separation.

[0047] The formation of irreversible promotion of liquid-liquid phase separation of cGAS and DNA refers to increasing the affinity of cGAS and auxiliary proteins, and promoting the liquid-liquid phase separation of cGAS and DNA, and then not returning to the state before increasing the affinity of cGAS and auxiliary proteins. For example, as demonstrated in the Examples, cGAS and auxiliary proteins are fused to FRB / FKBP, and dimerization is induced by rapamycin or its analogs to increase the affinity of cGAS and auxiliary proteins, thereby promoting the liquid-liquid phase separation of cGAS and DNA.

[0048] In some specific embodiments, regulating the liquid-liquid phase separation of cGAS and DNA includes converting the inhibition of liquid-liquid phase separation formation into a promotion of liquid-liquid phase separation formation, and also includes converting the state that has been converted to the state that promotes liquid-liquid phase separation formation to the state that inhibits liquid-liquid phase separation formation (that is, reversibly promoting the formation of liquid-liquid phase separation of cGAS and DNA).

[0049] In this specification, the term "binding strength", "binding affinity" or "affinity" refers to the strength of the binding interaction between a single biomolecule and its ligand or binding partner. Affinity is usually expressed using the equilibrium dissociation constant K D To measure and describe. DThe lower the value, the greater the affinity between the protein and its binding partner. "Binding affinity" can be measured by any method known in the art. In some specific embodiments, binding affinity can be quantified by dual-color fluorescence cross-correlation spectroscopy (dcFCCS).

[0050] In some optional embodiments, the amino acid sequence of cGAS is shown in SEQ ID NO:1.

[0051] In the present specification, the term "auxiliary protein" refers to any protein that can promote or inhibit the enzymatic activity of cGAS, for example, it may be a protein that promotes the enzymatic activity of cGAS by promoting the oligomerization and / or LLPS-induced aggregation phase of cGAS to accelerate the response of cGAS to dsDNA, such as PCBP1, G3BP1, etc.; or it may be a protein that inhibits the activity of cGAS by inhibiting the oligomerization and LLPS-induced aggregation of cGAS (referred to as auxiliary proteins that inhibit cGAS activity in the present specification), such as ORF9, ORF52 or VP22, etc.

[0052] In some optional embodiments, the auxiliary protein includes PCBP1, G3BP1, ORF9, ORF52 or VP22, but is not limited thereto, for example, any auxiliary protein known or unknown in the art that inhibits or promotes cGAS activity and / or liquid-liquid phase separation of DNA.

[0053] In some specific embodiments, the auxiliary protein includes PCBP1, whose amino acid sequence is shown in SEQ ID NO:2.

[0054] In some specific embodiments, the auxiliary protein includes G3BP1, whose amino acid sequence is shown in SEQ ID NO:3.

[0055] In some specific embodiments, the auxiliary protein includes ORF9, whose amino acid sequence is shown in SEQ ID NO:4.

[0056] In some specific embodiments, the auxiliary protein includes ORF52, whose amino acid sequence is shown in SEQ ID NO:5.

[0057] In some specific embodiments, the auxiliary protein includes VP22, whose amino acid sequence is shown in SEQ ID NO:6.

[0058] Multimerization domains and polymers

[0059] In some embodiments, the cGAS and the auxiliary protein are respectively bound to the multimerization domain of the multimer, and the affinity between cGAS and the auxiliary protein is regulated by regulating the multimerization domain to form a multimer.

[0060] In the present specification, the term "multimer" encompasses one or more dimers, trimers or higher order multimers.

[0061] In the present specification, "multimerization domain" refers to a molecule that preferentially interacts or associates with another molecule directly or through a multimerizing agent, wherein the interaction of different multimerization domains significantly contributes to or effectively promotes multimerization (i.e., forming a dimer, trimer or multipartite complex, which can be a homodimer, heterodimer, homotrimer, heterotrimer, homomultimer, heteromultimer).

[0062] In the present specification, the multimer is an inducible multimer, for example, under appropriate inducing conditions, the multimerization domains can be connected to form a multimer.

[0063] Suitable inducible multimeric multimerization domains include, but are not limited to, FKBP / FRB, which can be induced by, for example, rapamycin or AP21967, PYL / ABI, which can be induced by, for example, abscisic acid, GID1 / GAI, which can be induced by, for example, gibberellin or gibberellic acid, and pMag / nMag, which can be induced by, for example, blue light and / or temperature (Kawano, Fuun et al. "Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins." Nature communications vol. 6 6256. 24Feb. 2015, which is incorporated herein by reference), and other light-induced multimeric systems, such as the LOV2 domain of phototropin 1, phytochrome B, cryptochrome 2 (CRY2), FKF1, Vivid (VVD), UVR8 and EL222, etc. In other embodiments, variants of the above-mentioned multimerization domains, or other multimerization domains known in the art that can form multimers, may also be used. There are no particular limitations as long as they can achieve the effects of the present invention.

[0064] In some specific embodiments, the formation of multimers by the multimerization domains of the multimers is regulated by a multimerizing agent. "Multimerizing agent" refers to any molecule that is capable of binding to a multimerization domain, thereby bringing together at least two multimerization domains and any components thereby attached to the multimerization domains.

[0065] cGAS and accessory proteins fused to FRB / FKBP

[0066] In some embodiments, the cGAS and the accessory protein are fused to the FRB multimerization domain and the FKBP multimerization domain, respectively.

[0067] In some specific embodiments, the multimerization domain is associated with a multimerizing agent (e.g., a dimerizing agent). In some specific embodiments, the dimerizing agent is rapamycin or an analog thereof. For example, the multimerization domain is a pair selected from a FK506 binding protein (FKBP) multimerization domain and a FKBP-rapamycin binding (FRB) multimerization domain, or a variant thereof.

[0068] In some embodiments, the FRB multimerization domain is a polypeptide region capable of forming a ternary complex with the FKBP protein (FKBP multimerization domain) and rapamycin or its rapamycin analog. The FRB multimerization domain is present in many naturally occurring proteins, including mTOR proteins (also known as FRAP, RAPT1 or RAFT) from humans and other species; yeast proteins, including Tor1 and Tor2; and Candida FRAP homologs. Information about the nucleotide sequences, cloning and other aspects of these proteins is known in the art.

[0069] In some specific embodiments, the FRB multimerization domain used in the present invention generally contains at least 85 to 100 amino acid residues. In certain embodiments, the peptide sequence of the FRB multimerization domain includes (a) a naturally occurring peptide sequence spanning a region of at least a specified 93 amino acids of human mTOR or a corresponding region of a homologous protein; (b) a variant of a naturally occurring FRB, in which up to ten amino acids, or 1 to 5 amino acids, or 1 to 3 amino acids, or in some embodiments only one amino acid of the naturally occurring peptide has been deleted, inserted or replaced; (c) a peptide encoded by a nucleic acid molecule that can selectively hybridize with a DNA molecule encoding a naturally occurring FRB multimerization domain, or a peptide encoded by a DNA sequence that can selectively hybridize with a DNA molecule encoding a naturally occurring FRB multimerization domain (but due to the degeneracy of the genetic code).

[0070] In some specific embodiments, the amino acid sequence of the FRB multimerization domain is as shown in SEQ ID NO:7.

[0071] In some specific embodiments, the term "FK506 binding protein (FKBP) multimerization domain" refers to a FKBP polypeptide. FKBP is a cytosolic receptor for macrolides (such as FK506, FK520 and rapamycin) and is highly conserved between germ lines. For the purposes of the present invention, FKBP is a protein or protein domain that is capable of binding to rapamycin or an analog thereof and further forming a ternary complex with a protein or fusion protein containing FRB. The FKBP multimerization domain may also be referred to as a "rapamycin binding domain". Information on nucleotide sequences, cloning and other aspects of various FKBP species is known in the art. Homologous FKBP proteins in other mammalian species, yeast and other organisms are also known in the art and can be used in the methods disclosed herein. The size of the FKBP domain used in the present invention varies depending on the FKBP protein used.

[0072] The peptide sequences of the FKBP multimerization domain of the present invention include (a) a naturally occurring FKBP peptide sequence or a peptide sequence derived therefrom, derived from another human FKBP, derived from mouse or other mammalian FKBP, or derived from some other animal, yeast or fungal FKBP; (b) a variant of a naturally occurring FKBP sequence, in which up to ten amino acids, or 1 to 5 amino acids, or 1 to 3 amino acids, or in some embodiments only one amino acid of the naturally occurring peptide has been deleted, inserted or substituted; (c) a peptide sequence encoded by a nucleic acid molecule capable of selectively hybridizing with a DNA molecule encoding a naturally occurring FKBP, or a peptide sequence encoded by a DNA sequence capable of selectively hybridizing with a DNA molecule encoding a naturally occurring FKBP (but due to the degeneracy of the genetic code).

[0073] In some specific embodiments, the amino acid sequence of the FKBP multimerization domain is as shown in SEQ ID NO:8.

[0074] In some exemplary embodiments, the amino acid sequence of the fusion protein of cGAS and FRB (cGAS-FRB) is shown in SEQ ID NO:13.

[0075] In some exemplary embodiments, the amino acid sequence of the fusion protein of the auxiliary protein and FKBP is shown as SEQ ID NO: 18 (FKBP-ORF9), SEQ ID NO: 23 (FKBP-ORF52), or SEQ ID NO: 28 (FKBP-VP22).

[0076] cGAS and accessory proteins fused to pMag / nMag

[0077] In some embodiments, the cGAS and the auxiliary protein are fused to the pMag multimerization domain and the nMag multimerization domain, respectively. It is understood that the pMag multimerization domain or the nMag multimerization domain includes respective mutants. The pMag multimerization domain and the nMag multimerization domain are described in the literature Kawano, Fuun et al. "Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins." Nature communications vol. 6 6256. 24Feb. 2015:

[0078] The pMag multimerization domain may be selected from: pMag (I52R / M55R), pMagFast1 (I52R / M55R / I85V), pMagFast2 (I52R / M55R / I74V / I85V), pMagHigh1 (I52R / M55R / M135I / M165I) or variants thereof.

[0079] The nMag multimerization domain may be selected from: nMag (I52D / M55G), nMagFast1 (I52D / M55G / I85V), nMagFast2 (I52D / M55G / I74V / I85V), nMagHigh1 (I52D / M55G / M135I / M165I) or variants thereof.

[0080] In some specific embodiments, blue light is used as a multimerizing agent to induce the pMag multimerization domain and the nMag multimerization domain to form a dimer.

[0081] In some optional embodiments, the spectrum of the blue light is 470nm±20nm.

[0082] In some specific embodiments, the amino acid sequence of the pMag multimerization domain is as shown in SEQ ID NO:9.

[0083] In some specific embodiments, the nMag multimerization domain includes a nMagHigh1 multimerization domain, whose amino acid sequence is shown in SEQ ID NO:10.

[0084] In some exemplary embodiments, the amino acid sequence of the fusion protein of cGAS and pMag (cGAS-pMag) is shown in SEQ ID NO:16.

[0085] In some exemplary embodiments, the amino acid sequence of the fusion protein of the auxiliary protein and nMagHigh1 is shown in SEQ ID NO:21 (nMagHigh1-ORF9), SEQ ID NO:26 (nMagHigh1-ORF52), or SEQ ID NO:31 (nMagHigh1-VP22).

[0086] <Composition>

[0087] In the present invention, a composition is also provided, which includes a first fusion protein and a second fusion protein, wherein the first fusion protein includes cGAS and a first multimerization domain; the second fusion protein includes an auxiliary protein and a second multimerization domain; the first multimerization domain and the second multimerization domain form a multimer under the induction of a multimerization agent.

[0088] In some embodiments, the accessory protein includes an accessory protein that promotes cGAS activity or an accessory protein that inhibits cGAS activity.

[0089] In some optional embodiments, the auxiliary protein that promotes cGAS activity is selected from PCBP1 (SEQ ID NO: 2) or G3BP1 (SEQ ID NO: 3);

[0090] In some optional embodiments, the auxiliary protein that inhibits cGAS activity is selected from ORF9 (SEQ ID NO: 4), ORF52 (SEQ ID NO: 5) or VP22 (SEQ ID NO: 6).

[0091] In some optional embodiments, the composition further comprises a polymerizing agent.

[0092] In some embodiments, the cGAS, auxiliary protein, multimerization domain, multimerization agent, polymer, etc. are as described above in the <Method for regulating liquid-liquid phase separation of cGAS and DNA>.

[0093] In some exemplary embodiments, the first fusion protein includes a fusion protein of cGAS and FRB (cGAS-FRB), whose amino acid sequence is shown in SEQ ID NO:13.

[0094] In some exemplary embodiments, the first fusion protein includes a fusion protein of cGAS and pMag (cGAS-pMag), whose amino acid sequence is shown in SEQ ID NO:16.

[0095] In some exemplary embodiments, the second fusion protein includes a fusion protein of an auxiliary protein and FKBP, and its amino acid sequence is shown in SEQ ID NO: 18 (FKBP-ORF9), SEQ ID NO: 23 (FKBP-ORF52), or SEQ ID NO: 28 (FKBP-VP22).

[0096] In some exemplary embodiments, the second fusion protein includes a fusion protein of an auxiliary protein and nMag, and its amino acid sequence is shown in SEQ ID NO:21 (nMagHigh1-ORF9), SEQ ID NO:26 (nMagHigh1-ORF52), or SEQ ID NO:31 (nMagHigh1-VP22).

[0097] <Purpose>

[0098] The present invention also provides any of the following uses of the composition described above:

[0099] (a) Use in the preparation of a reagent for regulating liquid-liquid phase separation of cGAS and DNA;

[0100] (b) Use in the preparation of an agent for regulating the cGAS-STING pathway;

[0101] (c) Use in the preparation of drugs for preventing, treating and / or assisting in the treatment of diseases associated with the cGAS-STING pathway.

[0102] In some specific embodiments, regulating the cGAS-STING pathway includes promoting the cGAS-STING pathway, such as activating signal transduction downstream of cGAS-STING, specifically, increasing the phosphorylation levels of TBK1 and IRF3 and the expression level of IFN-β mRNA.

[0103] Example

[0104] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0105] The present invention proposes to use chemical induction or light induction to change the binding force between cGAS and auxiliary proteins, thereby changing the formation of phase separation and subsequent signal transduction, through in-depth research on molecular mechanisms.

[0106] 1. Materials and Methods

[0107] Plasmid construction

[0108] The full-length cGAS (amino acids 1-522) was cloned into the pET-28a vector and SUMO-tagged at the N-terminus. The FRB tag or pMag tag was fused to the C-terminus of cGAS. mCherry was further fused to the N-terminus of cGAS to form mCherry-cGAS, mCherry-cGAS-FRB, and mCherry-cGAS-pMag, respectively.

[0109] The coding sequences of G3BP1 and PCBP1 were cloned from a human cDNA library donated by Dr. Jizhou Li (Tsinghua University) and subsequently inserted into the pET-28a vector with a SUMO tag at the N-terminus.

[0110] The viral coat protein KSHV-ORF52 (also referred to as ORF52), HSV1-VP22 (also referred to as VP22), and VZV-ORF9 (also referred to as ORF9) sequences were synthesized by Sangon Biotech, and then cloned into the pET-28a vector with a SUMO tag at the N-terminus. The FKBP tag or nMagHigh1 tag was fused to the N-terminus of the viral coat protein. EGFP was further fused to the N-terminus of the viral coat protein to form EGFP-viral coat protein (EGFP-ORF9, EGFP-ORF52, EGFP-VP22), EGFP-FKBP-viral coat protein (EGFP-FKBP-ORF9, EGFP-FKBP-ORF52, EGFP-FKBP-VP22), and EGFP-nMagHigh1-viral coat protein (EGFP-nMagHigh1-ORF9, EGFP-nMagHigh1-ORF52, EGFP-nMagHigh1-VP22).

[0111] Protein expression and purification

[0112] cGAS protein was expressed in 2×Yeast Extract andTryptone (2×YT) medium by E. coli strain Rosetta (DE3) and induced with 200 μM isopropyl β-D-thiogalactoside (IPTG) at 18°C ​​for 19 hours. Cells expressing protein were collected and sonicated (ultrasonication 2 seconds, interval 4 seconds) in lysis buffer (20 mM HEPES pH 7.5, 400 mM NaCl, 30 mM imidazole, 10% glycerol, 1 mM Tris (2-carboxyethyl) phosphine (TCEP) and 1 mM PMSF) for 40 minutes. Subsequently, the clarified supernatant was incubated with Ni-NTA beads (GE Healthcare) at 4°C for 2 hours, and then the protein was eluted with 20 mM HEPES pH 7.5, 400 mM NaCl, 300 mM imidazole, 10% glycerol and 1 mM TCEP. Subsequently, the His6 SUMO tag was removed using SUMO protease (Ulp1) at a ratio of 1:20 (w / w) at 4°C overnight. TM The cleaved protein was purified using a Heparin HP column (GE Healthcare) and eluted with a 0.25 M to 1.5 M NaCl gradient in 20 mM HEPES pH 7.5, 10% glycerol, and 1 mM TCEP. The eluted protein was concentrated and then purified using a Superdex column in 20 mM HEPES pH 7.5, 150 mM KCl, 10% glycerol, and 1 mM TCEP. TM Size exclusion chromatography purification was performed using a 200Increase 10 / 300GL column (GE Healthcare). cGAS-FRB and cGAS-pMag were purified by the same procedure.

[0113] G3BP1 and PCBP1 proteins were expressed in Luria-Bertani (LB) medium by Escherichia coli strain BL21 (DE3) and induced with 500 μM IPTG for 16 hours at 16°C. Cells were collected and lysed in a solution containing 50mM HEPES 7.5in 250mM NaCl, 20mM imidazole, 1mMTCEP and 5% glycerol, and sonicated (sonication for 2 seconds, intervals of 4 seconds) for 30 minutes. Subsequently, the supernatant was loaded into Ni-NTA beads (GE Healthcare) and the protein was eluted with elution buffer (50mM HEPES 7.5, 250mM NaCl, 500mM imidazole, 1mM TCEP, 5% glycerol). His6 SUMO tag was cleaved by Ulp1 overnight at 4°C. The cleaved protein samples were lysed with HiTrap in a 50mM HEPES 7.5, 10% glycerol and 1mM TCEP solution. TM A heparin column (GE Healthcare) was treated with a linear NaCl gradient from 300 mM to 1 M. The eluted protein was purified by Superdex TM Size exclusion chromatography was performed using a 200Increase 10 / 300GL column (GE Healthcare) in 20 mM HEPES 7.5, 300 mM NaCl and 1 mM TCEP. Peak samples were concentrated, snap-frozen in liquid nitrogen and stored at -80°C.

[0114] The viral coat protein was expressed in LB medium by Escherichia coli strain Rosetta (DE3) and induced with 500 μM IPTG overnight at 18°C. Cells expressing His6 SUMO-tagged proteins were collected. Protease inhibitors (Roche) were added before lysis to inhibit protein degradation. The cells were harvested and lysed by ultrasound (ultrasound for 2 seconds, intervals of 4 seconds) for 30 minutes in 50mM Tris-HCl pH 7.5, 500mM NaCl, 20mM imidazole, 1mM TCEP and 5% glycerol. Subsequently, the supernatant was loaded into Ni-NTA beads (GE Healthcare) and the protein was eluted with elution buffer (50mM Tris-HCl pH7.5, 500mM NaCl, 500mM imidazole, 1mM TCEP, 5% glycerol). The His6 SUMO tag was cleaved by Ulp1 overnight at 4°C. The cleaved protein samples were purified by HiTrap TM The His6 SUMO tag and nucleic acid were removed by a linear NaCl gradient from 300 mM to 1 M using a heparin column (GE Healthcare). The eluted KSHV-ORF52 protein was purified by Superdex TMSize exclusion chromatography was performed on a 200Increase 10 / 300GL column (GE Healthcare) in 20 mM HEPES 7.5, 300 mM NaCl, and 1 mM TCEP solution. HSV1-VP22 and VZV-ORF9 proteins were isolated from Superdex 100 in buffers containing 600 mM and 500 mM NaCl, respectively. TM Eluted on a 200Increase 10 / 300GL column. Peak samples were concentrated, measured, and quickly frozen in liquid nitrogen before long-term storage at -80°C. The purification process of FKBP-virus coat protein (FKBP-ORF9, FKBP-ORF52, FKBP-VP22) and nMagHigh1-virus coat protein (nMagHigh1-ORF9, nMagHigh1-ORF52, nMagHigh1-VP22) was the same.

[0115] Protein labeling

[0116] cGAS protein was mixed with Alexa FluorTM 488 (AF488) NHS ester (ThermoFisherScientific) or Cyanine5 (Cy5) NHS ester (Lumiprobe) at a molar ratio (protein: dye) of 1:2 in buffer (20mM HEPES pH 7.5, 150mM KCl, 10% glycerol, 1mM TCEP) and incubated at 23°C for 20 minutes. G3BP1 and PCBP1 were mixed with AF488 in buffer (20mM HEPES pH 7.5, 300mM NaCl, 10% glycerol, 1mM TCEP) at a molar concentration ratio of 1:3 and incubated at 23°C for 2 hours. Virus coat proteins were labeled with AF488 at a molar concentration ratio of 1:5 in buffer (20mM HEPES pH 7.5, 300mM NaCl, 10% glycerol, 1mM TCEP) and incubated overnight at 4°C. Use Superdex TM The labeled proteins were separated from the excess free dye using a 200Increase 10 / 300GL column (GE Healthcare). The labeling efficiency was calculated based on the molar extinction coefficients as A280 (protein), A495 (AF488), and A652 (Cy5).

[0117] dsDNA labeling

[0118] 25 bp and 99 bp single-stranded DNA were synthesized by Sangon Biotech Co. The dsDNA molecules were annealed in a buffer (20 mM Tris-HCl pH 7.5, 50 mM NaCl) to form forward and reverse ssDNA.

[0119] Synthetic 5'-end NH2-modified DNA oligonucleotides (Sangon Biotech) were mixed with Cyanine5 NHS ester (Lumiprobe) at a molar ratio of 1:10 and incubated overnight at room temperature. The labeled DNA was separated from the excess free dye by ethanol precipitation. The dye labeling efficiency was calculated based on the molar extinction coefficient of DNA at 260 nm and the molar extinction coefficient of Cy5 at 652 nm. The labeling rate of all DNA oligonucleotides was approximately 100%.

[0120] In vitro phase separation of cGAS and dsDNA

[0121] Proteins were incubated with dsDNA in a final volume of 50 μL in a 96-well plate (Corning) pre-coated with 20 mg / mL BSA (Sigma). 10 μM 99 bp dsDNA (containing 200 nM Cy5-labeled dsDNA) and 10 μM cGAS (containing 200 nM AF488-labeled cGAS) were gently mixed in 5 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, and 1 mg / mL BSA to allow phase separation of cGAS from dsDNA. Time-lapse experiments were performed on a FV1200 (Olympus) confocal microscope using a 60× / 1.42NA oil objective. Images were acquired every 30 seconds over 20 minutes. Time-lapse images were processed using ImageJ.

[0122] Phase separation of cGAS and dsDNA in vitro affected by accessory proteins

[0123] In general, Cy5-cGAS and dsDNA were first incubated for 10 min in a 96-well plate (Corning) pre-coated with 20 mg / mL BSA (Sigma) to form phase separation, and the final volume was 45 μL, with a solution of 5 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM DTT, 1 mg / mL BSA, and then 5 μL of AF488-labeled auxiliary protein was added. The experiment was performed on a FV1200 (Olympus) confocal microscope using a 60× / 1.42NA oil objective. Images were collected every 30 seconds for 20 minutes. Time-lapse images were processed using ImageJ.

[0124] In vitro FRAP assay

[0125] Fluorescence recovery after photobleaching (FRAP) assays were performed using a FV1200 (Olympus) confocal microscope at room temperature. For FRAP of AF488-cGAS and 99 bp Cy5-dsDNA, a 2 μm diameter spot in a 10 μm droplet was photobleached using a 561 nm laser. Recovery images were acquired at 5 s intervals within 3 min after bleaching. The fluorescence intensity of the region of interest (ROI) was normalized to 1 before bleaching. Image intensity was measured by averaging the ROI, i.e., the real-time intensity of the background was subtracted from the intensity of the region of interest.

[0126] dcFCCS data analysis

[0127] The raw data of photon arrival time and FCCS curve were recorded within 5 min after mixing the samples. The dcFCCS measurement was repeated at least three times for each condition. The autocorrelation curves of AF488 and Cy5 detection channels were fitted by Equation 1:

[0128]

[0129] Where A is the amplitude of the autocorrelation curve, τ D is the diffusion time of the labeled molecule, T is the triplet fraction, τ tri is the relaxation time of the triplet state, and a is the ratio of the vertical radius to the horizontal radius of the laser. Here, A 488 and A 640 The amplitudes of the autocorrelation curves of the AF488 and Cy5 detection channels were calculated respectively. The cross-correlation curves were fitted using Eg.2:

[0130]

[0131] Among them A X is the amplitude of the cross-correlation curve, τ Dx is the diffusion time of the doubly labeled molecule.

[0132] Quantification of the hydrodynamic radius of condensates by dcFCCS

[0133] Obtain the dcFCCS curve. According to Equation 2, fit the dcFCCS curve to extract the diffusion time (τ Dx ), which can be used to quantify the hydrodynamic radius of heterocomplexes and condensates formed by AF488-cGAS and Cy5-dsDNA through Equation 3.

[0134]

[0135] We use AF488 as the standard. 488 In our measurements, Cd 488=0.65, which is used to calibrate the diffusion time extracted from the autocorrelation curve and the cross-correlation curve and calculated according to the published procedure. In our experiment, the relaxation time of the autocorrelation curve of AF488 under 488-nm laser excitation is about 0.09 ms.

[0136] Quantification of the proportion of components involved in condensation using dcFCCS

[0137] When the interaction between AF488-labeled molecules and Cy5-labeled molecules does not form aggregates or oligomers, the ratio of AF488-labeled molecules to form dual-labeled molecules can be calculated by the following formula:

[0138]

[0139] The ratio of AF640 labeled molecules to double labeled molecules can be calculated by the following formula:

[0140]

[0141] Cr 488 and Cr 640 is a correction factor representing the ratio of the overlapped excitation volume to the excitation volume of the 488 nm laser and the 640 nm laser. The correlation coefficient Cr used in this example was calibrated according to published procedures. 488 and Cr 640 ~0.52 and ~0.64 respectively.

[0142] Quantification of binding affinity by dcFCCS

[0143] DcFCCS experiments were performed in 20 mM Tris-HCl pH 7.5, 300 mM NaCl, and 5 mM MgCl2 solutions at 25 °C. 10 nM AF488-labeled accessory proteins were mixed with 20 nM Cy5-cGAS or 20 nM Cy5-dsDNA, and their binding affinities were quantified by dcFCCS. When necessary, 200 nM rapamycin was added. For the light-inducible system, the mixed samples were illuminated with 470 nm ± 20 nm blue light from a 1 W m-2 LED light source before measuring dcFCCS.

[0144] Assume that AF488-labeled A and Cy5-labeled B are mixed to form complex AB. According to formula 4, is the ratio of AF488-A to B to form a complex. Then, the binding constant (equilibrium dissociation constant) can be calculated using Equation 6.

[0145]

[0146] C A and CB are the total concentrations of AF488-labeled A molecules and Cy5-labeled molecules. [A], [B] and [AB] are the total concentrations of A molecules and Cy5-labeled molecules, respectively. AF488 Free molecules, B Cy5 Concentrations of free molecules and AB complexes.

[0147] In vitro cGAMP synthesis assay

[0148] cGAS and dsDNA were mixed with 1 mM ATP and 1 mM GTP in reaction buffer (20 mM Tris-HCl pH 7.5, 5 mM MgCl2) and incubated at 37°C for 2 hours. After incubation, the samples were heated at 95°C for 10 minutes and then centrifuged at 20,000 rpm for 10 minutes. The supernatant was diluted 5 times with 20 mM Tris-HCl pH 7.5 and loaded onto Mono Q TM 5 / 50 column (GE Healthcare). Gradient elution was performed in 20 mM Tris-HCl pH 7.5 solution with 0 to 0.4 M NaCl at a flow rate of 0.5 mL / min. The product cGAMP showed an absorbance peak at 256 nm. The yield of cGAMP was quantified using Unicorn 7.1 software (GE Healthcare).

[0149] If necessary, add auxiliary proteins at a concentration equivalent to that of cGAS. If necessary, add 200 nM rapamycin. For light-inducible systems, use 1 Wm -2 The mixed sample was irradiated with blue light from a light emitting diode light source (470nm±20nm).

[0150] In vitro phase separation of cGAS, accessory proteins, and dsDNA regulated by an inducible system

[0151] Cy5-cGAS, AF488-labeled accessory proteins, and dsDNA were incubated for 10 min in 5 mM Tris-HCl pH 7.5, 300 mM NaCl, and 1 mM DTT to form phase-separated aggregates on coverslips (ThermoFisher Scientific). 200 nM rapamycin was added when necessary. In the light-induced system, the mixed samples were exposed to 1 Wm during the 2-hour incubation. -2 Blue light irradiation from a light emitting diode light source (470 nm ± 20 nm). Experiments were performed on an AXR NSPARC (Nikon) confocal microscope using a 100× / 1.42NA oil objective. Image acquisition time was 30 min. Time-lapse images were processed using ImageJ.

[0152] Cell culture and plasmid transfection

[0153] HEK293T cells were cultured in Gibco-Double modified Eagle medium (DMEM) supplemented with 10% (v / v) fetal bovine serum (FBS), 2 mM l-glutamine, penicillin (100 U / mL) and streptomycin (100 mg / mL). The corresponding plasmids were transfected with polyethyleneimine mitochondria (PEI).

[0154] When using the rapamycin-induced cGAS phase separation and signaling system, the medium was replaced with fresh DMEM supplemented with 200 nM rapamycin. When using the light-induced dimerization system, the cells were cultured under a 470 nm ± 20 nm blue light source. After 48 hours, the cells were lysed for subsequent experiments.

[0155] Western blot analysis

[0156] HEK293T cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer containing a protease inhibitor cocktail and a phosphatase inhibitor. The lysate was centrifuged at 13,000 rpm for 10 min, and the supernatant was collected for measurement of protein concentration using the bicinchoninic acid (BCA) assay. After incubation with SDS loading buffer at 95°C for 10 min, protein samples were separated by 8% SDS polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membranes, incubated with blocking buffer containing 5% (w / v) milk and 0.1% Tween-20 (TBST) overnight at 4°C, and then incubated with primary antibodies for 2 h at room temperature and with secondary antibodies for 1 h at room temperature. Protein bands were visualized using a chemiluminescent substrate, and images were acquired using an iBright 1500 imaging system. Densitometric analysis was performed using ImageJ software.

[0157] RT-qPCR analysis

[0158] Cells were seeded into six-well plates and total RNA was extracted using HiPure Total RNA Kit (MGBIO). The diluted RNA was reverse transcribed using a cDNA synthesis kit (ThermoFisher Scientific). PCR amplification reactions were performed using specific primers and PCR MasterMix (ThermoFisher Scientific) using the cDNA obtained by reverse transcription as a template. Samples were analyzed using the Quantagene q225qPCR system (KUBO). All reagents were used according to the manufacturer's instructions. GAPDH (human) was used as an internal control.

[0159] 2. Experimental Results

[0160] (1) In the chemically induced design, FRB is fused to the C-terminus of cGAS and FKBP is fused to the N-terminus of the auxiliary protein. The fusion of FRB or FKBP has little effect on their interaction with dsDNA and the binding affinity between cGAS and auxiliary proteins such as viral tegument proteins. When rapamycin is added to the fused cGAS system, it induces FKBP and FRB to form dimers, thereby strengthening the interaction between cGAS-FRB and FKBP-auxiliary proteins and promoting the formation of phase separation ( Figure 1 A).

[0161] In the optically induced design, a cGAS phase separation and signal transduction system that can be regulated by light induction is formed by using regulatory proteins such as the pMag-nMagHigh1 pair that can form dimers by light induction. pMag is fused to the C-terminus of cGAS, and nMagHigh1 is fused to the N-terminus of the auxiliary protein. Irradiation with blue light can promote the formation of dimers between pMag and nMagHigh1, thereby promoting the binding of cGAS and auxiliary proteins, further promoting the formation of phase separation and downstream signal transduction ( Figure 1 B).

[0162] (2) During chemical induction, by fusing FRB to the C-terminus of cGAS and FKBP to the N-terminus of the accessory protein, the binding affinity between cGAS and the accessory protein increased by two orders of magnitude after the addition of rapamycin (Rapa) compared to the absence of rapamycin (No Rapa). Figure 2 In the in vitro phase transition experiment, the auxiliary protein will exclude cGAS from the condensed phase and inhibit the activity of cGAS. However, through the design of the present invention, cGAS-FRB is induced to re-enter the aggregated phase 2 minutes after the addition of rapamycin ( Figure 2 B) and increased the activity of cGAS by 2-5 times. In living cells, the addition of rapamycin induced cGAS to enter phase-separated puncta within 5-15 minutes, thereby activating downstream signal transduction ( Figure 2 C). Phosphorylation levels of TBK1 and IRF3 in cells ( Figure 2 D) and IFN-β mRNA expression levels were significantly increased after the addition of rapamycin ( Figure 3 ). This verifies that the method of the present invention can promote the phase separation of cGAS and the activation of downstream immune signals both in vitro and in cells by chemical induction.

[0163] During the light induction process, pMag was fused to the C-terminus of cGAS and nMaHigh to the N-terminus of the auxiliary protein. Through blue light irradiation, the binding of cGAS and its auxiliary protein can be regulated, thereby promoting the formation of phase separation and downstream signal transduction. In the in vitro phase transition experiment, cGAS entered the aggregation phase within 5 minutes through blue light irradiation ( Figure 4 In A), under 120 minutes of blue light irradiation (Blue Light), the activity of cGAS increased by 2-5 times compared with that without irradiation (No Light). Figure 4 In cells, blue light irradiation for 5-10 minutes can induce cGAS to enter phase transition aggregation points (puncta) ( Figure 4 C and Figure 4 D in the figure), and this phase transition is reversible. After 5 hours of blue light irradiation (Light off), cGAS separates from the phase transition aggregation point ( Figure 4 E).

[0164] The amino acid sequence involved in the present invention is:

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171] It should be noted that, although the technical solution of the present invention is introduced with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0172] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for regulating liquid-liquid phase separation of cGAS and DNA, wherein: The method comprises the steps of regulating the affinity between cGAS and an accessory protein; Optionally, the auxiliary protein includes an auxiliary protein that promotes cGAS activity or an auxiliary protein that inhibits cGAS activity; Optionally, the auxiliary protein that promotes cGAS activity is selected from PCBP1 (SEQ ID NO: 2) or G3BP1 (SEQ ID NO: 3); Optionally, the auxiliary protein that inhibits cGAS activity is selected from ORF9 (SEQ ID NO: 4), ORF52 (SEQ ID NO: 5) or VP22 (SEQ ID NO: 6).

2. The method according to claim 1, wherein: The regulating the liquid-liquid phase separation of cGAS and DNA includes irreversibly or reversibly promoting the formation of the liquid-liquid phase separation of cGAS and DNA.

3. The method according to claim 1 or 2, wherein: The cGAS and the auxiliary protein are respectively combined with the multimerization domain of the multimer, and the affinity between cGAS and the auxiliary protein is adjusted by adjusting the multimerization domain to form a multimer.

4. The method according to claim 3, wherein: The polymer is an induced polymer.

5. The method according to any one of claims 1 to 4, wherein: The cGAS and the auxiliary protein are fused to the FRB multimerization domain and the FKBP multimerization domain, respectively.

6. The method according to claim 5, wherein: Rapamycin or its analogue is used as a multimerizing agent to promote the formation of dimers between the FRB multimerization domain and the FKBP multimerization domain.

7. The method according to any one of claims 1 to 4, wherein: The cGAS and the auxiliary protein are fused to the pMag multimerization domain and the nMag multimerization domain, respectively.

8. The method according to claim 7, wherein: Blue light is used as a multimerizing agent to promote the formation of dimers between the pMag multimerization domain and the nMag multimerization domain.

9. A composition, wherein The composition includes a first fusion protein and a second fusion protein, wherein the first fusion protein includes cGAS and a first multimerization domain; The second fusion protein includes an accessory protein and a second multimerization domain; The first multimerization domain and the second multimerization domain form a multimer under the induction of a multimerization agent; Optionally, the auxiliary protein includes an auxiliary protein that promotes cGAS activity or an auxiliary protein that inhibits cGAS activity; Optionally, the auxiliary protein that promotes cGAS activity is selected from PCBP1 (SEQ ID NO: 2), G3BP1 (SEQ ID NO: 3); Optionally, the auxiliary protein that inhibits cGAS activity is selected from ORF9 (SEQ ID NO: 4), ORF52 (SEQ ID NO: 5) or VP22 (SEQ ID NO: 6); Optionally, the composition further comprises a polymerizing agent.

10. Any of the following uses of the composition according to claim 9: (a) Use in the preparation of a reagent for regulating liquid-liquid phase separation of cGAS and DNA; (b) Use in the preparation of an agent for regulating the cGAS-STING pathway; (c) Use in the preparation of drugs for preventing, treating and / or assisting in the treatment of diseases associated with the cGAS-STING pathway.

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