Ratio Fluorescent Probe Constructed Based on G Protein-Coupled Receptor and Its Construction Method

Abstract

The present invention discloses a ratio fluorescent probe constructed based on G protein-coupled receptors and its construction method. The present invention discovers that by introducing mutations at the same amino acid site (the third amino acid of the linker peptide segment) on the GRAB probe, the GRAB probe can have the property of excitation ratio type. These probes with the property of excitation ratio type can have high pH stability, are not restricted by the expression level of the probe itself and external hardware conditions, maintain high stability in fluorescence brightness, can correct fluorescence interference caused by movement, can sensitively detect the release of neurotransmitters, and have the potential to be applied to the in vivo quantitative detection of neurotransmitter concentration.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and specifically relates to a ratio-type 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 have been 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 spatio-temporal resolution and cell specificity (Wu et al., 2022). For example, although the classical microdialysis method can specifically detect neurotransmitters and neuromodulators, its sampling frequency is very low (once every 5 to 10 minutes), with low spatial resolution and no cell specificity (Chefer et al., 2009); fast-scan cyclic voltammetry has good time resolution but no cell specificity and selectivity (Kuhr and Wightman, 1986; Robinson et al., 2003). In recent years, the Li Yulong research group, as one of the teams that first developed fluorescence probes based on G protein-coupled receptor activation (GPCR Activation Based sensors, abbreviated as GRAB) internationally, has developed dozens of GRAB probes for detecting different types of neurotransmitters, including dopamine, serotonin, norepinephrine, endocannabinoids, and neuropeptides, etc. These novel genetically encoded fluorescent probes have well solved the defects of existing detection methods, can achieve high spatio-temporal resolution, and can specifically detect neurotransmitters and modulators in real time in specific cell types.

[0003] However, since these current neurotransmitter probes based on fluorescence intensity changes are affected by many factors during use, such as the expression level of chromophore numbers, fluorescence excitation intensity, and photobleaching phenomenon, the application scenarios of this type of probe that reflects neurotransmitter changes based on a single fluorescence intensity are mainly limited to judging the occurrence of neurotransmitter release events caused by neuronal activities, 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 to improve the excitation ratio-type properties of the probe. These probes with excitation ratio-type properties can have high pH stability, are not restricted by the expression level of the probe itself and external hardware conditions, maintain 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 applied to quantitatively detect the concentration of neurotransmitters in vivo. Summary of the Invention

[0005] To address the deficiencies of the prior art, the objective of the present invention is to develop a general strategy that can enhance the excitation ratio-type properties of GRAB probes.

[0006] To achieve the above objective, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, a ratio-type fluorescent probe constructed based on a G protein-coupled receptor is provided.

[0008] Furthermore, the ratio-type fluorescent probe includes a G protein-coupled receptor, a circularly permuted fluorescent protein, and a linker peptide segment; the linker peptide segment includes an N-terminal linker peptide and a C-terminal linker peptide; the circularly permuted fluorescent protein is inserted between the fifth transmembrane domain and the sixth transmembrane domain of the G protein-coupled receptor, and the N-terminus of the circularly permuted fluorescent protein is connected to the fifth transmembrane domain of the G protein-coupled receptor through the N-terminal linker peptide; the C-terminus of the circularly permuted fluorescent protein is connected to the sixth transmembrane domain of the G protein-coupled receptor through the C-terminal linker peptide.

[0009] Furthermore, the N-terminal linker peptide contains 5 amino acids, and the third amino acid of the N-terminal linker peptide is mutated.

[0010] Furthermore, the ratio-type fluorescent probe can be expressed on the cell membrane; and when the ratio-type fluorescent probe contacts the specific ligand of the G protein-coupled receptor, it can bind to it, resulting in a detectable change in the fluorescence intensity of the fluorescent probe.

[0011] Furthermore, the G protein-coupled receptor is of human origin.

[0012] Furthermore, the specific ligand includes neurotransmitters.

[0013] Furthermore, the neurotransmitters include NTS, SST, PACAP, NPY, dopamine, and / or serotonin.

[0014] Furthermore, the circularly permuted fluorescent protein is cpEGFP.

[0015] Furthermore, 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 site is mutated to glutamate.

[0016] Furthermore, 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 site is mutated to glutamate.

[0017] Furthermore, when the cpEGFP is linked to the G protein-coupled receptor that specifically binds to PACAP, its amino acid sequence is the sequence shown in SEQ ID NO.3.

[0018] Furthermore, when the cpEGFP is linked to the G protein-coupled receptor that specifically binds to NPY, its amino acid sequence is the sequence shown in SEQ ID NO.4.

[0019] Furthermore, when the cpEGFP is linked to the 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 site is mutated to valine.

[0020] Furthermore, when the cpEGFP is linked to the G protein-coupled receptor that specifically binds to serotonin, its amino acid sequence is the sequence shown in SEQ ID NO.6.

[0021] Furthermore, when the cpEGFP is linked to the G protein-coupled receptor that specifically binds to NTS, SST, PACAP, NPY or serotonin, the sequence before the third amino acid mutation of the N-terminal linker peptide is LEEGG.

[0022] Furthermore, when the cpEGFP is linked to the G protein-coupled receptor that specifically binds to NTS, SST or NPY, the third amino acid of the N-terminal linker peptide is mutated to leucine, and the sequence of the N-terminal linker peptide after mutation is LELGG.

[0023] Furthermore, when the cpEGFP is linked to the G protein-coupled receptor that specifically binds to PACAP, the third amino acid of the N-terminal linker peptide is mutated to leucine, isoleucine or methionine, and the sequence of the N-terminal linker peptide after mutation is any one of LELGG, LEIGG or LEMGG.

[0024] Furthermore, when the cpEGFP is linked to the G protein-coupled receptor that specifically binds to serotonin, the third amino acid of the N-terminal linker peptide segment is mutated to leucine or methionine, and the sequence of the N-terminal linker peptide after mutation is any one of LELGG or LEMGG.

[0025] Furthermore, when the cpEGFP is linked to the G protein-coupled receptor that specifically binds to dopamine, the sequence before the third amino acid mutation of the N-terminal linker peptide is LNSLI.

[0026] Furthermore, when the cpEGFP is linked to the G protein-coupled receptor that specifically binds to acetylcholine, the sequence before the third amino acid mutation of the N-terminal linker peptide is ETEGG.

[0027] Furthermore, when the cpEGFP is linked to the G protein-coupled receptor that specifically binds dopamine, the third amino acid of the N-terminal linker peptide mutates to leucine, alanine, or histidine, and the mutated sequence of its N-terminal linker peptide is any one of LNLLI, LNALI, and LNHLI.

[0028] The applicant's team of the present invention has previously established a fluorescence probe constructed based on G protein-coupled receptors. However, this probe that reflects neurotransmitter changes based on a single fluorescence intensity is restricted by many factors. Therefore, the applicant searched for mutation sites that can improve the excitation ratio-type 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 linker peptide), the excitation ratio-type performance can be improved on nearly ten types of GRAB probes. These probes with excitation ratio-type properties have high pH stability, are not restricted by the expression level of the probe itself and external hardware conditions, maintain high stability in fluorescence brightness, can correct fluorescence interference caused by movement, can sensitively detect the release of neurotransmitters, and have the potential to be applied to the in vivo quantitative detection of neurotransmitter concentration.

[0029] The principle of the ratio-type fluorescence probe constructed based on G protein-coupled receptors (also referred to as the ratio-type GRAB fluorescence probe in the present invention) of the present invention is as follows: The circularly permuted fluorescent protein is inserted between the fifth transmembrane domain and the sixth transmembrane domain of the GPCR. The binding of the ligand to the GPCR induces a conformational change of the GPCR, which in turn leads to a conformational change of the circularly permuted fluorescent protein, causing a change in the fluorescence signal intensity, thereby converting the ligand-induced conformational change of the GPCR into a change in the optical signal.

[0030] G protein-coupled receptors (GPCRs) are a class of seven-transmembrane proteins expressed on the cytoplasmic membrane. The main body of the GPCR protein consists of 7 α-helical structures spanning the cytoplasmic membrane. The N-terminal and 3 loops are located outside the cell, and the C-terminal and 3 loops are located inside the cell. The "G protein-coupled receptor (GPCR)" described herein is a large protein family of transmembrane receptors that sense extracellular molecules, activate intracellular signal transduction pathways, and ultimately activate cellular responses. Ligands that bind to 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 medicinal drugs. Based on sequence homology and functional similarity, GPCRs can be classified into at least 5 classes: class A rhodopsin-like, class B secretin-like, class C metabotropic / pheromone, class D fungal pheromone, and class E cAMP receptor.

[0031] Class A rhodopsin-like receptors include: amine receptors: acetylcholine, alpha-adrenergic receptor, beta-adrenergic receptor, dopamine, histamine, serotonin, octopamine, and trace amine; peptide receptors: angiotensin (NTS), bombesin, bradykinin, C5a anaphylatoxin, Fmet-leu-phe, AP J-like substance, interleukin-8, chemokine receptors (C-C chemokines, C-X-C chemokines, Β0ΝZ0 receptor (CXC6R), C-X3-C chemokines, and XC chemokines), CCK receptor, endothelin receptor, melanocortin receptor, neuropeptide Y (NPY) receptor, neurotensin receptor, opioid receptor, somatostatin (SST) receptor, tachykinin receptor (substance P (NK1), substance K (NK2), neuropeptide K (NK3), tachykinin-like 1, and tachykinin-like 2), vasopressin-like receptors (vasopressin, oxytocin, and Conopressin), galanin-like receptors (galanin, allatostatin, and GPCR54), protease-activated-like receptors (e.g., thrombin), orexin & neuropeptide FF, teleost urotensin II receptor, adrenomedullin (G10D) receptor, GPR37 / endothelin B-like receptor, chemokine receptor-like receptor, and neuropeptide U receptor; hormone protein receptors: follicle-stimulating hormone, luteinizing hormone-chorionic gonadotropin, thyroid-stimulating hormone, and gonadotropin; (Rhod) opsin receptors; olfactory receptors; prostanoid receptors: prostaglandin, prostacyclin, and thromboxane; nucleotide-like receptors: adenosine and purine receptors; cannabinoid receptors; platelet-activating factor receptors; gonadotropin-releasing hormone receptors; thyrotropin-releasing hormone & secretin receptors: thyrotropin-releasing hormone, growth hormone secretagogues, and growth hormone secretagogue-like; melatonin receptors; viral receptors; soluble sphingolipid (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 receptor, SREB, Mas proto-oncogene, RDC1, ORPH, LGR-like (hormone receptors), GPR, GPR45-like, cysteinyl leukotrienes, Mas-related receptors (MRGs), and GP40-like receptors.

[0032] Class B (secretin receptor family) of GPCRs includes polypeptide hormone receptors (calcitonin, corticotropin-releasing factor, gastric inhibitory polypeptide, glucagon, glucagon-like peptide-1, -2, growth hormone-releasing hormone, parathyroid hormone, PACAP, secretin, vasoactive intestinal peptide, urotensin, EMR1, latrophilin), molecules thought to mediate cell-cell interactions in the plasma membrane (brain-specific angiogenesis inhibitor (BAI)), and a group of Drosophila proteins that regulate the stress response and lifespan (Methuselah-like proteins).

[0033] 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 sensing, putative pheromone receptors, GABA-B receptors (the GABA-B receptor consists of two subunits (B1, B2) and is a dimer protein), and orphan GPRC5 receptors.

[0034] GPCRs are involved in various physiological processes, including vision, olfaction, behavioral and emotional regulation, immune system activity and inflammation regulation, autonomic nervous system transmission, cell density sensing, and many others. Inactive G proteins are known to bind to the receptor in their inactivated state. Once a ligand is recognized, the receptor or its subunits change conformation and thus mechanically activate the G protein, which dissociates from the receptor. Now the receptor can activate another G protein or switch back to its inactivated state. It is believed that receptor molecules exist in a conformational equilibrium between active and inactive biophysical states. Ligand binding to the receptor can shift the equilibrium towards the active receptor state.

[0035] G protein-coupled receptors that can be used in the present invention include, but are not limited to, acetylcholine receptors, dopamine D2 receptor (DRD2), serotonin receptor 6 (HTR6), angiotensin (NTS) receptor, neuropeptide Y (NPY) receptor, somatostatin (SST) receptor, PACAP receptor, which are well-known to those skilled in the art and whose sequences can be obtained through various means, such as querying publicly known databases.

[0036] Those skilled in the art can easily determine the N-terminus, transmembrane regions, intracellular loops, and C-terminus of G protein-coupled receptors, for example, based on their amino acid sequences and similarity to the transmembrane regions of known G protein-coupled receptors. Various bioinformatics methods can be used to determine the position and structure of transmembrane regions in a protein. For example, the BLAST program or the CLUSTALW program can be utilized for alignment and amino acid sequence comparison, which is routinely carried out in the art. Based on alignment with known G protein-coupled receptors containing transmembrane regions, those skilled in the art can predict the position and structure of transmembrane regions of other GPCRs. There are also many programs available for predicting the position and structure of transmembrane regions in a protein. For example, one or a combination of the following programs can be used: TMpred, which predicts transmembrane protein segments; TopPred, which predicts the topology of membrane proteins; PREDATOR, which predicts secondary structures from single and multiple sequences; TMAP, which predicts transmembrane regions of proteins from multiple aligned sequences; and AL0M2, which predicts transmembrane regions from a single sequence. According to the standard nomenclature, the numbering of transmembrane regions and intracellular loops is relative to the N-terminus of the GPCR.

[0037] The term "circularly permuted 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 breaking the protein at an arbitrary site to form a new C-terminus and N-terminus. The fluorescent protein itself has a chromophore center composed of three of its own 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 surrounding barrel-shaped structure of β-sheets. When the fluorescent protein is fused with the target protein, the pulling at the end of the fluorescent protein is difficult to cause changes in the environment around the chromophore and is difficult to change the fluorescence intensity of the fluorescent protein. The chromophore of the circularly permuted fluorescent protein is relatively close to the newly formed end. When it is connected to the target protein, the conformational change of the target protein will pull the end of the circularly permuted fluorescent protein, resulting in changes in the environment around the chromophore, thereby increasing or decreasing the fluorescence intensity of the fluorescent protein. Thus, the conformational change that occurs in the target protein is converted into a change in its fluorescence intensity, and thus it can be detected in real time by optical imaging methods. In a specific embodiment of the present invention, the circularly permuted fluorescent protein is circularly permuted enhanced green fluorescent protein (cpEGFP). The cpEGFP can be the cpEGFP from GCaMP6s or GCaMP6m (Chen, T.-W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295-300 (2013)), or the 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.

[0038] The ratio-type fluorescent probe based on G protein-coupled receptor constructed in the present invention (i.e., ratio-type GRAB fluorescent probe) 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 (such as HEK293T cells), and the expression morphology of the fluorescent protein in the cells can be analyzed. The protein expressed on the cell membrane is a very thin circle at the outermost periphery of the cell. The cell outline can be known by comparing the fluorescence channel with the bright field channel, and then analyzed. Probes that fail to be normally membrane-bound often aggregate inside the cell, showing aggregated signals inside the cell under the microscope. It can also be quantitatively measured by expressing another known cell membrane-localized protein and calculating the co-localization of the fluorescent probe signal with this protein.

[0039] The ratio-type fluorescent probe based on G protein-coupled receptor constructed in the present invention should be able to bind to the specific ligand of the G protein-coupled receptor when in contact therewith. As a result, the fluorescence intensity of the probe has a response amplitude at both 488 nm and 405 nm, and then the concentration change of the specific ligand is calculated through the Ex488 / 405 ratio. 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 fluorescence imaging is performed on the cells expressing the fluorescent probe. Continuous photographs are taken before and after adding the ligand, and the fluorescence response of the fluorescent probe to the specific ligand is detected by analyzing the change in fluorescence intensity recorded before and after adding the ligand.

[0040] The "ligand" or "specific ligand" of the G protein-coupled receptor described herein can be used interchangeably and refers to a molecule that can bind to and activate (or inhibit) the G protein-coupled receptor, including photosensitive compounds, odors, pheromones, hormones, and neurotransmitters. The binding of the G protein-coupled receptor to its ligand is highly specific. The ligand binds only to a specific receptor, and the receptor also binds only to a specific ligand structure. The specificity of the binding of the G protein-coupled receptor to its ligand means that the binding affinity of the G protein-coupled receptor to this ligand is significantly higher than the binding affinity to one or more other molecules. The "significant" 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.

[0041] The terms "linker peptide" or "linker peptide segment" used in this text are interchangeable and refer to a short peptide that links the third intracellular loop of a G-protein coupled receptor and a circularly permuted fluorescent protein. In the present invention, since the circularly permuted fluorescent protein is inserted into the third intracellular loop of the G-protein coupled receptor, the "linker peptide" described herein includes an N-terminal linker peptide located at the N-terminus of the circularly permuted fluorescent protein and a C-terminal linker peptide located at the C-terminus of the circularly permuted fluorescent protein. In the present invention, the role of the linker peptide is to help the fusion protein fold correctly and, at the same time, act as a bridge between the transmission of receptor conformational changes and changes in the brightness of the fluorescent protein. Therefore, the linker peptide used should be a linker peptide that can perform the said role.

[0042] The second aspect of the present invention provides a method for constructing a ratio-type GRAB fluorescent probe.

[0043] Further, the method includes mutating the third amino acid of the N-terminal linker peptide segment of the circularly permuted fluorescent protein linked to the G-protein coupled receptor.

[0044] Further, the circularly permuted fluorescent protein is cpEGFP, and the mutation includes mutating the third amino acid of the N-terminal linker peptide of cpEGFP to leucine.

[0045] Further, when the G-protein coupled receptor is a G-protein coupled receptor that specifically binds to PACAP, the mutation also includes mutating the third amino acid of the N-terminal linker peptide of cpEGFP to isoleucine or methionine.

[0046] Further, when the G-protein coupled receptor is a G-protein coupled receptor that specifically binds to dopamine, the mutation also includes mutating the third amino acid of the N-terminal linker peptide of cpEGFP to alanine or histidine.

[0047] Further, when the G-protein coupled receptor is a G-protein coupled receptor that specifically binds to serotonin, the mutation also includes mutating the third amino acid of the N-terminal linker peptide segment of cpEGFP to methionine.

[0048] Further, the method also includes modifying cpEGFP; the modification includes mutating the 127th site of cpEGFP.

[0049] Further, when the G-protein coupled receptor is a G-protein coupled receptor that specifically binds to NTS or SST, the mutation includes mutating the 127th site of cpEGFP to glutamate.

[0050] Further, when the G-protein coupled receptor is a G-protein coupled receptor that specifically binds to dopamine, the mutation includes mutating the 127th site of cpEGFP to valine.

[0051] The third aspect of the present invention provides a biomaterial.

[0052] Furthermore, the biomaterial includes:

[0053] 1) A polynucleotide that encodes the ratio-type fluorescent probe described in the first aspect of the present invention;

[0054] 2) An expression vector that contains the polynucleotide described in 1);

[0055] 3) A host cell that contains the polynucleotide described in 1) or the expression vector described in 2).

[0056] The term "expression vector" refers to an expression vector that can express a target protein in an appropriate host cell, and is a gene construct containing 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 the coding polynucleotide encoding the ratio-type GRAB fluorescent probe of the present invention or a fragment thereof. The recombinant vector can be transformed or transfected into a host cell.

[0057] The expression vector of the present invention can also be obtained by ligating (inserting) the polynucleotide of the present invention into an appropriate vector. There is no particular limitation on the vector into which the gene of the present invention will be inserted as long as it can replicate in the host. For example, plasmid vectors, phage vectors, viral vectors, etc. can be used. Specifically, commercially available expression vectors can be used, such as the pDisplay vector, which can be purchased from Invitrogen Corporation. 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 above examples.

[0058] The fourth aspect of the present invention provides any one of the following applications, and the application includes:

[0059] 1) The application of the method described in the second aspect of the present invention in constructing a ratio-type GRAB fluorescent probe;

[0060] 2) The application of the ratio-type fluorescent probe described in the first aspect of the present invention in detecting the concentration change of a specific ligand of a G protein-coupled receptor in a sample to be tested or a tissue to be tested;

[0061] 3) The application of the ratio-type GRAB probe constructed by the method described in the second aspect of the present invention in detecting the concentration change of a specific ligand of a G protein-coupled receptor in a sample to be tested or a tissue to be tested.

[0062] Furthermore, the specific ligand is a neurotransmitter.

[0063] Further, the neurotransmitters include NTS, SST, PACAP, NPY, dopamine, and / or serotonin.

[0064] Further, the detection includes qualitative and / or quantitative detection. Description of the Drawings

[0065] Figure 1 Showing the detection of the dynamic changes of neuropeptides and the pH stability of EGFP; wherein, A is a schematic diagram of the detection of neuropeptide release by a neuropeptide probe; B shows that the fluorescence brightness of EGFP changes with the change of pH;

[0066] Figure 2 Showing the chemical structure and spectrum of the Fura-2 calcium dye; wherein, A shows the chemical structure of the Fura-2 calcium dye and the calculation method of the fluorescence ratio; B shows the excitation spectrum of the Fura-2 calcium dye;

[0067] Figure 3 Showing the spectra of wild-type GFP and EGFP fluorescent proteins; wherein, A shows the spectrum of wild-type GFP, the solid line is the excitation spectrum, and the dashed line is the emission spectrum; B shows the spectrum of EGFP, the solid line is the excitation spectrum, and the dashed line is the emission spectrum;

[0068] Figure 4 Schematic diagram of the excitation spectrum model of a new generation of ratio-type neuropeptide probes; Left: Schematic diagram of the excitation spectrum of a non-ratio-type probe, Right: Excitation spectrum of an excitation ratio-type neuropeptide probe; The dashed line represents the probe not bound to the neuropeptide, and the solid line represents the probe bound to the neuropeptide;

[0069] Figure 5 Schematic diagram of the GFP spectrum and the occurrence of ESPT in GFP; wherein, A shows the excitation spectrum (solid line) and emission spectrum (dashed line) of GFP; B is a schematic diagram of the occurrence of ESPT in GFP, and the structures from left to right are Neutral form, State I, and Ionized form;

[0070] Figure 6 Schematic diagram showing the screening process of structure-guided ratio-type probes;

[0071] Figure 7Show the structural prediction and analysis of the NTS1.3 probe cpEGFP; among them, A shows the structure of the NTS1.3 probe cpEGFP predicted by Alphafold; B shows the spatial distribution of 15 sites within 6 Å around the S205 site. These 15 sites can be divided into 4 groups according to their spatial positions, namely the sites in the linker peptide region (dark blue), the sites adjacent to S205 (pink), the sites adjacent to E222 (light blue), and the sites close to the chromophore (green); C shows the occurrence frequency of each group of sites at different spatial distances from the S205 site from near to far.

[0072] Figure 8 Show the screening results of the ratio-type NTS1.3 probe; among them, A shows the spatial distribution of 15 sites within 6 Å around the S205 site in the NTS1.3 probe cpEGFP. These 15 sites can be divided into 4 groups according to their spatial positions, namely the sites in the linker peptide region (dark blue), the sites adjacent to S205 (pink), the sites adjacent to E222 (light blue), and the sites close to the chromophore (green); B shows the screening results of the four groups of amino acid sites; C shows the saturation mutagenesis screening results of the N-terminal linker peptide N3(E) site of the NTS1.3 probe cpEGFP.

[0073] Figure 9 Show the structural prediction and analysis of the NTS probe; among them, A shows the structural alignment of the NTS1.3 and NTS1.3-N3(E)L candidate probes. The gray color is the structure of the NTS1.3 probe cpEGFP predicted by Alphafold, and the green color is the structure of the NTS1.3-N3(E)L candidate probe cpEGFP predicted by Alphafold; B shows the relative spatial positions of the N3(E), S205, and E222 sites in the NTS1.3 probe cpEGFP (top); the relative spatial positions of the N3(E)L, S205, and E222 sites in the NTS1.3-N3(E)L candidate probe cpEGFP (bottom).

[0074] Figure 10 Show that the backbone carbon hydroxyl group of the S205 site is necessary for the occurrence of ESPT of the NTS probe; among them, 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 mutants of the S205 site of the NTS1.4 probe, the ΔF / F0 (488nm) and ΔF / F0 (405nm) of the mutants of the S205 site of the NTS1.4 probe (left), and the ΔR / R0 and maximum brightness of the mutants of the S205 site of the NTS1.4 probe (right).

[0075] Figure 11 Showing the screening results of sites near the chromophore of the NTS1.3 probe mutation; 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 mutating the R168, Q94, and R96 sites of the mutant NTS1.3.

[0076] Figure 12 Showing the screening results of sites related to the folding rate of the NTS1.3 probe mutation; among them, A is a display diagram of the amino acid sites where cpEGFP and sfGFP differ in sequence; B shows the summary of the repeated results of the mutant probes after mutating the S30 and E39 sites of the mutant NTS1.3.

[0077] Figure 13 Showing the development and optimization of the ratio-type SST probe; among them, A shows the structural alignment of the SST1.0 and SST2.1 candidate probes. The gray color is the cpEGFP structure of the SST1.0 probe predicted by Alphafold, showing the relative positions of the N3(E), S205, and E222 sites in space. The light blue color is the cpEGFP structure of the SST2.1 candidate probe predicted by Alphafold, showing the relative positions of the N3(E)L, S205, and E222 sites in space; B shows the comparison of the response amplitudes of SST2.1 and SST1.0; C is a display diagram of the amino acid sites where cpEGFP and sfGFP differ in sequence (left), and the summary of the repeated results of the mutant probes after mutating the S30 and E39 sites of the SST2.1 probe (middle and right).

[0078] Figure 14 Showing the optimization of the ratio-type PACAP probe; among them, A shows the structural alignment of the PACAP1.0 and PACAP1.0-N3(E)L candidate probes. The gray color is the cpEGFP structure of the PACAP1.0 probe predicted by Alphafold, showing the relative positions of the N3(E), S205, and E222 sites in space; B shows the cpEGFP structure of the PACAP1.0-N3(E)L candidate probe predicted by Alphafold, showing the relative positions of the N3(E)L, S205, and E222 sites in space; C shows the screening data of mutating the N3(E) site of the PACAP1.0 probe; D is the fluorescence response curves of the three candidate probes of PACAP-N3(E)I, PACAP-N3(E)L, and PACAP-N3(E)M to the PACAP38 concentration dependence. From left to right are the response amplitudes calculated under 488 nm excitation, the response amplitudes calculated under 405 nm excitation, and the response amplitudes calculated by the ratio.

[0079] Figure 15 Showing 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 represents the cpEGFP structure of the NPY1.0 probe predicted by Alphafold, showing the relative spatial positions of the N3(E), S205, and E222 sites (top). The light yellow color represents the cpEGFP structure of the NPY1.3 candidate probe predicted by Alphafold, showing the relative spatial positions of the N3(E)L, S205, and E222 sites (bottom); B shows the comparison of the response amplitudes of NPY1.0 and NPY1.3.

[0080] Figure 16 Showing the prediction and analysis of the GCG0.4 probe structure; among them, in Figure A, the green color represents the cpEGFP structure of the GCG0.4 probe predicted by Alphafold, showing the relative spatial positions of the N3(L), S205, and E222 sites; B is the fluorescence response curve of the GCG0.4 probe to the GCG concentration dependence. From left to right are the response amplitudes calculated under 488 nm excitation, the response amplitudes calculated under 405 nm excitation, and the response amplitudes calculated by the ratio.

[0081] Figure 17 Showing the development of the ratio-type DA probe; among them, A shows the excitation spectrum of the green dopamine probe DA3m. The dotted line indicates the absence of DA, and the solid line indicates the presence of 1 μM DA; B shows the screening results of mutating the N3(S) site of DA3m; C shows the screening results of mutating the S30 site of DA3m.

[0082] Figure 18 Showing the development of the ratio-type 5-HT probe; among them, 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 mutating the N3(E) site of 5-HT3.0.

[0083] Figure 19 Showing the development of the ratio-type ACh probe; among them, A shows the excitation spectrum of the green acetylcholine probe ACh4l. 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 their response amplitudes; C shows the fluorescence response curve of the ACh4l probe to the ACh concentration dependence. From left to right are the response amplitudes calculated under 488 nm excitation, the response amplitudes calculated under 405 nm excitation, and the response amplitudes calculated by the ratio.

[0084] Figure 20Spectral and pH stability detection of the NTS1.5 probe; among them, A shows the single-photon excitation (left) and emission (right) fluorescence spectra of the NTS1.5 probe. The dashed line indicates the absence of NTS, and the solid line indicates the presence of 1 μM NTS; B shows the single-photon excitation spectra of the NTS1.5 probe at different pH values; C is a statistical chart of the fluorescence brightness of the probe changing with pH.

[0085] Figure 21 It is shown that the ratio brightness of the NTS1.5 probe is not affected by the probe expression level and laser power changes; among them, A is a bar chart of Ex488 / 405, Ex488, and Ex405 of the NTS1.5 probe at different expression levels; B is a statistical chart of Ex488 / 405, Ex488, and Ex405 of the NTS1.5 probe at different expression levels; C is a bar chart of Ex488 / 405, Ex488, and Ex405 of the NTS1.5 probe at different expression levels; D is a statistical chart of Ex488 / 405, Ex488, and Ex405 of the NTS1.5 probe at different expression levels.

[0086] Figure 22 Spectral and pH stability detection of the SST2.2 probe; among them, A shows the single-photon excitation (left) and emission (right) fluorescence spectra of the SST2.2 probe. The dashed 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 spectra of the SST2.2 probe at different pH values; C is a statistical chart of the fluorescence brightness of the probe changing with pH.

[0087] Figure 23 It is shown that the ratio brightness of the SST2.2 probe is not affected by the probe expression level and laser power changes; among them, A is a bar chart of Ex488 / 405, Ex488, and Ex405 of the SST2.2 probe at different expression levels; B is a statistical chart of Ex488 / 405, Ex488, and Ex405 of the SST2.2 probe at different expression levels; C is a bar chart of Ex488 / 405, Ex488, and Ex405 of the SST2.2 probe at different expression levels; D is a statistical chart of Ex488 / 405, Ex488, and Ex405 of the SST2.2 probe at different expression levels. Detailed implementation manners

[0088] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0089] Example 1 General development strategy for ratio-type neuropeptide fluorescent probes

[0090] So far, the applicant's team has successfully developed a variety of neuropeptide probes with higher response amplitude and sensitivity in the early stage. Taking the NTS1.3 and SST2.0 probes as examples, the performance of the NTS1.3 probe has been preliminarily verified at the cell level, neuron level, acute brain slice and in vivo in mice in vitro culture, proving its potential to monitor the dynamic changes of NTS.

[0091] GRAB probes couple the conformational changes of GPCRs with the cyclic rearrangement of cpEGFP, that is, the brightness changes of cpEGFP are used 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 containing neuropeptides is about 5.5. When they 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 pH changes in the environment, and its fluorescence brightness will decrease with the decrease of pH ( Figure 1 B), which greatly affects the signal specificity of the probe to monitor neuropeptide release in vivo.

[0092] Roger Tsien developed the calcium dye Fura-2 as early as 1985 ( Figure 2 A), and its greatest feature is its excitation ratiometric property, that is, it has two excitation peaks at Ex340 nm and Ex380 nm ( Figure 2 B), and the fluorescence values under these two excitation lights at different calcium concentrations are used to calculate a standard curve for calcium quantitative research. However, the biggest drawback of Fura-2 is that both excitation lights are blue light, and long-term irradiation of cells or tissues will cause a certain degree of damage and affect the activity of cells or tissues.

[0093] According to the spectrum of wild-type GFP, wild-type GFP also has two excitation peaks at Ex405 nm and Ex488 nm ( Figure 3 A), and its fluorescent protein states correspond to the neutral state (Neutral form) and the ionized state (Ionized form) respectively. Among them, the Neutral form undergoes proton transfer in the excited state, which is called the excited state proton transfer (ESPT) process, and is transformed into the intermediate state State I, and then emits light and the fluorescence brightness can be detected by the outside world. Unfortunately, EGFP has introduced three mutations on and near the chromophore based on GFP to increase the brightness, but it has also lost the Ex405 nm excitation peak and only retains one Ex488 nm excitation peak ( Figure 3 B).

[0094] 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 affecting ESPT, restore the ESPT network of the probe, and promote the occurrence of ESPT. This excitation ratio-type probe has two excitation peaks at Ex405 nm and Ex488 nm, and after adding neuropeptide, it will cause a negative response (off response) at Ex405 and a positive response (on response) at Ex488 ( Figure 4 ). In this case, calculating the response using the Ex488 / 405 ratio will have a greater response 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 endow it with this excitation ratio-type property on the basis of the GRAB fluorescent probe previously studied by the applicant to increase the response amplitude and stability of the probe.

[0095] For candidate probes that already have a higher response amplitude, the next step is to rationally develop ratio-type neuropeptide probes based on structure guidance. According to the introduction in this article, for wild-type GFP, the fluorescence protein states corresponding to the two excitation peaks Ex405 and Ex488 of its excitation spectrum are the neutral state (Neutral form) and the ionized state (Ionized form) respectively ( Figure 5 A). Among them, in the Neutral form, proton transfer occurs in the excited state, that is, the ESPT process, and it is converted into the intermediate state StateI, and then emits light and the fluorescence brightness can be 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 backbone carbon hydroxyl group at the S205 site, the backbone 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 type, it is necessary to increase the response amplitude of the probe under 405 nm excitation, especially to increase the brightness of Ex405 in the unactivated state. According to the experience of previous studies, when the S205 and E222 sites in GFP are mutated to other amino acids, to a certain extent, it will affect the occurrence of ESPT in the fluorescent protein. For example, the E222Q mutation will completely block the ESPT of GFP because the backbone carbon carboxyl group at the 222 site is missing from 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 at the E222 site and its adjacent positions is directly changed, it is very likely to inhibit the occurrence of ESPT, which is not conducive to the development of ratio-type neuropeptide probes.

[0096] Therefore, this study selected to optimize the amino acid composition near the S205 site, aiming to screen for candidate probes with better ratiometric properties, promoting the occurrence of ESPT and having a larger response amplitude under Ex405. First, cpEGFP and its flanking peptide regions with a length of 5 amino acids at both ends were named and extracted as cpEGFP loop. Secondly, Alphafold3 was used for prediction. Finally, according to the prediction results, amino acid sites within 6 Å near the S205 site were analyzed in the structure and screened for saturation mutations, and their response amplitudes under Ex488, Ex405, and Ex488 / 405 were detected respectively, abbreviated as ΔF / F0(488), ΔF / F0 (405), and ΔR / R0 ( Figure 6 ).

[0097] Example 2 Development of ratiometric NTS probes

[0098] Taking the NTS1.3 probe as an example, it was found that there were a total of 15 amino acid sites within 6 Å of S205 ( Figure 7 A). These amino acid sites were divided into 4 groups according to their spatial positions, namely the linker peptide group (linker), the group of sites adjacent to the S205 site (sitesnear S205), the group of sites adjacent to the E222 site (sites near E222), and the chromophore group (sites near chromophore) ( Figure 7 B). Among them, the amino acids in the group of sites adjacent to the S205 site (sites near S205) and the group of sites adjacent to the E222 site (sites near E222) were relatively close to S205, appearing at a distance of 3.0 Å from S205. Further expanding the distance screening range, it was found that the amino acids in the linker peptide group (N-linker) gradually appeared ( Figure 7 C), and all these amino acids were located near the carboxyl and amine groups on the Cα of S205, that is, on the opposite side of the S205 hydroxyl group. It is worth mentioning that glutamate, the third amino acid of N-linker, numbered N3(E), corresponds to the number 146 in wild-type GFP. In the ESPT network of GFP, it can be found that site 146 is adjacent to the ESPT network in the spatial structure, suggesting that its conformational change is likely to affect the occurrence of ESPT.

[0099] These 15 amino acids are relatively close to the S205 site in space ( Figure 8A), it is very likely to promote the occurrence of the probe ESPT by changing its amino acid composition. Therefore, based on the NTS1.3 probe, saturation mutagenesis screening was carried out on these 15 sites. The probe was excited with light of wavelengths 488 nm and 405 nm respectively, and the response amplitude after adding 1 μM NTS was detected. The results showed that different mutations of the amino acids near the E222 site and the amino acid sites in the linker peptide region all improved the ΔF / F0(405) of the mutants to a certain extent. In contrast, the ΔF / F0(405) of the mutants at the amino acid sites in the linker peptide region was significantly higher than that of the control probe NTS1.3 ( Figure 8 B). These mutants were further repeatedly detected on cells to ensure the credibility of the signal enhancement. It was found that among the five sites in the linker peptide region, only when the glutamate at the N3(E) site was mutated to other amino acids, could the brightness and response amplitude of the candidate probe at 488 and 405 nm be improved to a certain extent. Especially when the glutamate at the N3(E) site was mutated to L (leucine), the brightness and response amplitude of the candidate probe at 488 and 405 nm had the greatest improvement. ( 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 linker peptide is LEEGG, and its C-terminal linker peptide is TGATR; NTS1.The sequence of the 3-probe is METDTLLLWVLLLWVPGSTGDTSLYKKVGTTGRLNSSAPGTPGTPAADPFQRAQAGLEEALLAPGFGNASGNASERVLAAPSSELDVNTDIYSKVLVTAVYLALFVVGTVGNTVTAFTLARKKSLQSLQSTVHYHLGSLALSDLLTLLLAMPVELYNFIWVHHPWAFGDAGCRGYYFLRDACTYATALNVASLSVERYLAICHPFKAKTLMSRSRTKKFISAIWLASALLAVPMLFTMGEQNRSADGQHAGGLVCTPTIHTATVKVVIQVNTFMSFIFPMVVISVLNTIIANKLTVMVRQAAEQLNGAPGEPAPAGPRDTDALDLEEGGNVYIKADKQKNGIKAAFNIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITWGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVGGEGEGDATEGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNTGATRRWRGREPNRVQALRHGVRVLRAVVIAFVVCWLPYHVRRLMFCYISDEQWTPFLYDFYHYFYMVTNALFYVSSTINPILYNLVSANFRHIFLATLACLCPVWRRRRKRPAFSRKADSVSSNHTLSSNATRETLY (SEQ ID NO.7).

[0100] To analyze the reason why the E (glutamic acid) to L (leucine) mutation at the N3 site of the NTS1.3 probe can significantly improve the ESPT of the probe, the sequences of 5 amino acids each of cpEGFP and its left and right linker peptides of the NTS1.3 probe and the NTS1.3-N3(E) candidate probe were extracted and their structural predictions were performed 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 linker peptide region of cpEGFP ( Figure 9A). The N-terminal linker peptide of cpEGFP in the NTS1.3-N3(E) candidate probe is closer to the chromophore cpEGFP side than that of the NTS1.3 probe, with an inward displacement of about 7.4 Å. Specifically, after NTS1.3 carries the N3(E)L mutation, the N-terminal linker peptide undergoes a large displacement towards the cpEGFP side. It is speculated that this may be due to the formation of chemical bonds between N3(E)L and certain amino acids on cpEGFP, resulting in an interaction. By searching within 3 Å of the N3(E)L site, it is found that N3(E)L can form two hydrogen bonds with the S205 site, and this interaction is not found in NTS1.3 ( Figure 9 B). In addition, a hydrogen bond formed between S205 and E222 can also 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 stabilizes the amino acid orientation of S205 by forming a hydrogen bond with the S205 site, making it easier to undergo ESPT. Since the NTS1.3-N3(E)L candidate probe has a large reaction amplitude at 405 and a large ratio reaction amplitude, it is named NTS1.4.

[0101] 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. To further verify the necessity of S205 for the occurrence of ESPT in the probe, a screening experiment was designed to mutate the S205 site based on the NTS1.4 probe, especially in the case of lacking the hydroxyl group of the main chain carbon, and to detect whether the ESPT of the probe would be affected, manifested as the change in the reaction amplitude of the probe under 405 excitation. Taking the mutation of S (serine) to A (alanine) as an example, predicting the cpEGFP of NTS1.4 and the NTS1.4-S205A mutant shows that the lack of the hydroxyl group of the main chain carbon at the 205 site does indeed disrupt the integrity of the ESPT network structure and is very likely to inhibit the occurrence of ESPT ( Figure 10 A). The reaction amplitude of S205A under Ex405 decreases significantly, and the reaction amplitude under Ex488 decreases slightly. In addition, compared with S (serine), amino acids lacking the hydroxyl group of the main chain carbon or replaced with other groups, such as T (threonine), C (cysteine), V (valine), and G (glycine), the reaction amplitudes of these mutant probes under Ex405 are significantly lower than that of NTS1.4 ( Figure 10 B). Therefore, S205 is necessary for the occurrence of ESPT in the probe. When S (serine) is mutated to other amino acids lacking the hydroxyl group of the main chain carbon, it will block the occurrence of ESPT in the probe, while N3(E)L plays an auxiliary role in the ESPT network.

[0102] For neuropeptide probes, L (leucine) at the N3 site indirectly promotes the occurrence of probe ESPT by forming a hydrogen bond with the side chain of S205. Whether there are amino acids in the cpEGFP of the probe that can directly affect the ESPT network remains to be discovered. To screen for these amino acids, based on Roger Tsien's description of the amino acid sites near the GFP chromophore ( Figure 11 A), sites R168, T203, Q94, and R96 that might directly affect the chromophore were selected for mutagenesis screening. Using NTS1.3 and SST2.0 as templates, it was hoped to obtain mutant probes with increased response amplitudes in both the Ex405 and Ex488 / 405 ratio responses. The results showed that whether saturating mutagenesis screening was performed on these four sites based on the NTS1.3 probe or the SST2.0 probe, although there were sites with increased response amplitudes under Ex405, the response amplitudes of these mutants under Ex488 were significantly reduced, resulting in a decreased ratio response ( Figure 11 B).

[0103] In addition to the sites near the chromophore directly affecting the ESPT network of cpEGFP, changes in the overall internal environment of the fluorescent protein (such as the folding of the fluorescent protein) are also likely to affect the ESPT network of cpEGFP. To further screen for sites that can promote the occurrence of probe ESPT, the cpEGFP used in the probe was sequence-aligned with superfolder GFP (sfGFP) developed by the Geoffrey S. Waldo research group, which has a faster folding efficiency, and the sites with amino acid differences in the two sequences were extracted ( Figure 12 A). At position 30, it is R (arginine) in sfGFP and S (serine) in cpEGFP, and at position 39, it is N (asparagine) in sfGFP and E (glutamic acid) in cpEGFP. The possible mechanisms by which mutations at these two sites improve the folding efficiency of the fluorescent protein are described in the article published on sfGFP. Taking S30 as an example, when S (serine) at position 30 is mutated to R (arginine), R (arginine) forms a complete network with structurally adjacent E17 and E32, which may then increase the folding rate of the fluorescent protein.

[0104] Therefore, using the NTS1.4 probe as a template, the amino acids corresponding to its 30th site (the 30th site of wild-type GFP, corresponding to the 127th site of cpEGFP) and 39th site (the 39th site of wild-type GFP, corresponding to the 136th site of cpEGFP) are G (glycine) and E (glutamic acid), respectively. Saturation mutagenesis screening was performed on these two sites respectively. The results showed that when the G (glycine) at the 30th site (the 127th site of cpEGFP) was mutated to E (glutamic acid), the response amplitudes of the mutant probe at 488, 405 nm and the ratio were significantly improved, and it was named NTS1.5 ( Figure 12 B). Thus, based on the N3(E)L mutation introducing the N-terminal linker peptide in the NTS1.3 probe and the G30E mutation in cpEGFP, the NTS1.5 probe with better ex-ratiometric characteristics was obtained.

[0105] Example 3 Extension of the ratiometric fluorescence probe strategy to other neuropeptide probes

[0106] Similarly, this study found that the N3(E)L mutation was also introduced during the optimization of the SST probe. The predicted structures of cpEGFP of the SST1.0 and SST2.1 probes showed that two hydrogen bonds could be formed between N3(E)L and the S205 site in cpEGFP of SST2.1, and this interaction was not found in SST1.0 ( Figure 13 A), and compared with SST1.0, both ΔF / F0 (488) and ΔF / F0 (405) of SST2.1 were significantly improved ( Figure 13 B). Using the SST2.1 probe as a template, the amino acids corresponding to its 30th and 39th sites (the 127th site and 136th site of cpEGFP) are S (serine) and E (glutamic acid), respectively. Saturation mutagenesis screening was performed on these two sites respectively. The results showed that when the S (serine) at the 30th site was mutated to E (glutamic acid), the response amplitudes of the mutant probe at Ex488, Ex405 and the Ex488 / 405 ratio were significantly improved, and it was named SST2.2 ( Figure 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 linker peptide is LEEGG, and its C-terminal linker peptide is TGAAA; SST2 after N3(E)L mutation.The sequence of 1 is METDTLLLWVLLLWVPGSTGDTSLYKKVGTTGEPLFPASTPSWNASSPGAASGGGDNRTLVGPAPSAGARAVLVPVLYLLVCAAGLGGNTLVIYVVLRFAKMKTVTNIYILNLAVADVLYMLGLPFLATQNAASFWPFGPVLCRLVMTLDGVNQFTSVFCLTVMSVDRYLAVVHPLSSARWRRPRVAKLASAAAWVLSLCMSLPLLVFADVQEGGTCNASWPEPVGLWGAVFIIYTAVLGFFAPLLVICLCYLLIVVKVRAAGVRVGCALDLELGGNVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITIGMDELYKGGTGGSMVRPGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATEGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNTGAAKRWRGRQLKVTWMVLVVVLVFAGCWLPFFTVNIVNLAVALPQEPASAGLYFFVVILSYANSCANPVLYGFLSDNFRQSFQKVLCLRKGSGAKDADATEPRPDRIRQQQEATPPAHRAAANGLMQTSKL (SEQ ID NO.8).

[0107] This study found that the N3(E)L mutation in the N-terminal linker peptide of the NTS probe cpEGFP greatly promoted the occurrence of the probe ESPT, and similar results were also obtained for the SST probe. Can this rule be extended to other neuropeptide probes, and thus large-scale development of ratiometric neuropeptide probes be carried out? Based on this hypothesis, the PACAP1.0 probe was selected as the mutation template. First, the structures of cpEGFP and its linker peptide sequence of the PACAP1.0 probe and the PACAP1.0-N3(E)L mutant were predicted, and the structures of the PACAP1.0 probe and the PACAP1.0-N3(E)L mutant were compared ( Figure 14A-B), it was found that the N3(E)L mutation could also form two hydrogen bonds with the side chain at the S205 site, and the hydrogen bond formed between S205 and E222 could be observed. These findings suggest that the PACAP1.0-N3(E)L mutation is very likely to enhance the ESPT of the PACAP1.0 probe. Therefore, experiments were designed to screen for saturation mutations at the N3(E) site of PACAP1.0. The results showed that when E (glutamic acid) was mutated to I (isoleucine), L (leucine), or M (methionine), the ΔF / F0 (488), ΔF / F0 (405), and ΔR / R0 of the mutants were significantly improved ( Figure 14 C). To further confirm which mutation had the best effect, the concentration-dependent curves of these three mutations were measured. The results showed that when E (glutamic acid) was mutated to L (leucine), the response amplitudes of the mutant under Ex405 and Ex488 were improved to the greatest extent, -35% and 1500% respectively, and the ratio response was close to 3000% ( Figure 14D). In summary, the enhancement of the N3(E)L mutation on the PACAP probe response is very similar to its enhancement on the NTS probe. In this study, the amino acid sequence of cpEGFP linked to the G protein-coupled receptor that specifically binds to PACAP is NVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN (SEQ ID NO.3); its N-terminal linker peptide is LEEGG, and its C-terminal linker peptide is TGAAA; PACAP1.The amino acid sequence of the 0 probe is METDTLLLWVLLLWVPGSTGDTSLYKKVGTTGMAGVVHVSLAALLLLPMAPAMHSDCIFKKEQAMCLEKIQRANELMGFNDSSPGCPGMWDNITCWKPAHVGEMVLVSCPELFRIFNPDQVWETETIGESDFGDSNSLDLSDMGVVSRNCTEDGWSEPFPHYFDACGFDEYESETGDQDYYYLSVKALYTVGYSTSLVTLTTAMVILCRFRKLHCTRNFIHMNLFVSFMLRAISVFIKDWILYAEQDSNHCFISTVECKAVMVFFHYCVVSNYFWLFIEGLYLFTLLVETFFPERRYFYWYTIIGWGTPTVCVTVWATLRLYFDDTGCWDMNDSTALWWVIKGPVVGSIMVNFVLFIGIIRILLQKLTSIPAPAGPRDTDALDLEEGGNVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNTGAAARWRASQYKRLARSTLLLIPLFGIHYTVFAFSPENVSKRERLVFELGLGSFQGFVVAVLYCFLNGEVQAEIKRKWRSWKVNRYFAVDFKHRHPSLASSGVNGGTQLSILSKSSSQIRMSGLPADNLAT (SEQ ID NO.9).

[0108] In addition, the same pattern was also observed for the new generation of NPY neuropeptide probes. The structural prediction results of cpEGFP and its linker peptide sequences of the NPY1.0 and NPY1.3 probes showed that the N3(E)L of the NPY1.3 probe could form a hydrogen bond with the side chain of S205 ( Figure 15 A), and its response amplitude under Ex405 was significantly improved compared to NPY1.0, by approximately -30% ( Figure 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 linker peptide is LEEGG, and its C-terminal linker peptide is TGAAA; before the N3(E)L mutation, NPY1.The amino acid sequence of probe 0 is METDTLLLWVLLLWVPGSTGDTSLYKKVGTTGNSTLFSQVENHSVHSNFSEKNAQLLAFENDDCHLPLAMIFTLALAYGAVIILGVSGNLALIIIILKQKEMRNVTNILIVNLSFSDLLVAITCLPFTFVYTLMDHWVFGEAMCKLNPFVQCVSITVSIFSLVLIAVERHQLIINPRGWRPNNRHAYVGIAVIWVLAVASSLPFLIYQVMTDEPFQNVTLDAYKDKYVCFDQFPSDSHRLSYTTLLLVLQYFGPLCFIFICYFKIYIRLKRRNNMMPPSRRGPDAVAAPPGGTERRPNGLGPERSAGPGGAEAEPLPTQLNGAPGEPAPAGPRDTDALDLEEGGNVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATEGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNTGAAARWRGRQNETKRINIMLLSIVVAFAVCWLPLTIFNTVFDWNHQIIATCNHNLLFLLCHLTAMMSTCVNPIFYGFLNKNFQRDLQFFFNFCDFRSRDDDYETIAMSTMHTDVSKTSLKQASPVAFKKINNNDDNEKI (SEQ ID NO.10).

[0109] Similarly, the amino acid corresponding to the third position of the N-terminal linker peptide of the GCG0.4 probe cpEGFP is L (leucine). This sequence may endow the GCG probe with the property of excitation ratio type and the possibility of ESPT occurring. Based on this hypothesis, structure prediction was performed on the cpEGFP of the GCG0.4 probe and its linker peptide sequence, 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 ( Figure 16A). Further detection of the concentration-dependent curve of the GCG0.4 probe revealed that as the concentration of exogenously added GCG increased, the response amplitude of the GCG0.4 probe gradually increased under Ex488 excitation, while the response amplitude gradually decreased under Ex405 excitation. The maximum response amplitudes of the GCG0.4 probe under Ex488 and Ex405 excitations were 600% and -45% respectively, and the maximum ratio response was approximately 1000% ( Figure 16 B).

[0110] 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), it can significantly increase the ΔF / F0 (405) and ΔR / R0 of the probe. This rule has been verified in multiple GRAB neuropeptide probes (including NTS, PACAP, SST, NPY, and GCG probes).

[0111] Example 4 Extension of the ratiometric fluorescence probe strategy to monoamine neurotransmitter probes

[0112] To systematically improve the ESPT of GRAB probes, in addition to neuropeptide probes, this study further extended the ratiometric probe development strategy to monoamine probes.

[0113] Dopamine is an important monoamine neurotransmitter that is widely present in the central nervous system (CNS) and the peripheral nervous system. The synthesis of dopamine starts from the amino acid tyrosine, which is converted into L-DOPA under the action of tyrosine hydroxylase, and then into dopamine under the action of dopa decarboxylase. The metabolism of dopamine mainly occurs through monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT), generating metabolites such as 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA).

[0114] As a neurotransmitter, dopamine transmits signals between neurons and affects a variety of physiological and psychological functions. Dopamine neurons are mainly concentrated in several regions of the midbrain, including the Substantia Nigra pars compacta (SNc), the Ventral Tegmental Area (VTA), and the Arcuate Nucleus. Dopamine neurons in these regions project to multiple regions of the whole brain through different pathways: (1) Nigrostriatal Pathway: Projects from the substantia nigra to the striatum and is mainly involved in motor control. The 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 the reward mechanism, 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 functions, 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 to regulate the hormone secretion of the hypothalamic-pituitary axis. Dopamine in this pathway inhibits the secretion of prolactin, and abnormal function may lead to hyperprolactinemia.

[0115] Therefore, the functions of dopamine in the central nervous system are complex and diverse, involving motor control, emotional regulation, reward mechanism, cognitive function, and endocrine regulation. Its distribution and functions in different neural pathways make it a key in the research of various neuropsychiatric diseases and drug addiction. Understanding these mechanisms of action of dopamine is crucial for developing effective treatment methods. Therefore, the detection of dopamine also requires more sensitive tools with high spatio-temporal resolution.

[0116] This study is based on the latest generation of green dopamine probe DA3m. Its excitation spectrum shows that DA3m has a very small response amplitude to DA under 400 nm excitation ( Figure 17 A), almost zero. Therefore, this study designed experiments to mutate the N3(S) and S30 sites (the 127th site of cpEGFP) of DA3m in order to obtain a better excitation ratio-type dopamine probe. The screening results show that when N3(S) of DA3m is mutated to L (leucine), A (alanine), and H (histidine), the ΔF / F0 (405) of these three mutant probes is significantly improved compared to DA3m, and the N3(S)H mutation has the greatest improvement in ΔF / F0 (405) and ΔR / R0 (Figure 18 B). Additionally, 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 ( Figure 17C). In the present invention, the amino acid sequence of cpEGFP linked to the G protein-coupled receptor that specifically binds to dopamine is YVYIKADKQKNGIKANFGIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGLMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKNDGFYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN (SEQ ID NO.5); Its N-terminal linker peptide is LNSLI, and its C-terminal linker peptide is NHDQL; The amino acid sequence of the dopamine probe DA3m is METDTLLLWVLLLWVPGSTGDTSLYKKVGTTGRTLNTSAMDGTGLVVERDFSVRILTACFLSLLILSTLLGNTLVCAAVIRFRHLRSKVTNFFVISLAVSDLLVAVLVMPWKAVAEIAGFWPFGSFCNIWVAFDIMCSTASILNLCVISVDRYWAISSPARYERKMTPKAAFILISVAWTLSVLISFIPVQLSWHKAKPTSPSDGNATSLAETIDNCDSSLSRTYAISSSVISFYIPVAIMIVLYTRIYRIAQKQLNSLIYVYIKADKQKNGIKANFGIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGLMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKNDGFYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNNHDQLKRETKVLKTLSVIMGVFVCCWLPFFILNCILPFCGSGETQPFCIDSNTFDVFVWFGWANSSLNPIIYAFNADFRKAFSTLLGCYRLCPATNNAIETVSINNNGAAMFSSHHEPRGSISKECNLVYLIPHAVGSSEDLKKEEAAGIARPLEKLSPALSVILDYDTDVSLEKIQPITQNGQHPT (SEQ ID NO.11).

[0117] Serotonin (5-HT) is another important monoamine neurotransmitter, which is widely present in the central nervous system (CNS) and the peripheral nervous system. Serotonin is converted from tryptophan to 5-hydroxytryptophan (5-HTP) by tryptophan hydroxylase, and then generated under the action of aromatic amino acid decarboxylase. The metabolism of serotonin is mainly carried out by monoamine oxidase (MAO) to generate 5-hydroxyindoleacetic acid (5-HIAA).

[0118] Serotonin plays a key role in regulating mood, appetite, sleep, memory, and learning. Serotonergic neurons are mainly located in several nuclei of the midbrain and brainstem, especially the Raphe Nuclei. These neurons are distributed in the raphe nucleus group, especially the dorsal raphe nucleus and the magnocellular nucleus, and they project to various regions of the brain, including the cortex, limbic system, and spinal cord. The specific functions of serotonin include (1) Mood regulation: It plays a key role in regulating mood and emotions. Clinical findings show that low levels of serotonin are associated with mood disorders such as depression and anxiety. Selective serotonin reuptake inhibitors (SSRIs) are commonly used antidepressant drugs that relieve symptoms by increasing the level of serotonin in the synaptic cleft. (2) Sleep regulation: It is involved 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 migraines. (5) Cognition and memory: The activity of serotonin in the prefrontal cortex and hippocampus is related to 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 large release of serotonin and blocks its reuptake, resulting in a significant increase in the level of serotonin in the synaptic cleft. This effect leads to intense feelings of pleasure and emotional resonance, but long-term use can deplete the serotonin reserves in cells, resulting in mood swings and cognitive impairment. Hallucinogens act by activating serotonin receptors (such as the 5-HT2A receptor), which in turn causes profound changes in perception, mood, and cognition, sometimes leading to psychotic-like symptoms.

[0119] To develop a sensitive 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 was very small under 400 nm excitation ( Figure 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 / F0 (405) of these two mutant probes was significantly improved compared with DA3m, and the N3(E)M mutation had the greatest improvement in ΔF / F0 (405) ( Figure 18B). In the present invention, the amino acid sequence of cpEGFP linked to the G protein-coupled receptor that specifically binds to dopamine is NVYIKADKQKNGIKAIFHIRHNIEDGGVQLAIHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN (SEQ ID NO.6); its N-terminal linker peptide is LEEGG, and its C-terminal linker peptide is TGAAA; the serotonin probe 5-HT3.The amino acid sequence of 0 is METDTLLLWVLLLWVPGSTGDTSLYKKVGTTGDKLDANVSSNEGFRSVEKVVLLTFLAVVILMAILGNLLVMVAVCRDRQLRKIKTNYFIVSLAFADLLVSVLVMPFGAIELVQDIWAYGEMFCLVRTSLDVYLCTASIFHLCCISLDRYYAICCQPLVYRNKMTPLRIALMLGGCWVLPMFISFLPIMQGWNNIGIVDVIEKRKFSHNSNSTWCVFMVNKPYAITCSVVAFYIPFLLMVLAYYRIYVVAKRGPDAVAAPPGGTERRPNGLGPERSAGPGGAEAEPLPTQLNGAPGEPAPAGPRDTDALDLEEGGNVYIKADKQKNGIKAIFHIRHNIEDGGVQLAIHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNTGAAARFRWRTETKAAKVLCVIMGCFCFCWAPFFVTNIVDPFIDYTVPEQVWTAFLWLGYINSGLNPFLYAFLNKSFRRAFLIILRCNYKVEKKPPVRQIPRVAATALSGRELNVNIYRHTNEPVIEKASDNEPGIEMQVENLELPVNPSSVVSERISSV (SEQ ID NO.12).

[0120] Acetylcholine (ACh) is widely present in the central nervous system and the 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 generate choline and acetic acid. Acetylcholine transmits signals between neurons and affects muscle contraction, memory, attention, and autonomic nerve function. Cholinergic neurons are mainly distributed in the basal forebrain (such as the medial septal nucleus and the basal nucleus of Meynert) and the brainstem (such as the locus coeruleus in the pons and the hypothalamus) in the central nervous system, and are mainly distributed in the autonomic nervous system (including the 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 and activates muscle fiber contraction. When the acetylcholine receptor malfunctions, it can lead to myasthenia gravis. (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 arousal: Acetylcholine regulates attention, arousal state, and cognitive function. Attention deficit hyperactivity disorder (ADHD) is related to abnormal acetylcholine function. (4) Autonomic nerve function: Acetylcholine regulates heart rate, digestion, and urinary function in the autonomic nervous system. Clinical correlation: Autonomic nerve dysfunction such as hypotension and arrhythmia is related to acetylcholine imbalance. Therefore, drugs that regulate acetylcholine levels can be used to treat corresponding diseases. For example, acetylcholinesterase (AChE) inhibitors can increase the acetylcholine level 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 cause muscle relaxation by blocking acetylcholine release and are used to treat muscle spasms and cosmetic plastic surgery.

[0121] To better discover and understand the functions and roles of acetylcholine, it is also necessary to develop sensitive excitation ratio-type ACh probes. This study found that the latest generation of green acetylcholine probe ACh4l is a good excitation ratio-type probe, and its excitation spectrum shows that the response amplitude of ACh4l to ACh under 400 nm excitation is relatively large ( Figure 19 A). To understand the reason why ACh4l has ratio-type characteristics, this study reviewed the optimization process of the ACh4l probe and found that ACh4l introduced 4 mutations on the basis of the previous generation of ACh3.0 probe version, namely N3(E)L, N4(G)N, N6.33R, and Y3F ( Figure 19B). The concentration-dependent curve of the ACh4l probe showed that as the ACh concentration increased, the ΔF / F0 (488) of the ACh4l probe gradually increased, the ΔF / F0(405) gradually decreased, and the ratio ΔR / R0 also gradually increased. Moreover, the response of the probe ΔR / R0 to 10 mM ACh was close to 1200%, significantly higher than 900% of its ΔF / F0 (488) ( Figure 19 C).

[0122] Example 5 Performance Characterization of the NTS1.5 Probe in HEK293T Cells

[0123] Ratiometric neuropeptide probes exhibit opposite-direction responses induced by binding neuropeptides under 488 and 405 nm excitation lights. Therefore, calculating with the Ex488 / 405 ratio will significantly enhance the response amplitude of the probe, providing the possibility for sensitive detection of the dynamic changes of neuropeptides. However, the advantages of ratiometric neuropeptide probes are not only higher response amplitudes but also can be used to correct the fluorescence value fluctuations caused by the influence of certain environmental factors (such as pH and excitation power in the environment). Because the probe itself has only one fluorescent protein containing one chromophore, when external factors (pH, excitation power, etc.) change, the fluorescence of the probe will change accordingly. But since the fluorescence of the ratiometric probe under 488 nm and 405 nm excitation both 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 fluorescence intensity changes caused by the changes in pH or excitation power, thereby improving the stability of the probe and ensuring the specificity of the probe detection signal.

[0124] First, the spectral properties of the NTS1.5 probe were characterized, and the results showed that the NTS1.5 probe had two excitation peaks at 400 nm and 505 nm respectively, and their corresponding emission peak was 525 nm ( Figure 20 A). To verify the pH stability of the NTS1.5 probe, the pH in the cell environment and inside the cell was changed, and the results showed that as the pH decreased, the fluorescence brightness of the probe under Ex488 and Ex405 also gradually decreased ( Figure 20 B). In contrast, the Ex488 / 405 ratio was less affected by different pH values. For example, when pH = 5, Ex488 was only 30% of the maximum brightness, but Ex488 / 405 was 80% of the maximum brightness, and its fluorescence brightness was significantly higher than Ex488 ( Figure 20 C). These results suggest that the NTS1.5 probe may be less affected by pH changes when detecting the dynamic changes of neuropeptide release in vivo.

[0125] In addition, the brightness of fluorescent proteins is also greatly affected by the probe expression level and laser power. For example, the higher the probe expression level, 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. Since the effects of probe expression level and laser power on Ex488 and Ex405 of cpEGFP are in the same direction, it is speculated that Ex488 / 405 may remain stable. To detect whether the fluorescence brightness of the NTS1.5 probe is affected by the probe expression level, NTS1.5 probes of 50 ng, 100 ng, 200 ng, and 300 ng were respectively expressed in HEK293T cells, and Ex488, Ex405, and Ex488 / 405 of the probe were detected at different expression levels. The experimental results showed that as the probe expression level increased, the fluorescence brightness under Ex488 and Ex405 also gradually increased, but the fluorescence brightness ratio of Ex488 / 405 remained stable ( Figure 21 A-B).

[0126] 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 excitation powers of 10%, 30%, 50%, 70%, and 90%, 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, the fluorescence brightness under 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 ( Figure 21 C-D).

[0127] Example 6 Performance Characterization of SST2.2 Probe in HEK293T Cells

[0128] Similarly, the spectral properties of the SST2.2 probe were characterized. The SST2.2 probe has two excitation peaks, at 400 nm and 505 nm respectively, and their corresponding emission peak is 525 nm ( Figure 22 A). To verify the pH stability of the SST2.2 probe, the pH in the cell environment and inside the cell was changed, and the results showed that as the pH decreased, the fluorescence brightness of the probe under 488 and 405 also gradually decreased ( Figure 22B). In contrast, the Ex488 / 405 ratio is less affected by different pH values. Even when pH = 3 and Ex488 is only 10% of the maximum brightness, Ex488 / 405 still remains 60% of the maximum brightness, and its fluorescence brightness is significantly higher than that of Ex488 ( Figure 22 C). These results suggest that the SST2.2 probe has good pH stability.

[0129] To detect whether the fluorescence brightness of the SST2.2 probe is affected by the probe expression level, 50 ng, 100 ng, 200 ng, and 300 ng of the SST2.2 probe were respectively expressed in HEK293T cells, and Ex488, Ex405, and Ex488 / 405 of the probe were detected at different expression levels. The experimental results showed that as the probe expression level increased, Ex488 and Ex405 also gradually increased, but Ex488 / 405 remained stable ( Figure 23 A - B). 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 excitation powers of 10%, 30%, 50%, 70%, and 90%, 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 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 ( Figure 23 C - D).

[0130] The above is an exemplary description of the present invention. It should be noted that without departing from the core of the present invention, any simple deformation, modification, or equivalent replacement that can be made by those skilled in the art without creative labor 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; 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; 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 the G protein-coupled receptor that specifically binds to serotonin, its amino acid sequence is the sequence shown in SEQ ID NO.6; 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; 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 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.

2. 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 connecting peptide to leucine; 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 dopamine, the sequence of the N-terminal connecting peptide before the third amino acid mutation is LNSLI.

3. The method according to claim 2, 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.

4. The method according to claim 2, characterized in that: When the G protein coupled receptor is a G protein coupled receptor that specifically binds to NTS or SST, the method further comprises mutating the 127 position of cpEGFP, wherein the mutation comprises mutating the 127 position of cpEGFP to glutamic acid; When the G protein-coupled receptor is a G protein-coupled receptor that specifically binds to dopamine, the method further comprises mutating position 127 of cpEGFP to valine.

5. A biomaterial, characterized in that: The biological material comprises: 1) A polynucleotide, characterized in that the polynucleotide encodes the ratiometric fluorescent probe according to claim 1; 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).

6. Any of the following applications, characterized in that: The applications include: 1) Use of the method according to any one of claims 2 to 4 in constructing a ratiometric GRAB fluorescent probe; 2) Use of the ratiometric fluorescent probe according to claim 1 in detecting changes in the concentration of specific ligands of G protein-coupled receptors 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 2 to 4 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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