Ratio-type fluorescent probe constructed based on G-protein coupled receptor and construction method of ratio-type fluorescent probe

Abstract

The invention discloses a ratio type fluorescent probe constructed based on a G protein coupled receptor and a construction method thereof. The invention finds that the GRAB probe has the property of excitation ratio type by introducing the mutation of the same amino acid site (connected with the third amino acid of the peptide fragment) on the GRAB probe. The probe with the excitation ratio type property can have higher pH stability, is not limited by the expression quantity of the probe and external hardware conditions, keeps higher stability of fluorescence brightness, can correct fluorescence interference caused by movement, can sensitively detect release of neurotransmitters, and can be used for detecting neurotransmitter release. The method has the potential of being applied to in-vivo quantitative detection of the neurotransmitter concentration.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a ratiometric fluorescent probe constructed based on G protein coupled receptors. Background Art

[0002] As the most important medium for signal transmission between neurons in the brain, the functions and modes of action of neurotransmitters have been widely studied and reported. In addition, although there are many studies on the real-time dynamic changes of these neurotransmitters in different brain regions of the central nervous system, most of the existing tools lack high spatiotemporal resolution and cell specificity (Wu et al., 2022). For example, although the classic microdialysis method can specifically detect neurotransmitters and neuromodulators, its sampling frequency is very low (once every 5 to 10 minutes), the spatial resolution is low and it is not cell specific (Chefer et al., 2009); although the fast scanning cyclic voltammetry has good time resolution, it has no cell specificity and selectivity (Kuhr and Wightman, 1986; Robinson et al., 2003). In recent years, as one of the teams that first developed GPCR Activation Based sensors (GRAB) in the world, Li Yulong's research group has developed dozens of GRAB probes for detecting different types of neurotransmitters, including dopamine, serotonin, norepinephrine, endocannabinoids, and neuropeptides. These new genetically encoded fluorescent probes have well solved the defects of existing detection methods, and can achieve high spatiotemporal resolution and real-time specific detection of neurotransmitters and modulators in specific cell types.

[0003] However, since these neurotransmitter probes based on changes in fluorescence intensity are affected by many factors during use, such as the expression level of chromophore number, fluorescence excitation intensity and photobleaching, the application scenarios of these probes that reflect neurotransmitter changes based on a single fluorescence intensity are mainly limited to judging the occurrence of neurotransmitter release events caused by neuronal activity, and cannot be further used to sensitively and accurately measure the concentration of neurotransmitter release.

[0004] Therefore, the present invention aims to develop a general strategy that can improve the excitation ratio properties of probes. These probes with excitation ratio properties can have high pH stability, are not limited by the expression level of the probe itself and external hardware conditions, maintain high stability of fluorescence brightness, can correct for fluorescence interference caused by movement, can sensitively detect the release of neurotransmitters, and have the potential to be used in the quantitative detection of neurotransmitter concentrations in vivo. Summary of the invention

[0005] In order to remedy the deficiencies of the prior art, the present invention aims to develop a general strategy that can improve the excitation ratiometric properties of GRAB probes.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides a ratiometric fluorescent probe constructed based on G protein coupled receptors.

[0007] Furthermore, the ratiometric fluorescent probe includes a G protein coupled receptor, a cyclically rearranged fluorescent protein and a connecting peptide; the connecting peptide includes an N-terminal connecting peptide and a C-terminal connecting peptide; the cyclically rearranged fluorescent protein is inserted between the fifth transmembrane region and the sixth transmembrane region of the G protein coupled receptor, and the N-terminus of the cyclically rearranged fluorescent protein is connected to the fifth transmembrane region of the G protein coupled receptor through the N-terminal connecting peptide; the C-terminus of the cyclically rearranged fluorescent protein is connected to the sixth transmembrane region of the G protein coupled receptor through the C-terminal connecting peptide.

[0008] Furthermore, the N-terminal connecting peptide comprises 5 amino acids, and the third amino acid of the N-terminal connecting peptide is mutated.

[0009] Furthermore, the ratiometric fluorescent probe can be expressed on a cell membrane; and when the ratiometric fluorescent probe contacts a specific ligand of the G protein coupled receptor, it can bind to the ligand, thereby causing a detectable change in the fluorescence intensity of the fluorescent probe.

[0010] Furthermore, the G protein coupled receptor is of human origin.

[0011] Furthermore, the specific ligand includes a neurotransmitter.

[0012] Furthermore, the neurotransmitters include NTS, SST, PACAP, NPY, dopamine and / or serotonin.

[0013] Furthermore, the cyclically rearranged fluorescent protein is cpEGFP.

[0014] Furthermore, when the cpEGFP is linked to the G protein-coupled receptor that specifically binds to NTS, its amino acid sequence is the sequence shown in SEQ ID NO.1, or the sequence after the 127th position thereof is mutated to glutamic acid.

[0015] Furthermore, when the cpEGFP is linked to a G protein-coupled receptor that specifically binds to SST, its amino acid sequence is the sequence shown in SEQ ID NO.2, or the sequence after its 127th position is mutated to glutamic acid.

[0016] Furthermore, when the cpEGFP is connected to the G protein-coupled receptor that specifically binds to PACAP, its amino acid sequence is the sequence shown in SEQ ID NO.3.

[0017] Furthermore, when the cpEGFP is connected to the G protein-coupled receptor that specifically binds to NPY, its amino acid sequence is the sequence shown in SEQ ID NO.4.

[0018] Furthermore, when the cpEGFP is linked to a G protein-coupled receptor that specifically binds to dopamine, its amino acid sequence is the sequence shown in SEQ ID NO.5, or the sequence after the 127th position thereof is mutated to valine.

[0019] Furthermore, when the cpEGFP is connected to a G protein-coupled receptor that specifically binds to serotonin, its amino acid sequence is the sequence shown in SEQ ID NO.6.

[0020] Furthermore, when the cpEGFP is connected to NTS, SST, PACAP, NPY or a G protein-coupled receptor that specifically binds to serotonin, the sequence of the N-terminal connecting peptide before the third amino acid mutation is LEEGG.

[0021] Furthermore, when the cpEGFP is connected to a G protein-coupled receptor that specifically binds to NTS, SST or NPY, the third amino acid of the N-terminal connecting peptide is mutated to leucine, and the sequence of the N-terminal connecting peptide after the mutation is LELGG.

[0022] Furthermore, when the cpEGFP is connected to the G protein-coupled receptor that specifically binds to PACAP, the third amino acid of the N-terminal connecting peptide is mutated to leucine, isoleucine or methionine, and the sequence of the mutated N-terminal connecting peptide is any one of LELGG, LEIGG or LEMGG.

[0023] Furthermore, when the cpEGFP is connected to a G protein-coupled receptor that specifically binds to serotonin, the third amino acid of the N-terminal connecting peptide segment is mutated to leucine or methionine, and the sequence of the N-terminal connecting peptide after mutation is any one of LELGG or LEMGG.

[0024] Furthermore, when the cpEGFP is connected to a G protein-coupled receptor that specifically binds to dopamine, the sequence of the N-terminal connecting peptide before the third amino acid mutation is LNSLI.

[0025] Furthermore, when the cpEGFP is connected to a G protein-coupled receptor that specifically binds to acetylcholine, the sequence of the N-terminal connecting peptide before the third amino acid mutation is ETEGG.

[0026] Furthermore, when the cpEGFP is connected to a G protein-coupled receptor that specifically binds to dopamine, the third amino acid of the N-terminal connecting peptide is mutated to leucine, alanine or histidine, and the sequence of the N-terminal connecting peptide after mutation is any one of LNLLI, LNALI and LNHLI.

[0027] The applicant team of the present invention has previously established a fluorescent probe based on G protein-coupled receptors, but this probe that reflects changes in neurotransmitters based on a single fluorescence intensity is limited by many factors; therefore, the applicant is looking for mutation sites that can improve the excitation ratio properties of the GRAB probe on this basis. The applicant found that by introducing mutations at the same amino acid site (the third amino acid of the N-terminal connecting peptide), the excitation ratio performance can be improved on nearly ten GRAB probes. These probes with excitation ratio properties can have high pH stability, are not limited by the expression level of the probe itself and external hardware conditions, maintain a high stability of fluorescence brightness, can correct fluorescence interference caused by movement, can sensitively detect the release of neurotransmitters, and have the potential to be used for in vivo quantitative detection of neurotransmitter concentrations.

[0028] The principle of the ratiometric fluorescent probe based on G protein coupled receptor (also referred to as ratiometric GRAB fluorescent probe in the present invention) of the present invention is: inserting the cyclically rearranged fluorescent protein between the fifth transmembrane region and the sixth transmembrane region of the GPCR, the binding of the ligand to the GPCR will induce the conformational change of the GPCR, thereby causing the conformational change of the cyclically rearranged fluorescent protein, causing the change of the fluorescence signal intensity, thereby converting the conformational change of the GPCR induced by the ligand into the change of the optical signal.

[0029] G protein-coupled receptors (GPCRs) are a class of seven transmembrane proteins expressed on the plasma membrane. The main body of the GPCR protein is composed of 7 segments of α-helical structures that span the plasma membrane. The N-terminus and 3 loops are located outside the cell, and the C-terminus and 3 loops are located inside the cell. The "G protein-coupled receptors (GPCRs)" described herein are a large protein family of transmembrane receptors that sense extracellular molecules, activate intracellular signal transduction pathways and ultimately activate cellular responses. The ligands that bind and activate these receptors include photosensitive compounds, odors, pheromones, hormones and neurotransmitters, and vary in size from small molecules to peptides to large proteins. GPCRs are involved in many diseases and are also the targets of about half of all modern pharmaceutical drugs. Based on sequence homology and functional similarity, GPCRs can be divided into at least 5 categories: class A rhodopsin-like, class B secretin-like, class C metabotropic / pheromone, class D fungal pheromone, and class E cAMP receptor.

[0030] Class A rhodopsin-like receptors include: amine receptors: acetylcholine, alpha adrenergic receptor, beta adrenergic receptor, dopamine, histamine, 5-hydroxytryptamine, octopamine and trace amines; peptide receptors: angiotensin (NTS), bombesin, bradykinin, C5a anaphylatoxin, Fmet-leu-phe, AP J-like substances, interleukin-8, chemokine receptors (CC chemokine, CXC chemokine, B0NZ0 receptor (CXC6R), C-X3-C chemokine and XC chemokine), CCK receptor, endothelin receptor, melanocortin receptor, neuropeptide Y (NPY) receptor, neurotensin receptor, opioid receptor, somatostatin (SST) receptor, tachykinin receptors (substance P (NK1), substance K (NK2), neuromodulatory peptide K (NK3), Tachykinin-like 1 and tachykinin-like 2), vasopressin-like receptors (vasopressin, oxytocin, and conopressin), galanin-like receptors (galanin, pleuronosine, and GPCR54), protease-activated-like receptors (e.g., thrombin), orexin & neuropeptide FF, urotensin II receptor, adrenomedullin (G10D) receptor, GPR37 / endothelin B-like receptor, chemokine receptor-like receptor, and neuromedin U receptor; hormone protein receptors: follicle-stimulating hormone hormones, luteinizing hormone-chorionic gonadotropin, thyrotropin and gonadotropins; (Rhod) opsin receptors; olfactory receptors; prostaglandin receptors: prostaglandins, prostacyclins and thromboxanes; nucleotide-like receptors: adenosine and purinergic receptors; cannabinoid receptors; platelet-activating factor receptors; gonadotropin-releasing hormone receptors; thyrotropin-releasing hormone & secretagogue receptors: thyrotropin-releasing hormone, growth hormone secretagogue and growth hormone secretagogue-like; melatonin receptors; viral receptors ; Lysosphingolipid & LPA (EDG) receptors; Leukotriene M receptors: Leukotriene B4 receptor BLT1 and Leukotriene M receptor BLT2; and Class A orphan / other receptors: Platelet ADP & KI01 receptors, SREB, Mas proto-oncogene, RDC1, ORPH, LGR-like (hormone receptor), GPR, GPR45-like, cysteinyl leukotrienes, Mas-related receptors (MRGs) and GP40-like receptors.

[0031] Class B (secretin receptor family) of GPCRs includes receptors for polypeptide hormones (calcitonin, corticotropin-releasing factor, gastric inhibitory peptide, glucagon, glucagon-like peptide-1, -2, growth hormone-releasing hormone, parathyroid hormone, PACAP, secretin, vasoactive intestinal polypeptide, uretic hormone, EMR1, latrophilin), molecules thought to mediate cell-cell interactions at the plasma membrane (brain-specific angiogenesis inhibitor (BAI)), and a group of Drosophila proteins that regulate stress responses and lifespan (Methuselah-like proteins).

[0032] Class C metabotropic glutamate / pheromone receptors include metabotropic glutamate, group I metabotropic glutamate, group II metabotropic glutamate, group III metabotropic glutamate, other metabotropic glutamate, extracellular calcium sensors, putative pheromone receptors, GABA-B receptor (GABA-B receptor consists of two subunits (B1, B2) and is a dimeric protein) and orphan GPRC5 receptor.

[0033] GPCRs are involved in a variety of physiological processes, including vision, smell, behavior and mood regulation, immune system activity and inflammation regulation, autonomic nervous system transmission, cell density sensing and many others. Known inactivated G proteins bind to receptors in their inactivated state. Once a ligand is recognized, the receptor or its subunits switch conformations and thus mechanically activate the G protein, which detaches from the receptor. The receptor can now activate another G protein, or switch back to its inactivated state. It is believed that the receptor molecule exists in a conformational equilibrium between active and inactivated biophysical states. Ligand binding to the receptor can shift the equilibrium toward an active receptor state.

[0034] The G protein-coupled receptors that can be used in the present invention include, but are not limited to, acetylcholine receptors, dopamine D2 receptors (DRD2), serotonin receptor 6 (HTR6), angiotensin (NTS) receptors, neuropeptide Y (NPY) receptors, somatostatin (SST) receptors, and PACAP receptors. These receptors are well known to those skilled in the art, and their sequences can be obtained through various means, such as querying well-known databases.

[0035] Those skilled in the art can easily determine the N-terminus, transmembrane region, intracellular loop and C-terminus of a G protein-coupled receptor, for example, based on its amino acid sequence and similarity to the transmembrane region of a known G protein-coupled receptor. Various bioinformatics methods can be used to determine the location and structure of the transmembrane region in a protein, for example, alignment and amino acid sequence comparison can be routinely performed in the art using the BLAST program or the CLUSTALW program. Based on the alignment with G protein-coupled receptors known to contain transmembrane regions, those skilled in the art can predict the location and structure of the transmembrane region of other GPCRs. There are also many programs that can be used to predict the location and structure of the transmembrane region in a protein. For example, one or a combination of the following programs can be used: TMpred, which predicts transmembrane protein fragments; TopPred, which predicts the topological structure of membrane proteins; PREDATOR, which predicts secondary structure from single and multiple sequences; TMAP, which predicts the transmembrane region of a protein from multiple aligned sequences; and AL0M2, which predicts the transmembrane region from a single sequence. According to standard nomenclature, the numbering of the transmembrane region and the intracellular loop is relative to the N-terminus of the GPCR.

[0036] The term "cyclically rearranged fluorescent protein" used herein is well known to those skilled in the art and refers to a fluorescent protein formed by connecting the molecular ends of the original fluorescent protein and then disconnecting the protein from any site to form a new carbon end and nitrogen end. The fluorescent protein itself has its own chromophore center composed of three amino acids, and the chemical reaction that occurs determines the spectral properties and fluorescence intensity of the fluorescent protein. Most of the chromophores of the fluorescent protein are located inside the protein and are protected by the barrel structure of the surrounding β-folding. When the fluorescent protein is fused with the target protein, the involvement of the end of the fluorescent protein is difficult to cause environmental changes around the chromophore, and it is difficult to change the fluorescence intensity of the fluorescent protein. The chromophore of the cyclically rearranged fluorescent protein is relatively close to the newly formed end. When it is connected to the target protein, the conformational changes of the target protein will involve the end of the cyclically rearranged fluorescent protein, resulting in changes in the environment around the chromophore, thereby increasing or decreasing the fluorescence intensity of the fluorescent protein, thereby converting the conformational changes of the target protein into changes in its fluorescence intensity, so that real-time detection can be performed by optical imaging methods. In a specific embodiment of the present invention, the circularly permutated fluorescent protein is circularly permutated enhanced green fluorescent protein (cpEGFP). cpEGFP can be cpEGFP from GCaMP6s or GCaMP6m (Chen, T.-W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295-300 (2013)), or cpEGFP from GECO1.2 (Zhao, Y. et al. An Expanded Palette of Genetically Encoded Ca 2+ Indicators. Science 333, 1888-1891 (2011)). Their sequences can be obtained from the NCBI database or the addgene database.

[0037] The ratiometric fluorescent probe (i.e., ratiometric GRAB fluorescent probe) constructed based on G protein coupled receptor of the present invention should be able to be expressed on the cell membrane. The method for detecting whether the probe can be expressed on the cell membrane is well known to those skilled in the art, for example, the probe can be expressed in cells (e.g., HEK293T cells), and the expression morphology of fluorescent protein in the cell is analyzed, and the protein expressed on the cell membrane is a very thin circle at the outermost periphery of the cell, and the cell contour can be obtained by comparing the fluorescent channel with the bright field channel, and then analyzed. Probes that fail to be normally on the membrane often gather in the cell, which is a clustering signal in the cell under a microscope. It is also possible to express another known cell membrane localized protein and perform quantitative measurement by calculating the co-localization of the fluorescent probe signal and the protein.

[0038] The ratiometric fluorescent probe constructed based on the G protein coupled receptor of the present invention should be able to bind to the specific ligand of the G protein coupled receptor when it contacts the specific ligand of the G protein coupled receptor, thereby causing the fluorescence intensity of the probe to have a response amplitude at 488 nm and 405 nm, and then calculating the concentration change of the specific ligand by the ratio Ex488 / 405. The method for detecting this is known to those skilled in the art, for example, the probe can be contacted with the specific ligand of the G protein coupled receptor, and then the cells expressing the fluorescent probe can be imaged for fluorescence, and continuous photography and recording are performed before and after the addition of the ligand, and the fluorescence intensity change recorded before and after the addition of the ligand is analyzed to detect whether the fluorescent probe has a fluorescent response to the specific ligand.

[0039] The "ligand" or "specific ligand" of the G protein-coupled receptor described herein are used interchangeably and refer to molecules that can bind to and activate (or inhibit) the G protein-coupled receptor, including photosensitive compounds, odors, pheromones, hormones and neurotransmitters. The binding of a G protein-coupled receptor to its ligand is highly specific, the ligand only binds to a specific receptor, and the receptor only binds to a specific ligand structure. The specificity of the binding of a G protein-coupled receptor to its ligand means that the binding affinity of the G protein-coupled receptor to the ligand is significantly higher than the binding affinity to one or more other molecules. The "significantly higher" in "significantly higher" can refer to statistical significance. The ligands that different G protein-coupled receptors can bind to, or the G protein-coupled receptors that different ligands can bind to, are well known to those skilled in the art.

[0040] The terms "connector peptide" or "connector peptide segment" used herein are used interchangeably and refer to a short peptide connecting the third intracellular loop of the G protein-coupled receptor and the cyclically rearranged fluorescent protein. In the present invention, since the cyclically rearranged fluorescent protein is inserted into the third intracellular loop of the G protein-coupled receptor, the "connector peptide" described herein includes an N-terminal connecting peptide located at the N-terminus of the cyclically rearranged fluorescent protein and a C-terminal connecting peptide located at the C-terminus of the cyclically rearranged fluorescent protein. In the present invention, the role of the connecting peptide is to help the fusion protein fold correctly, and at the same time, it plays a bridging role between the transmission of receptor conformational changes and changes in the brightness of the fluorescent protein. Therefore, the connecting peptide used should be a connecting peptide that can play the role described.

[0041] The second aspect of the present invention provides a method for constructing a ratiometric GRAB fluorescent probe.

[0042] Furthermore, the method comprises mutating the third amino acid of the N-terminal connecting peptide segment of the cyclically rearranged fluorescent protein connected to the G protein coupled receptor.

[0043] Furthermore, the cyclically rearranged fluorescent protein is cpEGFP, and the mutation includes mutating the third amino acid of the cpEGFP N-terminal connecting peptide to leucine.

[0044] Furthermore, when the G protein coupled receptor is a G protein coupled receptor that specifically binds to PACAP, the mutation further includes mutating the third amino acid of the cpEGFP N-terminal connecting peptide to isoleucine or methionine.

[0045] Furthermore, when the G protein coupled receptor is a G protein coupled receptor that specifically binds to dopamine, the mutation further includes mutating the third amino acid of the cpEGFP N-terminal connecting peptide to alanine or histidine.

[0046] Furthermore, when the G protein coupled receptor is a G protein coupled receptor that specifically binds to serotonin, the mutation further includes mutating the third amino acid of the cpEGFP N-terminal connecting peptide segment to methionine.

[0047] Furthermore, the method also includes modifying cpEGFP; the modification includes mutating site 127 of cpEGFP.

[0048] Furthermore, when the G protein-coupled receptor is a G protein-coupled receptor that specifically binds to NTS or SST, the mutation comprises mutating position 127 of cpEGFP to glutamic acid.

[0049] Furthermore, when the G protein-coupled receptor is a G protein-coupled receptor that specifically binds to dopamine, the mutation includes mutating position 127 of cpEGFP to valine.

[0050] A third aspect of the present invention provides a biomaterial.

[0051] Furthermore, the biomaterial comprises: 1) a polynucleotide encoding the ratiometric fluorescent probe according to the first aspect of the present invention; 2) an expression vector, the expression vector comprising the polynucleotide described in 1); 3) A host cell, wherein the host cell comprises the polynucleotide described in 1) or the expression vector described in 2).

[0052] The term "expression vector" refers to an expression vector capable of expressing a target protein in an appropriate host cell, and is a gene construct comprising operably linked basic regulatory elements, wherein the operably linked basic regulatory elements enable the inserted gene to be expressed. Preferably, the recombinant vector is constructed to carry a coding polynucleotide or a fragment thereof encoding the ratiometric GRAB fluorescent probe of the present invention. The recombinant vector can be transformed or transfected into a host cell.

[0053] The expression vector of the present invention can also be obtained by connecting (inserting) the polynucleotide of the present invention to an appropriate vector. As long as it can replicate in the host, there is no particular limitation on the vector into which the gene of the present invention will be inserted. For example, plasmid vectors, phage vectors, viral vectors, etc. can be used. Specifically, commercial expression vectors can be used, such as pDisplay vectors, which can be purchased from Invitrogen. In addition, animal viruses such as retroviruses, adenoviruses, and vaccinia viruses and insect viruses such as baculoviruses can be used. The plasmids that can be used in the present invention are not limited to the examples.

[0054] A fourth aspect of the present invention provides any one of the following applications, comprising: 1) Application of the method according to the second aspect of the present invention in constructing a ratiometric GRAB fluorescent probe; 2) Use of the ratiometric fluorescent probe described in the first aspect of the present invention in detecting changes in the concentration of a specific ligand of a G protein-coupled receptor in a sample or tissue to be tested; 3) Use of the ratiometric GRAB probe constructed by the method described in the second aspect of the present invention in detecting changes in the concentration of specific ligands of G protein-coupled receptors in a sample or tissue to be tested.

[0055] Furthermore, the specific ligand is a neurotransmitter.

[0056] Furthermore, the neurotransmitters include NTS, SST, PACAP, NPY, dopamine and / or serotonin.

[0057] Furthermore, the detection includes qualitative and / or quantitative detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Shows the detection of dynamic changes of neuropeptides and the pH stability of EGFP; A is a schematic diagram of the neuropeptide probe detecting neuropeptide release; B shows that the fluorescence brightness of EGFP changes with pH; Figure 2 The chemical structure and spectrum of Fura-2 calcium dye are shown; wherein A shows the chemical structure and fluorescence ratio calculation method of Fura-2 calcium dye; B shows the excitation spectrum of Fura-2 calcium dye; Figure 3 The spectra of wild-type GFP and EGFP fluorescent proteins are shown; wherein A shows the spectrum of wild-type GFP, the solid line is the excitation spectrum, and the dotted line is the emission spectrum; B shows the spectrum of EGFP, the solid line is the excitation spectrum, and the dotted line is the emission spectrum; Figure 4 Schematic diagram of the excitation spectrum model of the new generation ratiometric neuropeptide probe; left: schematic diagram of the excitation spectrum of the non-ratiometric probe, right: excitation spectrum of the ratiometric neuropeptide probe; the dotted line represents the probe without neuropeptide binding, and the solid line represents the probe with neuropeptide binding; Figure 5 GFP spectrum and schematic diagram of GFP undergoing ESPT; A shows the excitation spectrum (solid line) and emission spectrum (dashed line) of GFP; B is a schematic diagram of GFP undergoing ESPT, and the structures from left to right are Neutral form, State I and Ionized form respectively; Figure 6 Schematic showing the structure-guided ratiometric probe screening workflow; Figure 7 The structure prediction analysis of NTS1.3 probe cpEGFP is shown; A shows the structure of NTS1.3 probe cpEGFP predicted by Alphafold; B shows the spatial distribution of 15 sites within 6Å around the S205 site. These 15 sites can be divided into 4 groups according to their spatial positions, namely the site in the connecting peptide region (dark blue), the site next to S205 (pink), the site next to E222 (bright blue) and the site close to the chromophore (green); C shows the frequency of occurrence of each group of sites in the spatial distance from near to far with the S205 site as the center; Figure 8The results of ratiometric NTS1.3 probe screening are shown; A shows the spatial distribution of 15 sites within 6Å around the S205 site in the NTS1.3 probe cpEGFP. These 15 sites can be divided into four groups according to their spatial positions, namely the site in the connecting peptide region (dark blue), the site next to S205 (pink), the site next to E222 (bright blue), and the site close to the chromophore (green); B shows the screening results of four groups of amino acid sites; C shows the saturation mutation screening results of the N3 (E) site of the N-terminal connecting peptide of the NTS1.3 probe cpEGFP; Fig. 9 Displays the structure prediction and analysis of NTS probes; A shows the structural comparison of NTS1.3 and NTS1.3-N3(E)L candidate probes, gray is the NTS1.3 probe cpEGFP structure predicted by Alphafold, and green is the NTS1.3-N3(E)L candidate probe cpEGFP structure predicted by Alphafold; B shows the relative spatial positions of N3(E), S205 and E222 sites in NTS1.3 probe cpEGFP (top); the relative spatial positions of N3(E)L, S205 and E222 sites in NTS1.3-N3(E)L candidate probe cpEGFP (bottom); Fig.10 It shows that the main chain carbon hydroxyl group of the S205 site is necessary for the occurrence of NTS probe ESPT; A shows the relative spatial positions of the N3(E)L, S205 and E222 sites in the NTS1.4 probe cpEGFP (top), and the relative spatial positions of the N3(E)L, S205A and E222 sites in the NTS1.4 probe cpEGFP (bottom); B shows the screening results of the mutant S205 site of the NTS1.4 probe, and the ΔF / F of the mutant of the S205 site of the NTS1.4 probe 0 (488nm) and ΔF / F 0 (405 nm) (left), ΔR / R of mutants at the S205 site of the NTS1.4 probe 0 and maximum brightness (right); Fig.11 The results of screening for sites near the chromophore of the NTS1.3 probe mutation are shown; A is a schematic diagram of the spatial distribution of amino acid sites near the chromophore in the GFP_S65T fluorescent protein; B shows the summary of the results of all mutant probes after mutations in the R168, Q94 and R96 sites of NTS1.3; Fig.12The results of screening for sites related to NTS1.3 probe mutation and folding rate are shown; A is a display diagram of amino acid sites with differences in sequence between cpEGFP and sfGFP; B shows a summary of repeated results of mutant probes after mutation of S30 and E39 sites of NTS1.3; Fig.13 The development and optimization of ratiometric SST probes are shown in Figure A. A shows the structural alignment of candidate probes SST1.0 and SST2.1. Gray is the structure of the SST1.0 probe cpEGFP predicted by Alphafold, showing the relative spatial positions of the N3(E), S205 and E222 sites. Light blue is the structure of the SST2.1 candidate probe cpEGFP predicted by Alphafold, showing the relative spatial positions of the N3(E)L, S205 and E222 sites. B shows the comparison of the response amplitudes of SST2.1 and SST1.0. C is a display of the amino acid sites that differ between cpEGFP and sfGFP in sequence (left), and a summary of the repeated results of mutant probes after the S30 and E39 sites of the mutant SST2.1 probe (middle and right). Fig.14 The optimization of ratiometric PACAP probes is shown; A shows the structural alignment of PACAP1.0 and PACAP1.0-N3(E)L candidate probes, and the gray one is the PACAP1.0 probe cpEGFP structure predicted by Alphafold, showing the relative positions of N3(E), S205 and E222 sites in space; B shows the PACAP1.0-N3(E)L candidate probe cpEGFP structure predicted by Alphafold, showing the relative positions of N3(E)L, S205 and E222 sites in space; C shows the screening data of the N3(E) site of the mutant PACAP1.0 probe; D is the fluorescence response curve of the three candidate probes PACAP-N3(E)I, PACAP-N3(E)L and PACAP-N3(E)M in dependence on the concentration of PACAP38, from left to right are the response amplitude calculated under 488 nm excitation, the response amplitude calculated under 405 nm excitation and the response amplitude calculated by ratio; Fig.15 Shows the prediction and analysis of the NPY probe structure; A shows the structural alignment of the NPY1.0 and NPY1.3 probes. The gray color is the NPY1.0 probe cpEGFP structure predicted by Alphafold, showing the relative positions of the N3(E), S205 and E222 sites in space (top), and the light yellow color is the NPY1.3 candidate probe cpEGFP structure predicted by Alphafold, showing the relative positions of the N3(E)L, S205 and E222 sites in space (bottom); B shows the comparison of the response amplitudes of NPY1.0 and NPY1.3; Fig.16 Shows the prediction and analysis of the GCG0.4 probe structure; the green in A is the GCG0.4 probe cpEGFP structure predicted by Alphafold, showing the relative positions of N3(L), ​​S205 and E222 sites in space; B is the fluorescence response curve of the GCG0.4 probe dependent on GCG concentration, from left to right are the response amplitude calculated under 488 nm excitation, the response amplitude calculated under 405 nm excitation and the response amplitude calculated by ratio; Fig.17 The development of ratiometric DA probes is shown; A shows the excitation spectrum of the green dopamine probe DA3m, the dashed line indicates the absence of DA, and the solid line indicates the presence of 1 μM DA; B shows the screening results of the N3(S) site of mutant DA3m; C shows the screening results of the S30 site of mutant DA3m; Fig.18 The development of ratiometric 5-HT probes is shown; A shows the excitation spectrum of the green serotonin probe 5-HT3.0, the dotted line indicates the absence of 5-HT, and the solid line indicates the presence of 10 μM 5-HT; B shows the screening results of the N3(E) site of mutant 5-HT3.0; Fig.19 The development of ratiometric ACh probes is shown in Figure 1. A shows the excitation spectrum of the green acetylcholine probe ACh4l, where the dotted line indicates the absence of ACh and the solid line indicates the presence of 1 mM ACh. B shows the optimization process from ACh3.0 to ACh4l and the comparison of the response amplitudes of the two. C shows the fluorescence response curve of the ACh4l probe in dependence on ACh concentration, which, from left to right, are the response amplitude calculated under 488 nm excitation, the response amplitude calculated under 405 nm excitation, and the response amplitude calculated by ratio. Fig. 20 The spectrum and pH stability test of the NTS1.5 probe are shown; A shows the single-photon excitation (left) and emission (right) fluorescence spectra of the NTS1.5 probe, the dotted line indicates the absence of NTS, and the solid line indicates the presence of 1 μM NTS; B shows the single-photon excitation spectrum of the NTS1.5 probe at different pH values; C is a statistical graph showing the change in probe fluorescence brightness with pH; Fig.21It shows that the ratio brightness of the NTS1.5 probe is not affected by the changes in the probe expression level and laser power; wherein A is a bar graph of Ex488 / 405, Ex488 and Ex405 of the NTS1.5 probe at different expression levels; B is a statistical graph of Ex488 / 405, Ex488 and Ex405 of the NTS1.5 probe at different expression levels; C is a bar graph of Ex488 / 405, Ex488 and Ex405 of the NTS1.5 probe at different expression levels; D is a statistical graph of Ex488 / 405, Ex488 and Ex405 of the NTS1.5 probe at different expression levels; Fig. 22 The spectrum and pH stability test of the SST2.2 probe are shown; A shows the single-photon excitation (left) and emission (right) fluorescence spectra of the SST2.2 probe, the dotted line indicates the absence of SST-14, and the solid line indicates the presence of 1 μM SST-14; B shows the single-photon excitation spectrum of the SST2.2 probe at different pH values; C is a statistical graph showing the change in probe fluorescence brightness with pH; Fig.23 It shows that the ratio brightness of the SST2.2 probe is not affected by changes in the probe expression level and laser power; among them, A is a bar graph of Ex488 / 405, Ex488 and Ex405 of the SST2.2 probe at different expression levels; B is a statistical graph of Ex488 / 405, Ex488 and Ex405 of the SST2.2 probe at different expression levels; C is a bar graph of Ex488 / 405, Ex488 and Ex405 of the SST2.2 probe at different expression levels; D is a statistical graph of Ex488 / 405, Ex488 and Ex405 of the SST2.2 probe at different expression levels. DETAILED DESCRIPTION

[0059] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with specific embodiments.

[0060] Example 1 General development strategy for ratiometric neuropeptide fluorescent probes So far, the applicant team has successfully developed a variety of neuropeptide probes with higher response amplitudes and sensitivities. Taking NTS1.3 and SST2.0 probes as examples, the performance of NTS1.3 probe has been preliminarily verified at the in vitro cultured cell level, neuron level, acute brain slices and in vivo mice, proving its potential to monitor the dynamic changes of NTS.

[0061] The GRAB probe couples the conformational changes of GPCR with the cyclic rearrangement of cpEGFP, that is, it uses the brightness changes of cpEGFP to report the dynamic changes of GPCR ligands. In vivo, the pH value of the cytoplasm is 7-7.5, and the pH value of the vesicles encapsulating neuropeptides is about 5.5. When the vesicles fuse with the cell membrane to release neuropeptides, the pH value near the release site will fluctuate violently ( Figure 1 A). cpEGFP is very sensitive to changes in pH in the environment, and its fluorescence brightness decreases as the pH decreases ( Figure 1 B) greatly affects the signal specificity of the probe for monitoring neuropeptide release in vivo.

[0062] Roger Tsien developed the calcium dye Fura-2 as early as 1985 ( Figure 2 A), its biggest feature is that it has an excitation ratiometric property, that is, it has two excitation peaks, Ex340 nm and Ex380 nm ( Figure 2 B), and use the fluorescence values ​​under these two excitation lights at different calcium concentrations as a ratio, and then calculate a standard curve for quantitative calcium research. However, the biggest disadvantage of Fura-2 is that both excitation lights are blue light, and long-term exposure to cells or tissues will cause a certain degree of damage and affect the activity of cells or tissues.

[0063] According to the spectrum of wild-type GFP, wild-type GFP also has two excitation peaks, Ex405 nm and Ex488 nm ( Figure 3 A), the fluorescent protein states correspond to the neutral state (Neutral form) and the ionized state (Ionized form). Among them, the Neutral form undergoes proton transfer in the excited state, which is called the excited state proton transfer (ESPT) process, and is converted into the intermediate state State I, which then emits light and the fluorescence brightness is detected by the outside world. Unfortunately, EGFP introduces three mutations on and near the chromophore based on GFP to increase the brightness, but it also loses the Ex405 nm excitation peak and only retains an Ex488 nm excitation peak ( Figure 3 B).

[0064] Based on this, this study proposes a hypothesis, whether it is possible to predict and analyze the structure of the probe cpEGFP using Alphafold, find the key sites that affect ESPT, restore the ESPT network of the probe, and promote the occurrence of ESPT. This excitation ratio probe has two excitation peaks, Ex405 nm and Ex488 nm, and after the addition of neuropeptides, it will cause a negative response (off response) of Ex405 and a positive response (on response) of Ex488 ( Figure 4 ). In this case, the Ex488 / 405 ratio will have a larger reaction amplitude, and the ratio can also be used to correct signal fluctuations caused by pH, expression level, laser power, etc. Therefore, this study will further give the GRAB fluorescent probe, which was previously studied by the applicant, this excitation ratio property to increase the reaction amplitude and stability of the probe.

[0065] For candidate probes that already have higher response amplitudes, the next step is to develop ratiometric neuropeptide probes based on structure guidance. According to the introduction in this article, for wild-type GFP, the fluorescent protein states under the two excitation peaks Ex405 and Ex488 of its excitation spectrum correspond to the neutral form and the ionized form, respectively ( Figure 5 A). In the excited state, the Neutral form undergoes proton transfer, i.e., the ESPT process, and is converted to the intermediate state State I, which then emits light and is detected by the outside world. In this network, the transfer of protons depends on the integrity of the ESPT network, which is composed of the chromophore, the main chain carbon hydroxyl group at the S205 site, the main chain carbon carboxyl group at the E222 site, and water molecules ( Figure 5 B). In order to make the GRAB probe have the property of excitation ratio, it is necessary to increase the response amplitude of the probe under 405 nm excitation, especially to increase the brightness of Ex405 in the inactive state. According to the experience of previous studies, when the S205 and E222 sites in GFP are mutated to other amino acids, the occurrence of fluorescent protein ESPT will be affected to a certain extent. For example, the E222Q mutation will cause the ESPT of GFP to be completely blocked, because the main chain carbon carboxyl group at the 222 site is missing to participate in the ESPT network. In contrast, the S205V mutation will reduce the brightness of GFP, but GFP can still undergo ESPT. This suggests that the E222 site is essential for the occurrence of ESPT. If the amino acid composition of the E222 site and its adjacent positions is directly changed, it is likely to inhibit the occurrence of ESPT, which is not conducive to the development of ratio neuropeptide probes.

[0066] Therefore, this study chose to optimize the composition of amino acids near the S205 site in order to screen candidate probes with better ratio properties, promote the occurrence of ESPT, and have a larger response amplitude under Ex405. First, cpEGFP and its connecting peptide region with a length of 5 amino acids at both ends were named and extracted as cpEGFP loop, and then predicted using Alphafold3. Finally, based on the prediction results, the amino acid sites within 6Å near the S205 site were analyzed in the structure and saturation mutations were screened, and their response amplitudes under Ex488, Ex405, and Ex488 / 405 were tested, respectively, referred to as ΔF / F. 0 (488), ΔF / F 0 (405) and ΔR / R 0 ( Figure 6 ).

[0067] Example 2 Development of Ratio-Type NTS Probe Taking the NTS1.3 probe as an example, we found 15 amino acid sites within 6Å of S205 ( Figure 7 A). These amino acid sites are divided into four groups according to their spatial positions, namely the linker group, sites near S205, sites near E222, and sites near chromophore ( Figure 7 B). Among them, the amino acids in the sites near S205 and sites near E222 are closer to S205, appearing at 3.0Å from S205. Further expanding the distance screening range, it can be found that the amino acids in the connecting peptide group (N-linker) gradually appear ( Figure 7 C), and all of these amino acids are located near the carboxyl and amine groups on the Cα of S205, that is, on the opposite side of the hydroxyl group of S205. It is worth mentioning that glutamic acid, the third amino acid of the N-linker, is numbered N3(E), which corresponds to the number 146 of the wild-type GFP. In the ESPT network of GFP, it can be found that the 146 site is close to the ESPT network in the spatial structure, suggesting that its conformational changes are likely to affect the occurrence of ESPT.

[0068] These 15 amino acids are spatially close to the S205 site ( Figure 8A), it is very likely that by changing its amino acid composition, it promotes the occurrence of probe ESPT. Therefore, based on the NTS1.3 probe, these 15 sites were screened for saturation mutations, and the probes were excited with light at wavelengths of 488 nm and 405 nm, respectively, and their response amplitudes after the addition of 1 μM NTS were detected. The results showed that different mutations in the amino acids adjacent to the E222 site and the amino acid sites in the connecting peptide region increased the mutant's ΔF / F0(405) to a certain extent. In contrast, the ΔF / F0(405) of the mutants in the amino acid sites in the connecting peptide region was significantly higher than that of the control probe NTS1.3 ( Figure 8 B). These mutants were further tested repeatedly on cells to ensure the credibility of the signal enhancement. The results showed that among the five sites in the linker peptide region, only when the glutamic acid at the N3(E) site mutated to other amino acids, the brightness and response amplitude of the candidate probe at 488 and 405 nm could be improved to a certain extent. In particular, when the glutamic acid at the N3(E) site mutated to L (leucine), the brightness and response amplitude of the candidate probe at 488 and 405 nm were the largest ( Figure 8C). The amino acid sequence of cpEGFP linked to the G protein-coupled receptor that specifically binds to NTS in this study is NVYIKADKQKNGIKAAFNIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITWGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVGGEGEGDATEGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN (SEQ ID NO.1); its N-terminal connecting peptide is LEEGG, and its C-terminal connecting peptide is TGATR; NTS1.The sequence of the 3 probe is (SEQ ID NO.7). .

[0069] In order to analyze why the mutation of E (glutamic acid) to L (leucine) at the N3 site of the NTS1.3 probe can significantly improve the ESPT of the probe, the sequences of cpEGFP and its left and right connecting peptides of the NTS1.3 probe and the NTS1.3-N3(E) candidate probe were extracted and the structures were predicted using Alphafold. The predicted structures of the two probes are very similar, but there are significant conformational differences between the two probes in the N-terminal connecting peptide region of cpEGFP ( Fig. 9A). The N-terminal connecting peptide of cpEGFP of the NTS1.3-N3(E) candidate probe is closer to the chromophore cpEGFP side than the N-terminal connecting peptide of cpEGFP of the NTS1.3 probe, and is displaced inward by about 7.4Å. Specifically, after NTS1.3 carries the N3(E)L mutation, the N-terminal connecting peptide undergoes a large displacement toward the cpEGFP side. It is speculated that this may be caused by the chemical bond formed by N3(E)L and certain amino acids on cpEGFP, resulting in an interaction. By searching within 3Å of the N3(E)L site, it was found that N3(E)L can form two hydrogen bonds with the S205 site, while this interaction was not found in NTS1.3 ( Fig. 9 B). In addition, the hydrogen bond between S205 and E222 can be found in the predicted structure of NTS1.3-N3(E)L, which is also the structural basis for proton transfer in the ESPT network. The above results show that N3(E)L forms a hydrogen bond with the S205 site, stabilizes the amino acid orientation of S205, and makes it more prone to ESPT. Since the NTS1.3-N3(E)L candidate probe has a large response amplitude at 405 and a large ratio response amplitude, it is named NTS1.4.

[0070] The S205 site plays a key bridging role in the ESPT network. It is speculated that N3(E)L promotes the occurrence of ESPT by forming a hydrogen bond with the side chain of S205. In order to further verify the necessity of S205 for the occurrence of probe ESPT, a screening experiment was designed based on the mutation of the S205 site of the NTS1.4 probe, especially in the absence of the hydroxyl group of the main chain carbon, to detect whether the ESPT of the probe would be affected, which is manifested as a change in the response amplitude of the probe under 405 excitation. Taking the mutation of S (serine) to A (alanine) as an example, the cpEGFP of the NTS1.4 and NTS1.4-S205A mutants was predicted and it was found that the 205 site did destroy the integrity of the ESPT network structure after the lack of the hydroxyl group of the main chain carbon, and it is very likely to inhibit the occurrence of ESPT ( Fig.10 A). The response amplitude of S205A under Ex405 was significantly reduced, and the response amplitude under Ex488 was slightly reduced. In addition, compared with S (serine), amino acids lacking main chain carbon hydroxyl groups or replaced by other groups, such as T (threonine), C (cysteine), V (valine) and G (glycine), the response amplitudes of these mutant probes under Ex405 were significantly lower than those of NTS1.4 ( Fig.10 B). Therefore, S205 is essential for the occurrence of probe ESPT. When S (serine) mutates and lacks other amino acids of the main chain carbon hydroxyl group, the occurrence of probe ESPT will be blocked, while N3(E)L plays an auxiliary role in the ESPT network.

[0071] For the neuropeptide probe, L (leucine) at the N3 site indirectly promotes the occurrence of probe ESPT by forming a hydrogen bond with the S205 side chain. Whether there are amino acids in the probe's cpEGFP that can directly affect the ESPT network remains to be discovered. In order to screen these amino acids, based on Roger Tsien's description of the amino acid sites near the GFP chromophore ( Fig.11 A), R168, T203, Q94 and R96 sites that may directly affect the chromophore were selected for mutation screening, and NTS1.3 and SST2.0 were used as templates, in order to obtain mutant probes with improved response amplitudes in both Ex405 and Ex488 / 405 ratios. The results showed that whether it was based on the saturation mutation screening of these four sites based on the NTS1.3 probe or based on the SST2.0 probe, although there were sites with improved response amplitudes under Ex405, the response amplitudes of these mutants under Ex488 were significantly reduced, resulting in a decrease in the ratio response ( Fig.11 B).

[0072] In addition to the sites near the chromophore that directly affect the ESPT network of cpEGFP, changes in the overall environment of the fluorescent protein (such as the folding of the fluorescent protein) are also likely to affect the ESPT network of cpEGFP. In order to further screen out sites that can promote the occurrence of probe ESPT, the cpEGFP used in the probe and the superfolder GFP (sfGFP) with faster folding efficiency developed by Geoffrey S. Waldo's research group were compared to extract sites with amino acid differences in the two sequences ( Fig.12 A). Among them, position 30 is R (arginine) and S (serine) in sfGFP and cpEGFP respectively, and position 39 is N (asparagine) and E (glutamic acid) in sfGFP and cpEGFP respectively. The possible mechanism by which mutations at these two positions can improve the folding efficiency of fluorescent proteins is described in the articles published on sfGFP. Taking S30 as an example, when S (serine) at position 30 mutates to R (arginine), R (arginine) will form a complete network with structurally adjacent E17 and E32, which may increase the folding rate of fluorescent proteins.

[0073] Therefore, using the NTS1.4 probe as a template, the amino acids corresponding to positions 30 (position 30 of wild-type GFP, corresponding to position 127 of cpEGFP) and 39 (position 39 of wild-type GFP, corresponding to position 136 of cpEGFP) are G (glycine) and E (glutamate), respectively, and saturation mutation screening is performed on these two positions. The results show that when G (glycine) at position 30 (position 127 of cpEGFP) is mutated to E (glutamate), the mutant probe has a significant increase in the response amplitude at 488 and 405 nm and the ratio, and it is named NTS1.5 ( Fig.12 B). Thus, based on the N3(E)L mutation of the N-terminal linker peptide introduced into the NTS1.3 probe and the G30E mutation in cpEGFP, the NTS1.5 probe with better ex-ratiometric properties was obtained.

[0074] Example 3 Extending the ratiometric fluorescent probe strategy to other neuropeptide probes Similarly, this study found that the N3(E)L mutation was also introduced during the optimization of the SST probe. The predicted structures of the SST1.0 and SST2.1 probe cpEGFP showed that the N3(E)L in the SST2.1 cpEGFP could form two hydrogen bonds with the S205 site, while this interaction was not found in SST1.0 ( Fig.13 A), and SST2.1 has a ΔF / F 0 (488) and ΔF / F 0 (405) have significantly improved ( Fig.13 B). Using the SST2.1 probe as a template, the amino acids corresponding to positions 30 and 39 (positions 127 and 136 of cpEGFP) are S (serine) and E (glutamate), respectively. Saturation mutation screening was performed on these two positions. The results showed that when the S (serine) at position 30 was mutated to E (glutamate), the response amplitude of the mutant probe under Ex488, Ex405 and the Ex488 / 405 ratio was significantly improved, and it was named SST2.2 ( Fig.13C). In this study, the amino acid sequence of cpEGFP linked to the G protein-coupled receptor that specifically binds to PACAP is NVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITIGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATEGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN (SEQ ID NO.2); its N-terminal connecting peptide is LEEGG, and its C-terminal connecting peptide is TGAAA; SST2 after N3(E)L mutation.The sequence of 1 is (SEQ ID NO.8). .

[0075] This study found that the N3(E)L mutation in the N-terminal connecting peptide of the NTS probe cpEGFP greatly promoted the occurrence of probe ESPT, and similar results were obtained on the SST probe. Can this rule be generalized to other neuropeptide probes, and then develop ratiometric neuropeptide probes on a large scale? Based on this hypothesis, the PACAP1.0 probe was selected as a mutation template. First, the cpEGFP and connecting peptide sequences of the PACAP1.0 probe and the PACAP1.0-N3(E)L mutant were structurally predicted. The structures of the PACAP1.0 probe and the PACAP1.0-N3(E)L mutant were compared ( Fig.14AB), and found that the N3(E)L mutation can also form two hydrogen bonds with the side chain of the S205 site, and the hydrogen bond formed between S205 and E222 can be observed. These findings suggest that the PACAP1.0-N3(E)L mutation is likely to increase the ESPT of the PACAP1.0 probe. Therefore, an experiment was designed to perform saturation mutation screening on the N3(E) site of PACAP1.0. The results showed that when E (glutamate) mutated to I (isoleucine), L (leucine) or M (methionine), the mutant's ΔF / F 0 (488), ΔF / F 0 (405) and ΔR / R 0 There is a significant improvement ( Fig.14 C). In order to further confirm which mutation has the best effect, the concentration dependence curves of these three mutations were measured. The results showed that when E (glutamate) mutated to L (leucine), the response amplitude of the mutant under Ex405 and Ex488 was the largest, -35% and 1500% respectively, and the ratio response was close to 3000% ( Fig.14D). In summary, the improvement of the PACAP probe response by the N3(E)L mutation is very similar to that by the NTS probe. In this study, the amino acid sequence of the cpEGFP linked to the G protein-coupled receptor that specifically binds to PACAP is NVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN (SEQ ID NO.3); its N-terminal connecting peptide is LEEGG, and its C-terminal connecting peptide is TGAAA; PACAP1.The amino acid sequence of the 0 probe is (SEQ ID NO.9). .

[0076] In addition, the same pattern was found in the observation of the new generation of NPY neuropeptide probes. The structural prediction results of the cpEGFP and its connecting peptide sequence of NPY1.0 and NPY1.3 probes showed that N3(E)L of NPY1.3 probe can form a hydrogen bond with the S205 side chain ( Fig.15 A), and its response amplitude under Ex405 is significantly improved compared with NPY1.0, about -30% ( Fig.15B). In this study, the amino acid sequence of cpEGFP linked to the G protein-coupled receptor that specifically binds to NPY is NVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATEGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN (SEQ ID NO.4); its N-terminal connecting peptide is LEEGG, and its C-terminal connecting peptide is TGAAA; before N3(E)L mutation, NPY1.The amino acid sequence of the 0 probe is (SEQ ID NO.10). .

[0077] Similarly, the third amino acid corresponding to the N-terminal connecting peptide of the GCG0.4 probe cpEGFP is L (leucine). This sequence may give the GCG probe an excitation ratio property and the possibility of ESPT. Based on this hypothesis, the structure prediction of the cpEGFP and connecting peptide sequence of the GCG0.4 probe was performed, and it was found that N3 (L) can form two hydrogen bonds with the side chain of the S205 site, and the hydrogen bond formed between S205 and E222 was observed ( Fig.16A). Further testing of the concentration dependence curve of the GCG0.4 probe revealed that the response amplitude of the GCG0.4 probe under Ex488 stimulation gradually increased with the increase of the exogenously added GCG concentration, while the response amplitude under Ex405 stimulation gradually decreased. The maximum response amplitudes of the GCG0.4 probe under Ex488 and Ex405 stimulation were 600% and -45%, respectively, and the maximum ratio response was about 1000% ( Fig.16 B).

[0078] In summary, this study found that when the amino acid at the third position (N3) of the N-terminal linker peptide of the GRAB neuropeptide probe cpEGFP is L (leucine), the ΔF / F of the probe can be significantly increased. 0 (405) and ΔR / R 0 This rule has been verified in a variety of GRAB neuropeptide probes (including NTS, PACAP, SST, NPY and GCG probes).

[0079] Example 4: Extending the ratiometric fluorescent probe strategy to monoamine neurotransmitter probes In order to systematically improve the ESPT of GRAB probes, this study further expanded the ratiometric probe development strategy to monoamine probes in addition to neuropeptide probes.

[0080] Dopamine is an important monoamine neurotransmitter that is widely present in the central nervous system (CNS) and peripheral nervous system. The synthesis of dopamine starts from the amino acid tyrosine, which is converted into levodopa (L-DOPA) by the action of tyrosine hydroxylase, and then into dopamine by the action of dopa decarboxylase. The metabolism of dopamine is mainly carried out by monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT), producing metabolites such as 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA).

[0081] As a neurotransmitter, dopamine transmits signals between neurons, affecting a variety of physiological and psychological functions. Dopamine neurons are mainly concentrated in several areas of the midbrain, including the substantia nigra pars compacta (SNc), the ventral tegmental area (VTA), and the arcuate nucleus. Dopamine neurons in these areas project to multiple areas of the brain through different pathways: (1) Nigrostriatal Pathway: Projects from the substantia nigra to the striatum and is mainly involved in motor control. Degeneration of dopamine neurons in this pathway is the main cause of Parkinson's disease. (2) Mesolimbic Pathway: Projects from the ventral tegmental area to the nucleus accumbens, amygdala, and hippocampus. It is related to reward mechanisms, pleasure, and addiction. From the ventral tegmental area to the nucleus accumbens, amygdala, and hippocampus, this pathway plays an important role in drug addiction (such as cocaine and amphetamine). (3) Mesocortical Pathway: Projects from the ventral tegmental area to the prefrontal cortex, involves cognitive function, decision-making, and emotional regulation, and is closely related to the occurrence of schizophrenia and depression. (4) Tuberoinfundibular Pathway: Projects from the arcuate nucleus to the anterior pituitary gland, regulating hormone secretion of the hypothalamus-pituitary axis. Dopamine in this pathway inhibits the secretion of prolactin, and its dysfunction may lead to hyperprolactinemia.

[0082] Therefore, the functions of dopamine in the central nervous system are complex and diverse, involving motor control, emotion regulation, reward mechanism, cognitive function and endocrine regulation. Its distribution and function in different neural pathways make it the key to the study of various neuropsychiatric diseases and drug addiction. Understanding these mechanisms of action of dopamine is crucial for the development of effective treatments, so the detection of dopamine also requires more sensitive tools with high spatiotemporal resolution.

[0083] This study is based on the latest generation of green dopamine probe DA3m, whose excitation spectrum shows that DA3m has a small response amplitude to DA under 400 nm excitation ( Fig.17 A), is almost 0. Therefore, this study designed an experiment to mutate the N3(S) and S30 sites of DA3m (site 127 of cpEGFP) in order to obtain a better excitation ratio dopamine probe. The screening results showed that when the N3(S) of DA3m was mutated to L (leucine), A (alanine) and H (histidine), the ΔF / F of these three mutant probes was 0 (405) were significantly improved compared to DA3m, and the N3(S)H mutation had a significant effect on ΔF / F0 (405) and ΔR / R 0 The biggest improvement ( Fig.18 B). In addition, for the screening of the S30 site, it was found that the ΔF / F0 (405) and ΔR / R0 of the S30V (valine) mutant probe were significantly higher than those of DA3m ( Fig.17C). In the present invention, the amino acid sequence of cpEGFP connected to the G protein-coupled receptor that specifically binds to dopamine is YVYIKADKQKNGIKANFGIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGLMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKNDGFYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN (SEQ ID NO.5); its N-terminal connecting peptide is LNSLI, and its C-terminal connecting peptide is NHDQL; the amino acid sequence of the dopamine probe DA3m is (SEQ ID NO.11). .

[0084] Serotonin (5-HT) is another important monoamine neurotransmitter that is widely present in the central nervous system (CNS) and peripheral nervous system. Serotonin is converted from tryptophan to 5-hydroxytryptophan (5-HTP) by tryptophan hydroxylase, and then produced by aromatic amino acid decarboxylase. The metabolism of serotonin is mainly carried out by monoamine oxidase (MAO) to produce 5-hydroxyindoleacetic acid (5-HIAA).

[0085] Serotonin plays a key role in regulating mood, appetite, sleep, memory and learning. Serotonergic neurons are mainly located in several nuclei in the midbrain and brainstem, especially the raphe nuclei. These neurons are distributed in the raphe nuclei, especially the dorsal raphe nuclei and the giant cell nucleus, and they project to various areas of the brain, including the cortex, limbic system and spinal cord. The specific functions of serotonin include (1) Emotional regulation: It plays a key role in regulating mood and emotions. Clinically, low levels of serotonin are associated with mood disorders such as depression and anxiety. Selective serotonin reuptake inhibitors (SSRIs) are commonly used antidepressants that relieve symptoms by increasing the level of serotonin in the synaptic cleft. (2) Sleep regulation: It participates in regulating the sleep-wake cycle, especially the precursors of serotonin (such as melatonin) play an important role in promoting sleep. In addition, abnormal serotonin function can lead to sleep disorders, such as insomnia or irregular sleep. (3) Appetite control: It plays an important role in regulating appetite and satiety. Abnormal serotonin levels may lead to eating disorders such as anorexia or bulimia. (4) Pain regulation: It is involved in the regulation of pain signals in both the central and peripheral nervous systems. Serotonergic drugs can be used to relieve chronic pain and migraine. (5) Cognition and memory: Serotonin activity in the prefrontal cortex and hippocampus is associated with cognitive function, learning and memory formation. Serotonin dysfunction is associated with cognitive impairment and memory loss. (6) The role of serotonin in drug addiction is also very important. Many addictive drugs, such as MDMA (ecstasy) and hallucinogens, produce their effects by affecting the serotonin system: MDMA causes a significant increase in serotonin levels in the synaptic cleft by promoting the massive release of serotonin and preventing its reuptake. This effect leads to a strong sense of pleasure and emotional resonance, but long-term use can deplete serotonin reserves in cells, leading to depression and cognitive impairment. Hallucinogens work by activating serotonin receptors (such as the 5-HT2A receptor), which in turn cause profound changes in perception, mood, and cognition, sometimes leading to psychotic symptoms.

[0086] In order to develop a sensitive excitation ratiometric 5-HT probe, this study was based on the latest generation of green serotonin probe 5-HT3.0, whose excitation spectrum showed that the response amplitude of 5-HT3.0 to 5-HT under 400 nm excitation was very small ( Fig.18 A). Therefore, this study designed an experiment to mutate N3(E) of 5-HT3.0. The results showed that when N3(E) of 5-HT3.0 was mutated to M (methionine) and L (leucine), the ΔF / F of the two mutant probes was 0 (405) were significantly improved compared to DA3m, and the N3(E)M mutation had a significant effect on ΔF / F 0 (405) has the largest improvement ( Fig.18B). In the present invention, the amino acid sequence of cpEGFP connected to the G protein-coupled receptor specifically binding to dopamine is NVYIKADKQKNGIKAIFHIRHNIEDGGVQLAIHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN (SEQ ID NO.6); its N-terminal connecting peptide is LEEGG, and its C-terminal connecting peptide is TGAAA; the serotonin probe 5-HT3.The amino acid sequence of 0 is METDTLLLWVLLLWVPGSTGDTSLYKKVGTTGDKLDANVSSNEGFRSVEKVVLLTFLAVVILMAILGNLLVMVAVCRDRQLRKIKTNYFIVSLAFADLLVSVLVMPFGAIELVQDIWAYGEMFCLVRTSLDVYLCTASIFHLCCISLDRYYAICCQPLVYRNKMTPLRIALMLGGCWVLPMFISFLPIMQGWNNIGIVDVIEKRKFSHNSNSTWCVFMVNKPYAITCSVVAFYIPFLLMVLAYYRIYVVAKRGPDAVAAPPGGTERRPNGLGPERSAGPGGAEAEPLPTQLNGAPGEPAPAGPRDTDALDLEEGGNVYIKADKQKNGIKAIFHIRHNIEDGGVQLAIHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNTGAAARFRWRTETKAAKVLCVIMGCFCFCWAPFFVTNIVDPFIDYTVPEQVWTAFLWLGYINSGLNPFLYAFLNKSFRRAFLIILRCNYKVEKKPPVRQIPRVAATALSGRELNVNIYRHTNEPVIEKASDNEPGIEMQVENLELPVNPSSVVSERISSV (SEQ ID NO.12).

[0087] Acetylcholine (ACh) is widely present in the central nervous system and peripheral nervous system. Acetylcholine is synthesized from choline and acetyl coenzyme A under the action of choline acetyltransferase (ChAT). Its degradation is mainly catalyzed by acetylcholinesterase (AChE) to produce choline and acetic acid. Acetylcholine transmits signals between neurons, affecting muscle contraction, memory, attention and autonomic nervous function. Acetylcholinergic neurons are mainly distributed in the basal forebrain (such as the medial septal nucleus and Meynert basal nucleus) and brainstem (such as the locus coeruleus of the pons and hypothalamus) in the central nervous system, and are mainly distributed in the autonomic nervous system (including sympathetic and parasympathetic nerves) and motor nerve endings in the peripheral nervous system. The functions of acetylcholine include (1) muscle contraction: acetylcholine is released at the neuromuscular junction to activate muscle fiber contraction. When acetylcholine receptors are dysfunctional, myasthenia gravis will occur. (2) memory and learning: acetylcholine plays a key role in the hippocampus and cerebral cortex, promoting memory and learning. Clinically, it has been found that patients with Alzheimer's disease show a significant decrease in acetylcholine levels. (3) Attention and wakefulness: Acetylcholine regulates attention, wakefulness and cognitive function. Attention deficit hyperactivity disorder (ADHD) is associated with abnormal acetylcholine function. (4) Autonomic function: Acetylcholine regulates heart rate, digestion and urinary function in the autonomic nervous system. Clinical relevance: Autonomic dysfunction such as hypotension and arrhythmia are related to acetylcholine disorders. Therefore, drugs that regulate acetylcholine levels can be used to treat corresponding diseases. For example, acetylcholinesterase (AChE) inhibitors can increase acetylcholine levels in the synaptic cleft by inhibiting AChE activity, and are used to treat Alzheimer's disease and myasthenia gravis. Neurotoxins (such as botulinum toxin) can block the release of acetylcholine, causing muscle relaxation, and are used to treat muscle spasms and cosmetic surgery.

[0088] In order to better discover and understand the function and role of acetylcholine, it is also necessary to develop sensitive excitation ratio ACh probes. This study found that the latest generation of green acetylcholine probe ACh4l is a good excitation ratio probe. Its excitation spectrum shows that ACh4l has a large response amplitude to ACh under 400 nm excitation ( Fig.19 A). In order to understand why ACh4l has ratiometric properties, this study reviewed the optimization process of the ACh4l probe and found that ACh4l introduced four mutations based on the previous generation ACh3.0 probe version, namely N3(E)L, N4(G)N, N6.33R and Y3F ( Fig.19 B). The concentration dependence curve of ACh4l probe shows that as the ACh concentration increases, the ΔF / F of ACh4l probe increases. 0 (488) gradually increases, ΔF / F 0 (405) gradually decreases, and its ratio ΔR / R0 Also, the probe ΔR / R 0 The response to 10 mM ACh was close to 1200%, which was significantly higher than its ΔF / F 0 900% of (488) Fig.19 C).

[0089] Example 5 Characterization of the performance of NTS1.5 probe in HEK293T cells Ratio-type neuropeptide probes have the property of showing opposite reactions triggered by binding neuropeptides under 488 and 405 nm excitation light, so using the Ex488 / 405 ratio for calculation will significantly increase the probe's response amplitude, providing the possibility of sensitively detecting dynamic changes in neuropeptides. However, the advantage of ratio-type neuropeptide probes is not only that they have a higher response amplitude, but they can also be used to correct the fluctuation of fluorescence values ​​caused by certain environmental factors (such as pH and excitation power in the environment). Because the probe itself has only one fluorescent protein and contains one chromophore, when external factors (pH, excitation power, etc.) change, the fluorescence of the probe will change accordingly. However, since the fluorescence of the ratio-type probe under 488 nm and 405 nm excitation comes from the same chromophore, the influence of external factors on Ex488 and Ex405 is in the same direction. Therefore, using the Ex488 / 405 ratio may be beneficial to correct the changes in fluorescence intensity caused by changes in pH or excitation power, thereby improving the stability of the probe and ensuring the specificity of the probe detection signal.

[0090] First, the spectral properties of the NTS1.5 probe were characterized. The results showed that the NTS1.5 probe has two excitation peaks at 400 nm and 505 nm, and the corresponding emission peaks are both at 525 nm ( Fig. 20 A). In order to verify the pH stability of the NTS1.5 probe, the pH in the cell environment and inside the cell was changed. The results showed that as the pH decreased, the fluorescence brightness of the probe under Ex488 and Ex405 gradually decreased ( Fig. 20 B). In contrast, the ratio of Ex488 / 405 is less affected by different pH values. For example, at pH = 5, Ex488 is only 30% of the maximum brightness, but Ex488 / 405 is 80% of the maximum brightness, and its fluorescence brightness is significantly higher than Ex488 ( Fig. 20 C). These results suggest that the NTS1.5 probe may be less affected by pH changes when detecting dynamic changes in neuropeptide release in vivo.

[0091] In addition, the brightness of fluorescent proteins will also be greatly affected by the expression level of the probe and the laser power. For example, the higher the expression level of the probe, the higher the Ex488 and Ex405 of cpEGFP; the higher the laser power used in the experiment, the higher the Ex488 and Ex405 of cpEGFP. Because the effects of the probe expression level and the laser power on Ex488 and Ex405 of cpEGFP are in the same direction, it is speculated that Ex488 / 405 may remain stable. In order to detect whether the fluorescence brightness of the NTS1.5 probe will be affected by the expression level of the probe, 50 ng, 100 ng, 200 ng and 300 ng of the NTS1.5 probe were expressed in HEK293T cells, and Ex488, Ex405 and Ex488 / 405 at different expression levels of the probe were detected. The experimental results showed that with the increase in the expression level of the probe, the fluorescence brightness of Ex488 and Ex405 gradually increased, but the fluorescence brightness ratio of Ex488 / 405 remained stable ( Fig.21 AB).

[0092] In order to further verify whether the fluorescence brightness of the NTS1.5 probe is affected by the excitation power, 300 ng of the NTS1.5 probe was expressed in HEK293T cells, and the probe was excited with 10%, 30%, 50%, 70% and 90% excitation powers, and the Ex488, Ex405 and Ex488 / 405 of the probe were detected at different excitation power intensities. The experimental results showed that as the excitation power of the probe increased, the fluorescence brightness of Ex488 and Ex405 also increased proportionally, but the fluorescence brightness ratio of Ex488 / 405 remained relatively stable. For example, when the excitation power was 10%, Ex488 was 10% of the maximum brightness, but Ex488 / 405 was 70% of the maximum brightness, and its fluorescence brightness was significantly higher than that of Ex488 ( Fig.21 CD).

[0093] Example 6 Characterization of the performance of SST2.2 probe in HEK293T cells Similarly, the spectral properties of the SST2.2 probe are characterized. The SST2.2 probe has two excitation peaks at 400 nm and 505 nm, and the corresponding emission peaks are both at 525 nm ( Fig. 22 A). In order to verify the pH stability of the SST2.2 probe, the pH in the cell environment and in the cell was changed. The results showed that as the pH decreased, the fluorescence brightness of the probe at 488 and 405 also gradually decreased ( Fig. 22 B). In contrast, the ratio of Ex488 / 405 is less affected by different pH values. Even at pH=3, Ex488 has only 10% of its maximum brightness, but Ex488 / 405 still maintains 60% of its maximum brightness, and its fluorescence brightness is significantly higher than that of Ex488 ( Fig. 22 C). These results suggest that the SST2.2 probe has good pH stability.

[0094] In order to detect whether the fluorescence brightness of the SST2.2 probe is affected by the expression level of the probe, 50 ng, 100 ng, 200 ng and 300 ng of the SST2.2 probe were expressed in HEK293T cells, and Ex488, Ex405 and Ex488 / 405 at different expression levels of the probe were detected. The experimental results showed that as the expression level of the probe increased, Ex488 and Ex405 also gradually increased, but Ex488 / 405 remained stable ( Fig.23 AB). In order to further verify whether the fluorescence brightness of the SST2.2 probe is affected by the excitation power, 300 ng of the SST2.2 probe was expressed in HEK293T cells, and the probe was excited with 10%, 30%, 50%, 70% and 90% excitation powers, and Ex488, Ex405 and Ex488 / 405 of the probe were detected at different excitation power intensities. The experimental results showed that as the excitation power of the probe increased, Ex488 and Ex405 also increased proportionally, but Ex488 / 405 remained relatively stable. For example, when the excitation power was 10%, Ex488 was 10% of the maximum brightness, but Ex488 / 405 was 75% of the maximum brightness, and its fluorescence brightness was significantly higher than that of Ex488 ( Fig.23 CD).

[0095] The present invention is described above by way of example. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by those skilled in the art without inventive effort falls within the protection scope of the present invention.

Claims

1. A ratiometric fluorescent probe based on G protein-coupled receptors, characterized in that: The ratiometric fluorescent probe comprises a G protein coupled receptor, a cyclically rearranged fluorescent protein and a connecting peptide; the connecting peptide comprises an N-terminal connecting peptide and a C-terminal connecting peptide; the cyclically rearranged fluorescent protein is inserted between the fifth transmembrane region and the sixth transmembrane region of the G protein coupled receptor, and the N-terminus of the cyclically rearranged fluorescent protein is connected to the fifth transmembrane region of the G protein coupled receptor via the N-terminal connecting peptide; the C-terminus of the cyclically rearranged fluorescent protein is connected to the sixth transmembrane region of the G protein coupled receptor via the C-terminal connecting peptide; The N-terminal connecting peptide comprises 5 amino acids, and the third amino acid of the N-terminal connecting peptide is mutated.

2. The ratiometric fluorescent probe according to claim 1, characterized in that The ratiometric fluorescent probe can be expressed on the cell membrane; and the ratiometric fluorescent probe can bind to the specific ligand of the G protein-coupled receptor when in contact with it, thereby causing a detectable change in the fluorescence intensity of the fluorescent probe; The G protein-coupled receptor is of human origin; The specific ligand is a neurotransmitter; The neurotransmitters include NTS, SST, PACAP, NPY, dopamine and / or serotonin.

3. The ratiometric fluorescent probe according to any one of claims 1 to 2, characterized in that: The cyclically rearranged fluorescent protein is cpEGFP; When the cpEGFP is connected to the G protein-coupled receptor that specifically binds to NTS, its amino acid sequence is the sequence shown in SEQ ID NO.1, or the sequence after the 127th position thereof is mutated to glutamic acid; When the cpEGFP is connected to the G protein-coupled receptor that specifically binds to SST, its amino acid sequence is the sequence shown in SEQ ID NO.2, or the sequence after the 127th position thereof is mutated to glutamic acid; When the cpEGFP is connected to the G protein-coupled receptor that specifically binds to PACAP, its amino acid sequence is the sequence shown in SEQ ID NO.3; When the cpEGFP is connected to the G protein-coupled receptor that specifically binds to NPY, its amino acid sequence is the sequence shown in SEQ ID NO.4; When the cpEGFP is connected to a G protein-coupled receptor that specifically binds to dopamine, its amino acid sequence is the sequence shown in SEQ ID NO.5, or the sequence after its 127th position is mutated to valine; When the cpEGFP is connected to a G protein-coupled receptor that specifically binds to serotonin, its amino acid sequence is the sequence shown in SEQ ID NO.

6.

4. The ratiometric fluorescent probe according to claim 1, characterized in that When the cpEGFP is connected to NTS, SST, PACAP, NPY or a G protein-coupled receptor that specifically binds to serotonin, the sequence of the N-terminal connecting peptide before the third amino acid mutation is LEEGG; When the cpEGFP is connected to a G protein-coupled receptor that specifically binds to NTS, SST or NPY, the third amino acid of the N-terminal connecting peptide is mutated to leucine, and the sequence of the N-terminal connecting peptide after mutation is LELGG; When the cpEGFP is connected to the G protein-coupled receptor that specifically binds to PACAP, the third amino acid of the N-terminal connecting peptide is mutated to leucine, isoleucine or methionine, and the sequence of the N-terminal connecting peptide after mutation is any one of LELGG, LEIGG or LEMGG; When the cpEGFP is connected to a G protein-coupled receptor that specifically binds to serotonin, the third amino acid of the N-terminal connecting peptide segment is mutated to leucine or methionine, and the sequence of the N-terminal connecting peptide after mutation is any one of LELGG or LEMGG.

5. The ratiometric fluorescent probe according to claim 1, characterized in that When the cpEGFP is connected to a G protein-coupled receptor that specifically binds to dopamine, the sequence of the N-terminal connecting peptide before the third amino acid mutation is LNSLI; When the cpEGFP is connected to a G protein-coupled receptor that specifically binds to acetylcholine, the sequence of the N-terminal connecting peptide before the third amino acid mutation is ETEGG; When the cpEGFP is connected to a G protein-coupled receptor that specifically binds to dopamine, the third amino acid of the N-terminal connecting peptide is mutated to leucine, alanine or histidine, and the sequence of the N-terminal connecting peptide after mutation is any one of LNLLI, LNALI and LNHLI.

6. A method for constructing a ratiometric GRAB fluorescent probe, characterized in that: The method comprises mutating the third amino acid of the N-terminal connecting peptide segment of the cyclically rearranged fluorescent protein connected to the G protein coupled receptor; The cyclically rearranged fluorescent protein is cpEGFP; The mutation includes mutating the third amino acid of the cpEGFP N-terminal linker peptide to leucine.

7. The method according to claim 6, characterized in that When the G protein-coupled receptor is a G protein-coupled receptor that specifically binds to PACAP, the mutation further comprises mutating the third amino acid of the cpEGFP N-terminal connecting peptide to isoleucine or methionine; When the G protein-coupled receptor is a G protein-coupled receptor that specifically binds to dopamine, the mutation further comprises mutating the third amino acid of the cpEGFP N-terminal connecting peptide to alanine or histidine; When the G protein coupled receptor is a G protein coupled receptor that specifically binds to serotonin, the mutation further comprises mutating the third amino acid of the cpEGFP N-terminal connecting peptide segment to methionine.

8. The method according to claim 6, characterized in that The method further comprises modifying cpEGFP; the modification comprises mutating site 127 of cpEGFP; When the G protein-coupled receptor is a G protein-coupled receptor that specifically binds to NTS or SST, the mutation comprises mutating position 127 of cpEGFP to glutamic acid; When the G protein-coupled receptor is a G protein-coupled receptor that specifically binds to dopamine, the mutation comprises mutating position 127 of cpEGFP to valine.

9. A biomaterial, characterized in that: The biological material comprises: 1) A polynucleotide, characterized in that the polynucleotide encodes the ratiometric fluorescent probe according to any one of claims 1 to 6; 2) An expression vector, characterized in that the expression vector comprises the polynucleotide described in 1); 3) A host cell, characterized in that the host cell contains the polynucleotide described in 1) or the expression vector described in 2).

10. Any of the following applications, characterized in that: The applications include: 1) Use of the method according to any one of claims 7 to 9 in constructing a ratiometric GRAB fluorescent probe; 2) Use of the ratiometric fluorescent probe according to any one of claims 1 to 6 in detecting changes in the concentration of a specific ligand of a G protein-coupled receptor in a sample or tissue to be tested; 3) Use of the ratiometric GRAB probe constructed by the method according to any one of claims 7 to 9 in detecting changes in the concentration of a specific ligand of a G protein-coupled receptor in a sample or tissue to be tested; The specific ligands include neurotransmitters; The neurotransmitters include NTS, SST, PACAP, NPY, dopamine and / or serotonin; The detection includes qualitative and / or quantitative detection.

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