Fusion protein, related molecule thereof, method therefor, and use thereof

Abstract

Provided is a fusion protein, comprising a stimulator of interferon genes (STING) component and a response component, wherein the STING component comprises at least one STING protein fragment capable of specifically binding to a specific ligand, and the response component, in response to the specific binding, directly or indirectly generates a detectable signal.

Description

A fusion protein and its related molecules, methods and applications Cross-referencing

[0001] This application claims priority to Chinese application No. 202510330457.7, filed on March 18, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This specification relates to the field of biotechnology, and in particular to a fusion protein and its applications. Background Technology

[0003] Cyclic GMP-AMP (cGAMP) acts as a danger signaling molecule in cells, mediating the innate immune response triggered by cell-free DNA in the cytoplasm. Under normal conditions, DNA, the genetic material of most organisms, is encased within organelles such as the nucleus, mitochondria, or chloroplasts and does not circulate freely in the cytoplasm. However, under the influence of external factors, such as leakage of chromosomal or mitochondrial DNA, or when cells are infected by viruses or bacteria, cell-free DNA appears in the cytoplasm. This is a danger signal for the cell, triggering the cGAS-cGAMP-STING signaling pathway, a mechanism of innate immune response. Specifically, the free DNA first activates cyclic GMP-AMP synthase (cGAS), which catalyzes the synthesis of cGAMP using adenosine triphosphate (ATP) and guanosine triphosphate (GTP) as substrates. Subsequently, the generated cGAMP binds to the stimulator of interferon genes (STING) on the endoplasmic reticulum. Activated STING migrates from the endoplasmic reticulum to the Golgi apparatus and intermediate structure (ERGIC), recruiting downstream proteins TANK-binding kinase 1 (TBK1) and IκB kinase (IKK). These activate interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB) transcription factor, respectively, upregulating the expression of type I interferon (IFN-I) and inflammatory cytokines. Furthermore, similar proteins to cGAS and STING are widely present in eukaryotic and prokaryotic cells, demonstrating the high evolutionary conservation of the cGAS-cGAMP-STING signaling pathway. cGAMP signaling also plays an important role in pathologies such as viral infection, tumorigenesis, and neuroinflammation. During viral infection, cGAS detects related DNA synthesis and activates antiviral immune responses. Tumor cells contain abundant free DNA, leading to increased cGAMP signaling; early activation of the cGAS-cGAMP-STING signaling pathway can play a role in immune surveillance. In the nervous system, increased cGAMP signaling in microglia triggers neuroinflammation and is associated with various neurodegenerative diseases. Despite its significant role in the development of many diseases, current technologies for detecting cGAMP signaling are extremely limited.

[0004] Currently, methods for detecting cGAMP mainly include liquid chromatography-mass spectrometry (LC-MS), nucleic acid aptamer-coupled fluorescent dye method, multi-stage enzyme-coupled bioluminescence method, and fluorescent protein-based fluorescence resonance energy transfer probe method. Among these, LC-MS allows for quantification of isolated samples using mass spectrometry, but suffers from low sampling frequency, poor spatial resolution, high equipment requirements, and does not meet high-throughput needs. Nucleic acid aptamer-coupled fluorescent dye method utilizes specific aptamers to bind to cGAMP, triggering fluorescence changes for detection, but suffers from poor molecular specificity, high cost, and susceptibility to environmental influences. Multi-stage enzyme-coupled bioluminescence method indirectly reflects cGAMP content by converting cGAMP with multiple enzymes, but is susceptible to inherent AMP / ATP interference, cumbersome detection process, and poor specificity. Fluorescent protein-based fluorescence resonance energy transfer probe method links fluorescent proteins to both ends of a STING protein, utilizing conformational changes when the fluorescent protein binds to cGAMP to generate fluorescence resonance energy transfer to monitor the cGAMP signal; however, it has a small dynamic monitoring range, poor signal-to-noise ratio, and is difficult for real-time in vivo monitoring.

[0005] Therefore, it is necessary to develop detection methods and reagents for cGAMP, especially for the cGAS-cGAMP-STING signaling pathway, in order to achieve high specificity, high sensitivity and high throughput screening of the cGAS-cGAMP-STING signaling pathway and related substances. Summary of the Invention

[0006] This specification provides one or more embodiments of a fusion protein comprising: a Stimulator of interferon genes (STING) component, the STING component comprising at least one STING protein fragment; and a response component; wherein the response component is directly or indirectly connected to the STING component, the STING component is capable of specifically binding to a specific ligand, and the response component generates a detectable signal directly or indirectly in response to the specific binding.

[0007] In some embodiments, the fusion protein further includes a connecting member, through which the responsive member is connected to the STING member.

[0008] In some embodiments, the complete STING is a dimer structure, and the STING protein fragment includes one of the dimers, a portion of a dimer, or a mutant of a dimer.

[0009] In some embodiments, the STING protein fragment comprises the amino acid sequence shown in SEQ ID NO.3.

[0010] In some embodiments, the responsive component is linked to a STING protein fragment.

[0011] In some embodiments, the specific ligand includes a signaling molecule and a STING agonist.

[0012] In some embodiments, the response component includes a responder, which is capable of undergoing a conformational change in response to specific binding, and the responder can generate a detectable signal directly or indirectly in response to the conformational change.

[0013] In some embodiments, the linker includes at least one linker peptide that links the STING protein fragment and the response component.

[0014] This specification provides one or more embodiments of a polynucleotide encoding the above-described fusion protein.

[0015] This specification provides an expression vector for the above-described polynucleotide through one or more embodiments.

[0016] This specification provides one or more embodiments of a host cell comprising the above-described fusion protein, the above-described polynucleotide, and / or the above-described expression vector.

[0017] This specification provides one or more embodiments for the application of the above-described fusion protein in the detection of cGAMP levels or cGAS activity.

[0018] This specification provides one or more embodiments for the application of the above-described fusion protein in the preparation of reagents or kits for detecting cGAMP content or cGAS activity.

[0019] This specification provides one or more embodiments for the application of the above-described fusion protein in the preparation of a kit for detecting cGAS-STING pathway agonists and inhibitors.

[0020] This specification provides one or more embodiments for the application of the above-described fusion protein in screening drugs for the treatment of diseases related to the cGAS-STING pathway.

[0021] This specification provides one or more embodiments of a method for screening cGAS-STING pathway agonists and inhibitors using the above-described fusion protein. The method includes: expressing the fusion protein in vivo or in vitro; establishing a standard signal intensity; reacting the fusion protein with a candidate and measuring a detectable signal intensity, wherein an increase in the detectable signal intensity based on the standard signal intensity indicates that the candidate is a cGAS-STING pathway agonist, and a decrease in the detectable signal intensity based on the standard signal intensity indicates that the candidate is a cGAS-STING inhibitor.

[0022] One or more embodiments of this specification also provide a kit for identifying candidate active substances that can affect the cGAS-STING pathway, the kit comprising: the aforementioned fusion protein or the aforementioned host cell; and a detection reagent used to measure the signal intensity or change of a detectable signal generated by the candidate contacting the fusion protein or the host cell.

[0023] One or more embodiments of this specification also provide a reagent for detecting cGAMP levels, cGAS activity, or cGAS-STING pathway agonists and inhibitors, the reagent comprising: the above-described fusion protein or the above-described host cell.

[0024] One or more embodiments of this specification also provide a reagent for detecting mitochondrial DNA leakage, the reagent comprising: the above-described fusion protein or the above-described host cell.

[0025] One or more embodiments of this specification also provide a reagent for detecting DNA virus infection, the reagent comprising: the above-described fusion protein or the above-described host cell. Attached Figure Description

[0026] This specification will be further illustrated by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings.

[0027] Figure 1 is a schematic diagram of a fusion protein according to some embodiments of this specification;

[0028] Figure 2 shows the single-photon spectrum (a) and two-photon spectrum (b) of the fusion protein according to some embodiments of this specification;

[0029] Figure 3 is a graph showing the binding affinity of the fusion protein to cGAMP according to some embodiments of this specification;

[0030] Figure 4 is a graph showing the in vitro detection results of STING agonist for fusion protein pairs according to some embodiments of this specification;

[0031] Figure 5 is a graph showing the real-time detection results of cGAMP generated by cGAS catalysis in vitro using the fusion protein shown in some embodiments of this specification.

[0032] Figure 6 is a graph showing the detection results of cGAMP in cells for the fusion protein according to some embodiments of this specification;

[0033] Figure 7 is a graph showing the detection results of HT-DNA in cells by the fusion protein according to some embodiments of this specification;

[0034] Figure 8 is a graph showing the detection results of cGAMP production in cells after mitochondrial DNA leakage by the fusion protein according to some embodiments of this specification;

[0035] Figure 9 is a graph showing the detection results of cGAMP production in cells after DNA virus infection by the fusion protein according to some embodiments of this specification.

[0036] Figure 10 is a graph showing the detection results of STING agonist in a live brain slice of the fusion protein according to some embodiments of this specification;

[0037] Figure 11 is a graph showing the detection results of the fusion protein against STING agonist in live animals according to some embodiments of this specification; and

[0038] Figure 12 is a graph showing the detection results of cGAMP content in human serum samples by the fusion protein according to some embodiments of this specification. Detailed Implementation

[0039] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort.

[0040] The following are the meanings of some commonly used abbreviations and English words in this instruction manual:

[0041] cGAMP, cyclic GMP-AMP, cyclic guanosine monophosphate-adenosine monophosphate;

[0042] cGAS, cyclic GMP-AMP synthase;

[0043] ATP, adenosine triphosphate;

[0044] GTP, guanosine triphosphate;

[0045] STING, stimulator of interferon genes;

[0046] ERGIC, ER-Golgi intermediate compartment, Golgi apparatus and intermediate structure;

[0047] TBK1, TANK-binding kinase 1;

[0048] IKK, IκB kinase;

[0049] IRF3, interferon regulatory factor 3;

[0050] NF-κB, nuclear factor kappa B;

[0051] IFN-I, type I interferon;

[0052] AMP, adenosine monophosphate;

[0053] c-di-GMP, cyclic diguanosine monophosphate;

[0054] c-di-AMP, cyclic diadenosine monophosphate;

[0055] cpGFP, circularly permuted green fluorescent protein;

[0056] cpYFP, ​​circularly permuted yellow fluorescent protein;

[0057] cpRFP, circularly permuted red fluorescent protein;

[0058] cpBFP, circularly permuted blue fluorescent protein;

[0059] cpEGFP, circularly permuted enhanced green fluorescent protein;

[0060] cpEYFP, circularly permuted enhanced yellow fluorescent protein;

[0061] cpiRFP, circularly permuted infrared fluorescent protein;

[0062] split-GFP, a split-green fluorescent protein;

[0063] split-YFP, a split-yellow fluorescent protein;

[0064] split-RFP, a split-red fluorescent protein;

[0065] spit-BFP, split-blue fluorescent protein;

[0066] split-EGFP, a split-enhanced green fluorescent protein;

[0067] split-EYFP, split-enhanced yellow fluorescent protein;

[0068] split-iRFP, split-infrared fluorescent protein;

[0069] 6XHis tag, hexa histidine-tag;

[0070] T7 tag, T7 epitope tag, T7 label;

[0071] Xpress tag;

[0072] IRES: internal ribosome entry site;

[0073] RBS: ribosome-binding site;

[0074] PBS, phosphate-buffered saline;

[0075] AAV, adeno-associated virus;

[0076] HSV-1, Herpes Simplex Virus type 1;

[0077] VACV, Vaccinia Virus.

[0078] As used herein, "fusion protein" refers to a complex polypeptide, that is, a continuous amino acid sequence composed of two or more polypeptides. Fusion proteins can be artificially prepared using recombinant nucleic acid methods, for example, by recombining polynucleotide sequences encoding two or more polypeptides and expressing them in host cells. The polynucleotides can be RNA or DNA sequences.

[0079] As used herein, a "specific ligand" is a molecule that binds selectively only to a specific target molecule (such as a protein, receptor, or other molecule). In this specification, "specific ligands" primarily include molecules that selectively bind to the STING receptor.

[0080] As used herein, "signaling molecules" refer to certain chemical molecules within an organism that can transmit information between or within cells, regulate cellular physiological activities and functions, coordinate the activities of various cells in a multicellular organism, and enable the organism as a whole to respond to environmental changes. "Signaling molecules" can specifically bind to signaling molecule receptors inside or on the cell surface, initiating intracellular signal transduction processes, thereby enabling the cell to respond accordingly to the signal. The basic scheme of this invention, in addition to being applied to signaling molecules and their receptors, may also be applicable to other combinations of molecules and receptors.

[0081] This specification provides a fusion protein comprising: a Stimulator of interferon genes (STING) component, the STING component comprising at least one STING protein fragment; and a response component; wherein the response component is directly or indirectly connected to the STING component, the STING component is capable of specifically binding to a specific ligand, and the response component generates a detectable signal directly or indirectly in response to the specific binding.

[0082] STING constructs refer to the portions of fusion proteins derived from the STING receptor.

[0083] As used herein, a “STING construct” is derived from a naturally occurring STING receptor and has the same or similar structure and / or sequence as the STING receptor. In some embodiments, the STING receptor from which the STING construct is derived may be from different organisms; for example, the STING receptor may be derived from mammals; or, for example, the STING receptor may be of mouse or human origin.

[0084] In some embodiments, the complete STING is a dimer, and the STING protein fragment includes one of the dimers, a portion of a dimer, or a mutant of a dimer.

[0085] In some embodiments, the STING component may include a first STING protein fragment and a second STING protein fragment. For example, the first STING protein fragment and the second STING protein fragment are respectively one and the other of a dimer form of the STING receptor. Alternatively, the first STING protein fragment and the second STING protein fragment are respectively a portion of one and the other of a dimer form of the STING receptor. In some embodiments, the first STING protein fragment and the second STING protein fragment are identical. In some embodiments, the first STING protein fragment and the second STING protein fragment are different.

[0086] In some embodiments, the STING component may include a STING protein fragment. In some embodiments, the STING component includes only a STING protein fragment.

[0087] In some embodiments, the STING protein fragment may include the entire NCBI reference sequence NP_082537.1, a portion thereof, or a mutant thereof (but retaining the function of specifically binding to cGAMP). In some embodiments, the STING protein fragment may include the entire PDB reference sequence 4YP1_A, a portion thereof, or a mutant thereof (but retaining the function of specifically binding to cGAMP).

[0088] In some embodiments, the STING protein fragment may include the entire, a portion, or a mutant of the GenBank sequence AVQ94753.1 (but retaining the function of specifically binding to cGAMP). In some embodiments, the STING protein fragment may include the entire, a portion, or a mutant of the PDB (Reference Sequence) 6DXG_A (but retaining the function of specifically binding to cGAMP).

[0089] In some embodiments, the STING protein fragment includes the cGAMP binding domain of STING, a portion thereof, or a mutant thereof.

[0090] In some embodiments, the amino acid sequence of the STING protein fragment has at least 80% similarity to SEQ ID NO.3. In some embodiments, the amino acid sequence of the STING protein fragment has at least 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, 99%, 100%, or any value within the range formed by any two of the above values, similarity to SEQ ID NO.3.

[0091] In some embodiments, the amino acid sequence of the fusion protein of this specification has at least 80% similarity to SEQ ID NO.1 or SEQ ID NO.2. In some embodiments, the fusion protein of this specification comprises an amino acid sequence having at least 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, 99%, 100%, or any value within the range formed by any two of the above values, similar to SEQ ID NO.1. In some embodiments, the fusion protein of this specification comprises an amino acid sequence having at least 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, 99%, 100%, or any value within the range formed by any two of the above values, similar to SEQ ID NO.2.

[0092] The STING component can specifically bind to a specific ligand. In some embodiments, the specific ligand may include a signaling molecule, a small molecule compound, or other molecule capable of specifically binding to the STING component. In some embodiments, the specific ligand may be a signaling molecule. In some embodiments, the specific ligand may be a molecule having a similar structure and / or sequence to a naturally occurring or artificially synthesized signaling molecule. In some embodiments, the signaling molecule may be derived from different organisms, such as mammals. For example, the signaling molecule may be mouse-derived or human-derived.

[0093] In some embodiments, the signaling molecule may be selected from at least one of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), cyclic diguanosine monophosphate (c-di-GMP), and cyclic diadenosine monophosphate (c-di-AMP).

[0094] In some embodiments, when the signaling molecule is selected from at least one of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), cyclic diguanosine monophosphate (c-di-GMP), and cyclic diadenosine monophosphate (c-di-AMP), the STING component may include a receptor or a portion of a receptor that specifically binds to cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), cyclic diguanosine monophosphate (c-di-GMP), and cyclic diadenosine monophosphate (c-di-AMP).

[0095] In some embodiments, when the specific ligand is cGAMP, the STING component may include a domain that specifically binds to cGAMP, a portion thereof, or a mutant thereof (but retains the function of specifically binding to cGAMP). For example, the STING component may include a first polymer and / or a second polymer, which may be the same or different.

[0096] In some embodiments, the STING component may be part of a receptor that specifically binds to cGAMP. For example, the STING component may include a portion of a first polymer and / or a portion of a second polymer, wherein the portion of the first polymer and the portion of the second polymer may be the same or different.

[0097] In some embodiments, the specific ligand may be a STING agonist. For example, STING agonists may include DMXAA (Vadimezan), ADU-S100 (MIW815), MK-1454, SR-717, G10, diABZI, MSA-2, SN-011, SB 11285, E7766, IACS-8803, MK-2118, TDI-01, BI-1387446, GSK3745417, BMS-986301, and XMT-2056. This specification does not impose specific limitations on the selection of the specific ligand.

[0098] A response component is a molecule in a fusion protein that can respond to conformational changes and convert those changes into a detectable signal. In some embodiments, the response component is linked to a STING protein fragment.

[0099] In some embodiments, the STING component includes a first STING protein fragment and a second STING protein fragment, and the response component includes a responder connected to the C-terminus of the first STING protein fragment and the N-terminus of the second STING protein fragment.

[0100] In some embodiments, the STING component includes a STING protein fragment, and the responsive component includes a first responder and a second responder, wherein the first responder is connected to the N-terminus of the STING protein fragment, and the second responder is connected to the C-terminus of the STING protein fragment.

[0101] In some embodiments, when a specific ligand specifically binds to the STING member, the STING member undergoes a conformational change, and in response to this conformational change, the response member can directly or indirectly generate a detectable signal.

[0102] In some embodiments, the detectable signal is a light signal, a chemical signal, or a thermal signal. In some embodiments, the detectable signal is a light signal, and the responder is selected from at least one of fluorescent proteins, luciferases, and protein tags that can covalently bind to fluorescent ligands. For example, the responder can be a fluorescent protein, a cyclically rearranged fluorescent protein, a luciferase or a cyclically rearranged luciferase, a protein tag that can covalently bind to fluorescent ligands, or a cyclically rearranged protein tag that can covalently bind to fluorescent ligands. In some embodiments, the fluorescent protein is selected from cyclically rearranged fluorescent proteins. In some embodiments, the cyclically permuted fluorescent protein is selected from at least one of the following: cyclically permuted green fluorescent protein (cpGFP), cyclically permuted yellow fluorescent protein (cpYFP), cyclically permuted red fluorescent protein (cpRFP), cyclically permuted blue fluorescent protein (cpBFP), cyclically permuted enhanced green fluorescent protein (cpEGFP), cyclically permuted enhanced yellow fluorescent protein (cpEYFP), and cyclically permuted infrared fluorescent protein (cpiRFP). In some embodiments, the cyclically permuted fluorescent protein is selected from cpGFP, which includes at least one of cpCitrine, cpmCitrine, cpmGold, cpYPet, cpFOLD6, cpmNeonGreen, cpStayGold, cpmBaojin, or cpmClover3. In some embodiments, the fluorescent protein undergoing cyclic rearrangement is cpmClover3.

[0103] In some embodiments, the fluorescent protein is selected from split fluorescent proteins. In some embodiments, the split fluorescent protein is selected from at least one of split-green fluorescent protein (Split-GFP), split-yellow fluorescent protein (Split-YFP), split-red fluorescent protein (Split-RFP), split-blue fluorescent protein (Split-BFP), split-enhanced green fluorescent protein (Split-EGFP), split-enhanced yellow fluorescent protein (Split-EYFP), and split-infrared fluorescent protein (Split-iRFP). In some embodiments, the splitting fluorescent protein is selected from Split-GFP, which includes Split-Citrine, Split-mCitrine, Split-mGold, Split-YPet, Split-FOLD6, split-NeonGreen, split-StayGold, split-mBaojin, or Split-mClover3. In some embodiments, the splitting fluorescent protein is Split-mClover3.

[0104] In some embodiments, the response component can indirectly generate a detectable signal in response to the specific binding of a specific ligand to the STING component. For example, the signal can be amplified before detection using techniques such as enzyme-linked reactions, cascade reactions, biotin-avidin systems, or nucleic acid amplification.

[0105] In some embodiments, the fusion protein further includes a connecting member through which the responsive member is connected to the STING member. In some embodiments, the responsive member is connected to the STING member via the connecting member.

[0106] A linker is a plurality of amino acid residues or small molecule compounds or combinations thereof used to connect the STING member and the responsive member. In some embodiments, the linker is essential. In other embodiments, the linker may be omitted, i.e., the responsive member and the STING member may be directly linked.

[0107] In some embodiments, the linker includes at least one linker peptide that links the STING protein fragment and the response component.

[0108] In some embodiments, the linker includes a first linker peptide and a second linker peptide. In some embodiments, the length of the first linker peptide or the second linker peptide is 3-10 amino acids. In some embodiments, the length of the first linker peptide is 3 amino acids, and the length of the second linker peptide is 9 amino acids. In some embodiments, the length of the first linker peptide is 9 amino acids, and the length of the second linker peptide is 3 amino acids. In some embodiments, the amino acid sequence of one of the first linker peptide and the second linker peptide is GEF, and the amino acid sequence of the other is LRGGGSGPC (SEQ ID NO. 5).

[0109] In some embodiments, the STING component includes a first STING protein fragment and a second STING protein fragment, and the responsive component includes a responder connected to the C-terminus of the first STING protein fragment via a first linker peptide and to the N-terminus of the second STING protein fragment via a second linker peptide. For example, the STING component includes a first cGAMP-binding domain and a second cGAMP-binding domain, and the responsive component includes cpmClover3, which is connected to the C-terminus of the first cGAMP-binding domain via the first linker peptide and to the N-terminus of the second cGAMP-binding domain via the second linker peptide. The amino acid sequence of the first linker peptide is GEF, and the amino acid sequence of the second linker peptide is LRGGGSGPC (SEQ ID NO. 5).

[0110] In some embodiments, the linker includes a first linker peptide or a second linker peptide. In some embodiments, the length of the first linker peptide or the second linker peptide is 3-10 amino acids. In some embodiments, the length of the first linker peptide or the second linker peptide is 3 amino acids or 9 amino acids. In some embodiments, the amino acid sequence of the first linker peptide or the second linker peptide is GEF or LRGGGSGPC (SEQ ID NO. 5). For example, the STING component includes a first cGAMP-binding domain and a second cGAMP-binding domain, and the responsive component includes cpmClover3, which is linked to the C-terminus of the first cGAMP-binding domain via the first linker peptide or the second linker peptide, and cpmClover3 is directly linked to the N-terminus of the second cGAMP-binding domain. The amino acid sequence of the first linker peptide is GEF. As another example, cpmClover3 is directly linked to the C-terminus of the first cGAMP-binding domain, and cpmClover3 is linked to the N-terminus of the second cGAMP-binding domain via the first linker peptide or the second linker peptide. The amino acid sequence of the first linker peptide or the second linker peptide is LRGGGSGPC (SEQ ID NO. 5).

[0111] In some embodiments, the responsive component includes a first responder and a second responder. The first responder is linked to the N-terminus of the STING protein fragment via a first linker peptide, and the second responder is linked to the C-terminus of the STING protein fragment via a second linker peptide. For example, the responsive component includes a first Split-mClover3 and a second Split-mClover3. The first Split-mClover3 is linked to the N-terminus of the cGAMP-binding domain via the first linker peptide, and the second Split-mClover3 is linked to the C-terminus of the cGAMP-binding domain via the second linker peptide. The amino acid sequence of the first linker peptide is LRGGGSGPC (SEQ ID NO. 5), and the amino acid sequence of the second linker peptide is GEF.

[0112] In some embodiments, the linker includes a first linker peptide or a second linker peptide. In some embodiments, the length of the first linker peptide or the second linker peptide is 3-10 amino acids. In some embodiments, the length of the first linker peptide or the second linker peptide is 9 amino acids or 3 amino acids. In some embodiments, the amino acid sequence of the first linker peptide or the second linker peptide is LRGGGSGPC (SEQ ID NO. 5) or GEF. For example, the responsive member includes a first Split-mClover3 and a second Split-mClover3, wherein the first Split-mClover3 is linked to the N-terminus of the cGAMP-binding domain via the first linker peptide or the second linker peptide, and the second Split-mClover3 is directly linked to the C-terminus of the cGAMP-binding domain. The amino acid sequence of the first linker peptide or the second linker peptide is LRGGGSGPC (SEQ ID NO. 5). As another example, the second Split-mClover3 is directly linked to the C-terminus of the cGAMP-binding domain, and the first Split-mClover3 is linked to the N-terminus of the cGAMP-binding domain via the first linker peptide or the second linker peptide. The amino acid sequence of the first linker peptide or the second linker peptide is GEF.

[0113] In some embodiments, the fusion protein further includes a tag that may include chemical groups or peptides. This tag can be used for a variety of functions, such as protein purification, signal regulation, and protein localization.

[0114] In some embodiments, the label is a protein purification label, which includes at least one of a hexa histidine-tag (6XHis tag), a T7 epitope tag (T7 tag), and an Xpress tag. This specification does not impose specific limitations on the choice of protein purification label.

[0115] Figure 1 is a schematic diagram of a fusion protein according to some embodiments of this specification. The fusion protein constructed according to this specification will be illustrated using Figure 1 as an example. A STING component (such as a cGAMP-binding domain, STING CBD) and a responsive component (such as cpmClover3) are linked by linker peptides (such as a first linker peptide and a second linker peptide). For example, the STING component includes a first cGAMP-binding domain and a second cGAMP-binding domain, and the responsive component includes cpmClover3, which is linked to the C-terminus of the first cGAMP-binding domain via a first linker peptide and to the N-terminus of the second cGAMP-binding domain via a second linker peptide. As another example, the responsive component includes a first split-mClover3 and a second split-mClover3, where the first split-mClover3 is linked to the N-terminus of the cGAMP-binding domain via a first linker peptide and the second split-mClover3 is linked to the C-terminus of the cGAMP-binding domain via a second linker peptide. Specific ligands (such as cGAMP) bind specifically to STING constructs (such as STING CBD), and response constructs (such as cpmClover3 or split-mClover3) generate detectable signals in response to the specific binding of the specific ligands to the STING constructs (such as STING CBD). The degree of activation of the STING construct (such as STING CBD) receptor by the specific ligands (e.g., cGAMP) is determined by detecting the intensity or changes of the signal.

[0116] The embodiments in this specification also provide a polynucleotide encoding the above-described fusion protein.

[0117] The embodiments of this specification also provide an expression vector containing the above-described polynucleotides.

[0118] As used herein, an "expression vector" refers to a gene construct capable of expressing a target protein in a host cell. The expression vector carries polynucleotides encoding the STING and response components of the fusion protein. In some embodiments, the expression vector may also contain operatively linked basic regulatory elements, such as promoters, cis-elements (e.g., enhancers, splice sequences, IRES, RBS, etc.).

[0119] In some embodiments, the expression vector can also be obtained by inserting the aforementioned polynucleotides into a suitable vector. For example, inserting plasmid vectors, phage vectors, and viral vectors. Another example is inserting animal viruses such as retroviruses, adenoviruses, adeno-associated viruses, and vaccinia viruses, or insect viruses such as baculoviruses. This specification does not impose specific limitations on the choice of vector.

[0120] Expression vectors can transform or transfect host cells to express polynucleotides.

[0121] This specification also provides a host cell comprising the above-described fusion protein, the above-described polynucleotide, and / or the above-described expression vector. In some embodiments, the host cell may include mammalian cells, such as mouse cells or human cells. In some embodiments, the host cell may include prokaryotic cells, such as Escherichia coli, Bacillus subtilis, lactic acid bacteria, etc. This specification does not impose specific limitations on the selection of the host cell.

[0122] This specification also provides a transgenic animal comprising the above-described fusion protein, the above-described polynucleotide, the above-described expression vector, and / or the above-described host cell. In some embodiments, the transgenic animal may include vertebrates, such as zebrafish, mice, rhesus monkeys, and marmosets. In some embodiments, the transgenic animal may include invertebrates, such as fruit flies, octopuses, and nematodes. This specification does not impose specific limitations on the selection of transgenic animals.

[0123] The embodiments in this specification also provide an application of the fusion protein in detecting cGAMP content or cGAS activity.

[0124] The embodiments in this specification also provide the application of a fusion protein in the preparation of reagents or kits for detecting cGAMP content or cGAS activity.

[0125] This specification also provides a method for screening cGAS-STING pathway agonists and inhibitors using fusion proteins. The method includes: expressing the fusion protein in vivo or in vitro; establishing a standard signal intensity; and reacting the fusion protein with a candidate, measuring the detectable signal intensity. An increase in the detectable signal intensity based on the standard signal intensity indicates that the candidate is a cGAS-STING pathway agonist, and a decrease in the detectable signal intensity based on the standard signal intensity indicates that the candidate is a cGAS-STING inhibitor.

[0126] Candidates can be small molecule compounds or large biomolecules. In some embodiments, candidates can activate, inhibit, or have no effect on the cGAS-STING pathway.

[0127] In some embodiments, establishing a standard signal intensity includes: purifying the fusion protein, forming a mixture with ATP, GTP, DNA, and MgCl2, and detecting the detectable signal intensity as the standard signal intensity.

[0128] In some embodiments, the signal strength of the detectable signal is enhanced by at least 20% to 300%, and this enhancement is used to determine the candidate as a cGAS-STING pathway agonist. In some embodiments, the signal strength of the detectable signal is enhanced by at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, at least 200%, at least 220%, at least 240%, at least 260%, at least 280%, or at least 300%, and this enhancement is used to determine the candidate as a cGAS-STING pathway agonist. That is, only when a preset signal enhancement threshold is reached can the candidate be determined as a cGAS-STING pathway agonist.

[0129] In some embodiments, the signal strength of the detectable signal is attenuated by at least 5% to 99%, and this attenuation is used to determine the candidate as a cGAS-STING pathway inhibitor. In some embodiments, the signal strength of the detectable signal is attenuated by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, and this attenuation is used to determine the candidate as a cGAS-STING pathway inhibitor. That is, only when a preset signal attenuation threshold is reached can the candidate be determined as a cGAS-STING inhibitor.

[0130] In some embodiments, the detectable signal is a fluorescence signal. For example, the fluorescence signal intensity is enhanced by 20% to 300%, and this is used to identify the candidate as a cGAS-STING pathway agonist. Alternatively, the fluorescence signal intensity is weakened by 20% to 300%, and this is used to identify the candidate as a cGAS-STING pathway inhibitor.

[0131] This specification also provides an example of the use of a fusion protein in screening drugs for treating diseases related to the cGAS-STING pathway. In some embodiments, the disease may include at least one of neurological diseases, viral infections, bacterial and fungal infections, autoimmune diseases, tumors, and aging.

[0132] In some embodiments, neurological diseases include, but are not limited to: amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, multiple sclerosis, stroke, ischemia / reperfusion brain injury, epilepsy, frontotemporal dementia, traumatic brain injury, and Alzheimer's disease.

[0133] In some embodiments, viral infections include, but are not limited to: herpes simplex virus, herpesvirus, hepatitis B virus, hepatitis C virus, influenza virus, measles virus, human immunodeficiency virus, dengue virus, West Nile virus, Zika virus, poliovirus, mouse encephalitis virus, and coronavirus.

[0134] In some embodiments, bacterial and fungal infections include, but are not limited to: Mycobacterium tuberculosis, Listeria, Salmonella, Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Helicobacter pylori, Cryptococcus neoformans, and Aspergillus.

[0135] In some embodiments, autoimmune diseases include, but are not limited to: systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, psoriasis, multiple sclerosis, ankylosing spondylitis, autoimmune hepatitis, and inflammatory bowel disease.

[0136] In some embodiments, the tumor includes, but is not limited to: breast cancer, lung cancer, colorectal cancer, prostate cancer, melanoma, liver cancer, pancreatic cancer, gastric cancer, and glioblastoma.

[0137] cGAS-STING pathway-related diseases also include other diseases, but this instruction manual does not make specific limitations.

[0138] This specification also provides an example of the application of a fusion protein in the preparation of a kit for detecting cGAS-STING pathway agonists and inhibitors. The kit includes the aforementioned fusion protein or the aforementioned host cells, and detection reagents. The detection reagents can be used to measure the signal intensity or change in signal intensity of a detectable signal generated by the candidate substance contacting the fusion protein or host cells. For example, the detection reagents may include PBS buffer or cell culture medium. The detection reagents may also include other reagents for detecting the signal intensity of the detectable signal; this specification does not impose specific limitations.

[0139] This specification also provides a kit for identifying candidate active substances targeting the STING receptor. The kit includes the aforementioned fusion protein or the aforementioned host cells, and detection reagents. The detection reagents can be used to measure the signal intensity or change in signal intensity of a detectable signal generated by the candidate substance contacting the fusion protein or host cells. For example, the detection reagents may include PBS buffer or cell culture medium. The detection reagents may also include other reagents for detecting the signal intensity of the detectable signal; this specification does not impose specific limitations.

[0140] The embodiments of this specification also provide a reagent for detecting cGAMP content, cGAS activity, or cGAS-STING pathway agonists and inhibitors, the reagent comprising: the above-mentioned fusion protein or the above-mentioned host cell.

[0141] The embodiments of this specification also provide a reagent for detecting mitochondrial DNA leakage, the reagent comprising: the above-described fusion protein or the above-described host cell.

[0142] The embodiments of this specification also provide a reagent for detecting DNA virus infection, the reagent comprising: the above-described fusion protein or the above-described host cell.

[0143] The embodiments described in this specification have at least the following beneficial effects:

[0144] (1) Utilizing the characteristic of the STING receptor specifically binding to its specific ligand, a reporter system that can efficiently screen signaling molecule drugs is provided;

[0145] (2) A fusion protein is constructed by connecting STING components and response components with a linker peptide containing a specific sequence. The STING component can bind specifically to a specific ligand, and the response component generates a detectable signal directly or indirectly in response to the specific binding, making the specific binding of the STING receptor to its specific ligand visible. The whole reporter system has the advantages of being simple and intuitive.

[0146] (3) The fusion proteins constructed in the examples of this specification are used for screening signaling molecule drugs. They have advantages such as high detection sensitivity, high signal-to-noise ratio, and good stability, and their application prospects are promising. Examples

[0147] The following examples are more specific descriptions related to some of the above examples. Some content in these examples may be replaced or combined with corresponding content in other examples to form new examples. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies. The quantitative experiments in the following examples were all performed in triplicate, and the results were averaged. It should be understood that the following examples are for better explanation of this specification and are not intended to limit this specification. Example 1: cGAMP fluorescent probe amino acid sequence

[0148] Figure 1 is a schematic diagram of the design of a cGAMP fluorescent probe according to some embodiments of this specification.

[0149] For Strategy 1 in Figure 1, the amino acid sequence of the cGAMP fluorescent probe is SEQ ID NO.1, and the nucleotide sequence of the cGAMP fluorescent probe is SEQ ID NO.10. In SEQ ID NO.1, the underlined amino acids represent the components of the connecting peptides; the space between the two connecting peptides is a cyclically rearranged green fluorescent protein sequence (the bolded portion not italicized); the front end of the N-terminal connecting peptide and the rear end of the C-terminal connecting peptide are the cGAMP-binding domains of the STING protein, and the amino acid sequences of the cGAMP-binding domains of the STING protein are as follows: SEQ ID NO.3 (LNVAHGLAWSYYIGYLRLILPGLQARIRMFNQLHNNMLSGAGSRRLYILFPLDCGVPDNLSVVDPN IRFRDMLPQQNIDRAGIKNRVYSNSVYEILENGQPAGVCILEYATPLQTLFAMSQDAKAGFSREDRLE QAKLFCRTLEEILEDVPESRNNCRLIVYQEPTDGNSFSLSQEVLRHIRQE) or SEQ ID NO.4 (FNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPN) As shown in the figure: IRFLDKLPQQTGDRAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLE QAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQE).

[0150] SEQ ID NO.1:

[0151] The underlined region in SEQ ID NO.1 represents the components of the linker peptides; the first linker peptide sequence is GEF, and the second linker peptide sequence is SEQ ID NO.5 (LRGGGSGPC). The bolded, non-italicized amino acid sequence between the two linkers is the cyclically rearranged green fluorescent protein sequence SEQ ID NO.6. The front end of the N-terminal linker peptide and the rear end of the C-terminal linker peptide are the cGAMP-binding domains of the STING protein. The sequence of the STING protein's cGAMP-binding domain is SEQ ID NO.3 and its mutant. The bolded, italicized amino acid sequence is the tag SEQ ID NO.9 required for protein purification; this part is linked to the STING protein's cGAMP-binding domain by LRGGGSGSG.

[0152] In some embodiments, the nucleotide sequence encoding the first linker peptide is GGTGAGTTC; the nucleotide sequence encoding the second linker peptide is SEQ ID NO.12 (CTGCGCGGCGGCGGCAGCGGCCCTTGT); the nucleotide sequence encoding the cyclically rearranged green fluorescent protein is SEQ ID NO.13; and the nucleotide sequence encoding the cGAMP-binding domain of the STING protein is SEQ ID NO.14 (TTAAATGTTGCCCACGGGCTGGCCTGGTCATACTACATTGGGTACTTGCGGTTGATCTTACCAGGGCTCCAGGCCCGGATCCGAATGTTCAATCAGCTACATAACAACATGCTCAGTGGTGCAGGGAGCCGAAGACTGTACATCCTCTTTCCATTGGACTGTGGGGTGCCTGACAACCTGAGTGTAGTTGACCCCAACATTCGATTCCGAGATATGCTGCCCCAGCAAAACATCGACCGTGCTGGCATCAAGAATCGGGTTTATTCCAACAGCGTCTACGAGATTCTGGAGAACGGACAGCCAGCAGGCGTCTGTATCCTGGAGTACGCCACCCCCTTGCAGACCCTGTTTGCCATGTCACAGGATGCCAAAGCTGGCTTCAGTCGGGAGGATCGGCTTGAGCAGGCTAAACTCTTCTGCCGGACACTTGAGGAAATCCTGGAAGATGTCCCCGAGTCTCGAAATAACTGCCGCCTCATTGTCTACCAAGAACCCACAGACGGAAACAGTTTCTCACTGTCTCAGGAGGTGCTCCGGCACATTCGTCAGGAA) or SEQ ID NO.15(); The nucleotide sequence encoding tag SEQ ID NO. 9 is SEQ ID NO. 16 (ATGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCCG). In some embodiments, SEQ ID NO. 14 and SEQ ID NO. 15 may be interchanged.

[0153] For Strategy 2, the amino acid sequence of the cGAMP fluorescent probe is SEQ ID NO.2, and the nucleotide sequence of the cGAMP fluorescent probe is SEQ ID NO.11.

[0154] SEQ ID NO.2:

[0155] The underlined region in SEQ ID NO.2 represents the components of the linker peptides; the first linker peptide is sequence SEQ ID NO.5 (LRGGGSGPC), and the second linker peptide sequence is GEF. The region between the two linkers is the cGAMP-binding domain of the STING protein, SEQ ID NO.3. The bolded, non-italicized regions at the front of the N-terminal linker peptide and the rear of the C-terminal linker peptide are the split green fluorescent protein sequences SEQ ID NO.7 and SEQ ID NO.8, respectively. The bolded, italicized amino acid sequence is the tag SEQ ID NO.9 required for protein purification.

[0156] In some embodiments, the nucleotide sequence encoding the first linker peptide is GGTGAGTTC; the nucleotide sequence encoding the second linker peptide is SEQ ID NO.12; the nucleotide sequence encoding the cGAMP-binding domain of the STING protein is SEQ ID NO.14; and the nucleotide sequence encoding the splitting green fluorescent protein is SEQ ID NO.14. NO.17(ATGGTATCTAAAGGAGAAGAATTATTTACCGGTGGTGGTGCCGATCTTAGTGGAACTGGATGGCGACGTGAACGGCCATAAGTTTTCTGTGCCGGAGAAGGCGAAGGCGATGCGACCAACGGTAAACTGACCCTGAAATTTATTTGCACCACCGGTAAACTGCCGGTGCCGTGGCCGACGCTGGTGACGACCTTTGGCTATGGCGTAGCGTGCTTTA GCCGTTATCCGGATCACATGAAACAGCATGATTTTTTTAAAAGCGCGATGCCGGAAGGCTATGTGCAGGAACGCACCATCAGCTTCAAAGATGATGGCACCTATAAAACCAGGGCCGAAGTGAAATTCGAAGGCGATACCTTAGTTAATCGCATTGAACTGAAAGGTATTGATTTCAAAGAAGATGGCAATATTCTGGGCCATAAACTGGAATACAAC) and SEQ ID NO.18 (CACTACGTCTATATCACGGCCGACAAGCAGAAGAACTGCATCAAGGCTAACTTCAAGATCCGCCACAACGTTGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCCATCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATTACACATGGCATGGACGAGCTGTACAAG); The nucleotide sequence encoding tag SEQ ID NO.9 is SEQ ID NO.16. Example 2: Excitation and emission spectra of purified cGAMP fluorescent probe.

[0157] A cGAMP fluorescent probe (hereinafter referred to as the probe, corresponding to Strategy 1; however, Strategy 2 can achieve essentially the same effect) was expressed in *E. coli*, and the cells were collected after culturing at room temperature for 72 hours. The cells were then sonicated in phosphate-buffered saline (PBS) at pH 7.2, purified using a Ni Sepharose 6Fast Flow packing material (Cytiva), and dissolved in PBS buffer at pH 7.2 using a 30 kDa ultrafiltration tube (Merck). The probe concentration was determined using a micro-spectrophotometer. The probe solution was aliquoted into six wells of a 96-well plate, with each well containing 98 μL of 1.75 μM probe solution. Two μL of PBS was added to three wells, and 2 μL of 500 μM cGAMP solution (final concentration 10 μM) was added to the other three wells. The excitation and emission spectra of the probe were then detected using a BioTek multi-mode microplate reader. As shown in Figure 2, in the presence of saturated concentration (10 μM) cGAMP, the purified probe exhibited an excitation peak at 490 nm and an emission peak at 510 nm. Comparing the fluorescence intensity at 510 nm, it was observed that the fluorescence intensity of the probe in the presence of saturated concentration cGAMP increased 22-fold compared to the control group containing PBS buffer. The probe was aliquoted into 3.5 cm cell culture dishes, each containing 980 μL of 1.75 μM probe solution. Fluorescence intensity at different excitation wavelengths was detected using two-photon microscopy. Subsequently, 20 μL of 500 μM cGAMP solution (final concentration 10 μM) or 20 μL of PBS buffer was added to each dish, and fluorescence intensity at different excitation wavelengths was again detected using two-photon microscopy. As shown in Figure 2, in the presence of saturated concentration cGAMP, the purified probe exhibited a two-photon excitation peak at 930 nm. Example 3: Affinity of cGAMP fluorescent probe to cGAMP

[0158] The purified probe (concentration 1.75 μM) from Example 2 was mixed with cGAMP solutions of different concentrations to obtain concentration-dependent curves. As shown in Figure 3, under 480 nm laser excitation, the half-maximal effective concentration (affinity) of the cGAMP fluorescent probe for cGAMP was approximately 216 nM, meeting the requirements for most in vitro detection and cell applications. Example 4: In vitro detection of STING agonists using cGAMP fluorescent probes.

[0159] The purified probe (concentration 1.75 μM) from Example 2 was mixed with different concentrations of cGAMP analogs (100 μM), as shown in Figure 4. The fluorescence detection results of the cGAMP fluorescent probe for different STING agonists were obtained. The fluorescence intensity enhancement of the STING agonists ADU-S100, SR717, MSA-2, diABZL, DMXAA, and CMA using the cGAMP fluorescent probe was approximately 2000%, 500%, 15%, 110%, 160%, and 20%, respectively. Example 5: Real-time detection of cGAMP generated by cGAS catalysis using the cGAMP fluorescent probe.

[0160] The purified probe (concentration 1.75 μM) from Example 2 was mixed with ATP, GTP, DNA, and MgCl2. Fluorescence intensity was detected using a microplate reader. Then, purified cGAS protein (concentration 10 μM) was added, as shown in Figure 5. Real-time detection results of cGAMP production catalyzed by cGAS using the cGAMP fluorescent probe were obtained. The results showed that the fluorescence intensity gradually increased with the duration of the enzymatic reaction, reaching a plateau at approximately 2 hours, with a maximum fluorescence intensity change of approximately 2400%. Example 6: Detection of cGAMP in cells using the cGAMP fluorescent probe.

[0161] The cGAMP fluorescent probe was constructed into cells via lentiviral infection to form a stable cell line expressing the cGAMP fluorescent probe. Cells were then seeded into 96-well plates and cultured in colorless cell culture medium after cell adhesion. Fluorescence intensity was detected using an Opera high-throughput screening system (PerkinElmer), with single-photon excitation wavelengths of 460-490 nm and emission wavelengths of 500-550 nm. Simultaneously, cGAMP (cGAMP + lipo3000) was delivered to the cells in the 96-well plates using a Lipofectamine 3000 kit. The signal change amplitude (ΔF / F0) of the probe after stimulation with 100 μg cGAMP (Taoshu Biotechnology Co., Ltd.) is shown in Figure 6. As can be seen from Figure 6, the dynamic detection range of the cGAMP fluorescent probe in cells is approximately 8-12. Example 7: Detection of cGAS activity in cells using the cGAMP fluorescent probe.

[0162] Cell lines stably expressing the cGAMP fluorescent probe from Example 6 were seeded into 96-well plates. After cell adhesion, the cells were cultured in colorless cell culture medium. Fluorescence intensity was detected using an Opera high-throughput screening system (PerkinElmer), with single-photon excitation wavelengths of 460-490 nm and emission wavelengths of 500-550 nm. HT-DNA (Merck) was simultaneously delivered to the cells in the 96-well plates using a Lipofectamine 3000 kit. The signal change amplitude (ΔF / F0) of the probe after cell stimulation with HT-DNA is shown in Figure 7. As can be seen from Figure 7, the signal change amplitude of the cGAMP fluorescent probe increases with increasing HT-DNA concentration, with a maximum dynamic detection range of approximately 2-3. Example 8: Detection of cGAMP production induced by mitochondrial DNA leakage in cells using cGAMP fluorescent probes.

[0163] Cell lines stably expressing the cGAMP fluorescent probe from Example 6 were seeded into 96-well plates. After cell adhesion, the cells were cultured in colorless cell culture medium. Fluorescence intensity was detected using an Opera high-throughput screening instrument (PerkinElmer), with single-photon excitation wavelengths of 460-490 nm and emission wavelengths of 500-550 nm. Simultaneously, cells were treated with three compounds—ABT737 (Taoshu Biotechnology), S63845 (Taoshu Biotechnology), and Q-VD-OPh (Taoshu Biotechnology)—to induce mitochondrial damage and mitochondrial DNA leakage. The changes in probe signal (ΔF / F0) after treatment with these compounds are shown in Figure 8. As can be seen from Figure 8, the signal change of the cGAMP fluorescent probe increases with increasing treatment time, with a maximum dynamic detection range of approximately 60%. Example 9: Detection of cGAMP production induced by DNA virus infection in cells using the cGAMP fluorescent probe.

[0164] Cell lines stably expressing the cGAMP fluorescent probe from Example 6 were seeded into 96-well plates. After cell adhesion, the cells were cultured in colorless cell culture medium. Fluorescence intensity was detected using an Opera high-throughput screening system (PerkinElmer), with single-photon excitation wavelengths of 460-490 nm and emission wavelengths of 500-550 nm. Double-stranded DNA viruses, such as HSV-1 (Brinkkes) or VCV (Brinkkes), were used to infect the cells. The change in probe signal (ΔF / F0) after cell infection with DNA viruses is shown in Figure 9. As can be seen from Figure 9, the signal change of the cGAMP fluorescent probe increases with increasing infection time, with a maximum dynamic detection range of approximately 40%. Example 10: Detection of STING agonists in live brain slices using cGAMP fluorescent probes.

[0165] First, adeno-associated virus (AAV) carrying the cGAMP gene was precisely injected into a specific region of the mouse brain. After 2-3 weeks, ensuring that viral expression reached a stable state, the mice were deeply anesthetized, and live brain slices were prepared under sterile conditions. These brain slices were incubated in artificial cerebrospinal fluid to simulate the in vivo environment. Two-photon microscopy was used to monitor the brain slices in real time and observe the dynamic changes of the cGAMP fluorescent probe. During this process, the STING agonist (SR717, purchased from Taoshu Biotechnology Co., Ltd.) was added to the artificial cerebrospinal fluid, and the signal change amplitude (ΔF / F0) of the cGAMP fluorescent probe was recorded. The detection results are shown in Figure 10. As can be seen from Figure 10, the dynamic detection range of the cGAMP fluorescent probe for detecting the STING agonist in live brain slices is approximately 0.5-1. Example 11: Detection of STING agonist by cGAMP fluorescent probe in live animals.

[0166] AAV virus carrying the cGAMP gene was precisely injected into the cerebral cortex of mice. After 2-3 weeks, ensuring that viral expression reached the expected level, the mice were deeply anesthetized, and their skulls were removed under aseptic conditions. The cerebral cortex region of the mice was monitored in real time using two-photon microscopy to observe changes in the fluorescence signal of the cGAMP fluorescent probe. During this process, the STING agonist (SR717, purchased from Taoshu Biotechnology Co., Ltd.) was applied to the cerebral cortex region of the mice, and the signal change amplitude (ΔF / F0) of the cGAMP fluorescent probe was recorded. The detection results are shown in Figure 11. As can be seen from Figure 11, the dynamic detection range of the cGAMP fluorescent probe is approximately 1-1.5. Therefore, this example verifies the effectiveness of the cGAMP fluorescent probe in detecting STING agonists in the in vivo environment. Example 12: Detection of cGAMP content in human serum samples using cGAMP fluorescent probe.

[0167] In the field of biomedical research, accurate detection of cGAMP levels is crucial for understanding the pathogenesis and treatment of certain diseases. This embodiment uses E. coli lysate expressing the cGAMP fluorescent probe, or the purified cGAMP fluorescent probe from Example 2, mixed with human serum samples. Two-photon microscopy was used to detect the fluorescence signal generated after mixing the cGAMP fluorescent probe with the human serum sample, as shown in Figure 12. The results indicate that the cGAMP fluorescent probe can detect the cGAMP level in human serum samples. Therefore, based on the cGAMP fluorescent probe, researchers hope to assess an individual's health status and even provide early warnings of potential health risks. The application of this technology not only provides a new tool for clinical diagnosis but also opens up new possibilities for personalized medicine and precision treatment.

[0168] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0169] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0170] Furthermore, unless expressly stated in the claims, the order of processing elements and sequences, the use of numbers and letters, or other names described in this specification are not intended to limit the order of the processes and methods described herein. Although some inventive embodiments that are currently considered useful have been discussed by way of various examples in the foregoing disclosure, it should be understood that such details are for illustrative purposes only, and the appended claims are not limited to the disclosed embodiments. Rather, the claims are intended to cover all modifications and equivalent combinations that conform to the substance and scope of the embodiments described herein.

[0171] Similarly, it should be noted that, in order to simplify the description disclosed herein and thus aid in the understanding of one or more embodiments of the invention, the foregoing description of embodiments in this specification may sometimes combine multiple features into a single embodiment, drawing, or description thereof. However, this method of disclosure does not imply that the subject matter of this specification requires more features than those mentioned in the claims. In fact, the embodiments contain fewer features than all the features of a single embodiment disclosed above.

[0172] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0173] For each patent, patent application, patent application publication, and other material, such as articles, books, specifications, publications, and documents, referenced in this specification, the entire contents of which are incorporated herein by reference. This excludes historical application documents that are inconsistent with or conflict with the content of this specification, as well as documents that limit the broadest scope of the claims in this specification (currently or subsequently appended to this specification). It should be noted that in the event of any inconsistency or conflict between the descriptions, definitions, and / or terminology used in the supplementary materials to this specification and the content of this specification, the descriptions, definitions, and / or terminology used in this specification shall prevail.

[0174] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

A fusion protein, characterized in that, The fusion protein includes: An interferon gene activator (STING) component, wherein the STING component comprises at least one STING protein fragment; and Response components; The response component is directly or indirectly connected to the STING component, the STING component is capable of specifically binding to a specific ligand, and the response component generates a detectable signal directly or indirectly in response to the specific binding. The fusion protein according to claim 1, characterized in that, The fusion protein further includes a connecting member, through which the responsive member is connected to the STING member. The fusion protein according to claim 1 or 2 is characterized in that, The complete STING is a dimer, and the STING protein fragment includes one dimer, a portion of a dimer, or a mutant of a dimer. The fusion protein according to any one of claims 1-3 is characterized in that, The STING component comprises two STING protein fragments, which may be identical or different. The fusion protein according to any one of claims 1-3 is characterized in that, The STING component includes a STING protein fragment. The fusion protein according to any one of claims 1-5 is characterized in that, The STING protein fragment is of human or animal origin. The fusion protein according to any one of claims 1-6 is characterized in that, The STING protein fragment includes the cGAMP binding domain of STING, or a portion thereof, or a mutant thereof. The fusion protein according to any one of claims 1-7 is characterized in that, The amino acid sequence of the STING protein fragment has at least 80% similarity to SEQ ID NO.

3. The fusion protein according to any one of claims 1-7 is characterized in that, The STING protein fragment includes the amino acid sequence shown in SEQ ID NO.

3. The fusion protein according to any one of claims 1-9 is characterized in that, The response component is connected to the STING protein fragment. The fusion protein according to any one of claims 1-10 is characterized in that, The STING component includes a first STING protein fragment and a second STING protein fragment, and the response component includes a responder connected to the C-terminus of the first STING protein fragment and the N-terminus of the second STING protein fragment. The fusion protein according to claim 11 is characterized in that, The amino acid sequence of the fusion protein has at least 80% similarity to SEQ ID NO.

1. The fusion protein according to any one of claims 1-10 is characterized in that, The STING component includes a STING protein fragment, and the response component includes a first responder and a second responder, wherein the first responder is connected to the N-terminus of the STING protein fragment, and the second responder is connected to the C-terminus of the STING protein fragment. The fusion protein according to claim 13 is characterized in that, The amino acid sequence of the fusion protein has at least 80% similarity to SEQ ID NO.

2. The fusion protein according to any one of claims 1-14 is characterized in that, The specific ligands include signaling molecules and STING agonists. The fusion protein according to any one of claims 1-15 is characterized in that, The signaling molecule is selected from at least one of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), cyclic diguanosine monophosphate (c-di-GMP), and cyclic diadenosine monophosphate (c-di-AMP). The fusion protein according to any one of claims 1-15 is characterized in that, The STING agonists include: DMXAA (Vadimezan), ADU-S100 (MIW815), MK-1454, SR-717, G10, diABZI, MSA-2, SN-011, SB 11285, E7766, IACS-8803, MK-2118, TDI-01, BI-1387446, GSK3745417, BMS-986301, and XMT-2056. The fusion protein according to any one of claims 1-17 is characterized in that, The response component includes a responder, which, in response to the specific binding, is capable of undergoing a conformational change, and in response to the conformational change, the responder can directly or indirectly generate a detectable signal. The fusion protein according to any one of claims 1-18 is characterized in that, The detectable signal is an optical signal, a chemical signal, or a thermal signal. The fusion protein according to any one of claims 1-19 is characterized in that, The detectable signal is an optical signal, and the responder is selected from at least one of fluorescent protein, luciferase, and protein tag that can covalently bind to a fluorescent ligand. The fusion protein according to any one of claims 1-20 is characterized in that, The fluorescent protein is selected from cyclically rearranged fluorescent proteins. The fusion protein according to any one of claims 1-21 is characterized in that, The cyclically rearranged fluorescent protein is selected from at least one of the following: cyclically rearranged green fluorescent protein (cpGFP), cyclically rearranged yellow fluorescent protein (cpYFP), cyclically rearranged red fluorescent protein (cpRFP), cyclically rearranged blue fluorescent protein (cpBFP), cyclically rearranged enhanced green fluorescent protein (cpEGFP), cyclically rearranged enhanced yellow fluorescent protein (cpEYFP), and cyclically rearranged infrared fluorescent protein (cp infrared fluorescent protein, cpiRFP). The fusion protein according to any one of claims 1-21 is characterized in that, The fluorescent protein of the cyclic rearrangement is selected from cpGFP, which includes at least one of cpCitrine, cpmCitrine, cpmGold, cpYPet, cpFOLD6, cpmNeonGreen, cpStayGold, cpmBaojin, or cpmClover3. The fusion protein according to claim 23 is characterized in that, The fluorescent protein involved in the cyclic rearrangement is cpmClover3. The fusion protein according to claim 20 is characterized in that, The fluorescent protein is selected from split fluorescent proteins. The fusion protein according to claim 25 is characterized in that, The split fluorescent protein is selected from at least one of split green fluorescent protein (split-GFP), split yellow fluorescent protein (split-YFP), split red fluorescent protein (split-RFP), split blue fluorescent protein (split-BFP), split enhanced green fluorescent protein (split-EGFP), split enhanced yellow fluorescent protein (split-EYFP), and split-infrared fluorescent protein (split-iRFP). The fusion protein according to claim 25 or 26 is characterized in that, The split fluorescent protein is selected from split-GFP, which includes at least one of split-Citrine, split-mCitrine, split-mGold, split-YPet, split-FOLD6, split-NeonGreen, split-StayGold, split-mBaojin, or split-mClover3. The fusion protein according to any one of claims 25-27 is characterized in that, The fluorescent protein involved in the splitting is split-mClover3. The fusion protein according to claim 1 or 2 is characterized in that, The linker includes at least one linker peptide that links the STING protein fragment and the response component. The fusion protein according to any one of claims 1-29 is characterized in that, The connecting component includes a first connecting peptide and a second connecting peptide. The fusion protein according to any one of claims 1-30 is characterized in that, The STING component includes a first STING protein fragment and a second STING protein fragment, and the response component includes a responder, which is connected to the C-terminus of the first STING protein fragment via a first linker peptide and to the N-terminus of the second STING protein fragment via a second linker peptide. The fusion protein according to any one of claims 1-30 is characterized in that, The response component includes a first responder and a second responder, wherein the first responder is linked to the N-terminus of the STING protein fragment via a first linker peptide, and the second responder is linked to the C-terminus of the STING protein fragment via the second linker peptide. The fusion protein according to any one of claims 1-32 is characterized in that, The length of the first linker peptide or the second linker peptide is 3-10 amino acids. The fusion protein according to any one of claims 1-33 is characterized in that, The first linker peptide is 3 amino acids long, and the second linker peptide is 9 amino acids long. The fusion protein according to any one of claims 1-33 is characterized in that, The first linker peptide is 9 amino acids long, and the second linker peptide is 3 amino acids long. The fusion protein according to any one of claims 1-35 is characterized in that, The amino acid sequence of one of the first linker peptide and the second linker peptide is GEF, and the amino acid sequence of the other is SEQ ID NO.

5. The polynucleotide encoding the fusion protein of claims 1-36. An expression vector comprising the polynucleotide of claim 37. A host cell comprising the fusion protein of claims 1-36, the polynucleotide of claim 37, and / or the expression vector of claim 38. The host cell of the expression vector according to claim 39 is characterized in that, The host cell is a prokaryotic cell. The host cell of the expression vector according to claim 39 or 40 is characterized in that, The host cell is a mammalian cell. The host cell of the expression vector according to any one of claims 39-41 is characterized in that, The host cell is a human-derived cell. The use of the fusion protein described in claims 1-36 in the detection of cGAMP content or cGAS activity. The use of the fusion protein described in claims 1-36 in the preparation of reagents or kits for detecting cGAMP content or cGAS activity. The use of the fusion protein described in claims 1-36 in the preparation of a kit for detecting cGAS-STING pathway agonists and inhibitors. The use of the fusion protein described in claims 1-36 in screening drugs for the treatment of diseases related to the cGAS-STING pathway. The application according to claim 46 is characterized in that, The diseases mentioned include at least one of the following: neurological diseases, viral infections, bacterial and fungal infections, autoimmune diseases, tumors, and aging. The application according to claim 47 is characterized in that, The neurological diseases mentioned include, but are not limited to: amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, multiple sclerosis, stroke, ischemic / reperfusion brain injury, epilepsy, frontotemporal dementia, traumatic brain injury, and Alzheimer's disease. The application according to claim 47 is characterized in that, The viral infections mentioned include, but are not limited to: herpes simplex virus, herpesvirus, hepatitis B virus, hepatitis C virus, influenza virus, measles virus, human immunodeficiency virus, dengue virus, West Nile virus, Zika virus, poliovirus, mouse encephalitis virus, and coronavirus. The application according to claim 47 is characterized in that, The bacterial and fungal infections include, but are not limited to: Mycobacterium tuberculosis, Listeria, Salmonella, Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Helicobacter pylori, Cryptococcus neoformans, and Aspergillus. The application according to claim 47 is characterized in that, The autoimmune diseases mentioned include, but are not limited to: systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, psoriasis, multiple sclerosis, ankylosing spondylitis, autoimmune hepatitis, and inflammatory bowel disease. The application according to claim 47 is characterized in that, The tumors include, but are not limited to: breast cancer, lung cancer, colorectal cancer, prostate cancer, melanoma, liver cancer, pancreatic cancer, gastric cancer, and glioblastoma. The method for screening cGAS-STING pathway agonists and inhibitors using the fusion protein described in claims 1-36 is characterized in that... The method includes: The fusion protein is expressed in vivo or in vitro; Establish standard signal strength; The fusion protein is reacted with the candidate, and the intensity of the detectable signal is measured. If the intensity of the detectable signal increases based on the standard signal intensity, the candidate is a cGAS-STING pathway agonist. If the intensity of the detectable signal decreases based on the standard signal intensity, the candidate is a cGAS-STING inhibitor. The method according to claim 53 is characterized in that, The establishment of the standard signal intensity includes: purifying the fusion protein, forming a mixture with ATP, GTP, DNA, and MgCl2, and detecting the detectable signal intensity as the standard signal intensity. The method according to claim 53 is characterized in that, The detectable signal is a fluorescence signal. A kit for identifying candidate active substances that can affect the cGAS-STING pathway, characterized in that, The kit includes: The fusion protein according to any one of claims 1-36 or the host cell according to any one of claims 39-42; The detection reagent is used to determine the signal intensity or change of a detectable signal generated when the candidate comes into contact with the fusion protein or the host cell. A reagent for detecting cGAMP levels, cGAS activity, or cGAS-STING pathway agonists and inhibitors, characterized in that... The reagents include: The fusion protein according to any one of claims 1-36 or the host cell according to any one of claims 39-41. A reagent for detecting mitochondrial DNA leakage, characterized in that, The reagents include: The fusion protein according to any one of claims 1-36 or the host cell according to claim 37. A reagent for detecting DNA virus infection, characterized in that, The reagents include: The fusion protein of claims 1-36 or the host cell of claim 37.

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