Screening method, system and equipment for key mutation sites for improving excitation ratio type property of GRAB probe

Abstract

The invention discloses a screening method, system and equipment for key mutation sites for improving the excitation ratio type property of a GRAB probe. Probe structure simulation is carried out in combination with Alphafold, and potential amino acid sites capable of improving the excitation ratio type property are excavated through structure and amino acid network analysis. In combination with a high-throughput screening method, efficient screening of candidate probes is achieved, and finally it is found that the N3 site can be used for improving the excitation ratio type property of the probes. The invention provides a design thought for preparing the excitation ratio type GRAB probe.

Description

Technical Field

[0001] The present invention belongs to the field of bioinformatics, and in particular, relates to a screening method, system and device for improving the ratiometric properties of GRAB probe excitation key amino acid sites. Background Art

[0002] The GRAB probe couples the conformational changes of GPCRs with the cyclic rearrangement of cpEGFP, that is, it uses the brightness changes of cpEGFP to report the dynamic changes of GPCR ligands. However, since these neurotransmitter probes based on fluorescence intensity changes are affected by many factors during use, such as the number of chromophores, fluorescence excitation intensity, and photobleaching, the application scenarios of this 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 activity, and cannot be further used to sensitively and accurately measure the concentration of neurotransmitter release.

[0003] In addition, in vivo, the pH value of the cytoplasm is 7-7.5, and the pH value of the vesicles encapsulating neuropeptides is about 5.5. When they fuse with the cell membrane to release neuropeptides, the pH value near the release site will fluctuate dramatically. cpEGFP is very sensitive to changes in pH in the environment, and its fluorescence brightness will decrease with the decrease of pH, which greatly affects the signal specificity of the probe in monitoring neuropeptide release in vivo.

[0004] Therefore, it is very important to develop excitation ratio probes by finding key amino acid sites that can improve the excitation ratio properties of GRAB probes. This excitation ratio probe has two excitation peaks, Ex405nm and Ex488nm, and the ligands trigger negative and positive reactions under these two excitation peaks, respectively. Therefore, the reaction calculated by the Ex488 / 405 ratio will have a larger reaction amplitude, and the ratio can also be used to correct signal fluctuations caused by pH, expression level, laser power, etc. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a screening method, system and device for key amino acid sites that improve the excitation ratio properties of GRAB probes.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides a method for screening key mutation sites for improving the ratiometric properties of GRAB probe excitation.

[0007] Further, the method comprises: Structure prediction: Alphafold was used to predict the structure of the loop region of the cyclically rearranged fluorescent protein in the GRAB probe; the loop region of the cyclically rearranged fluorescent protein is the cyclically rearranged fluorescent protein in the GRAB probe and its connecting peptide region with a length of 5 amino acids at both ends; Analyze amino acid sites: Based on the structural prediction results, analyze the amino acid sites within 6Å around the cyclically rearranged fluorescent protein S205 site in the structure; Saturation mutagenesis: Saturation mutagenesis of amino acid sites within 6Å of the cyclically rearranged fluorescent protein S205 site; Screening of key mutation sites: The amino acid sites that can significantly improve the response amplitude of the fluorescent probe under Ex405 and Ex488 / 405 after mutation are selected as the key mutation sites for improving the excitation ratio properties of the GRAB probe.

[0008] Furthermore, the step of screening key mutation sites includes screening using a high-throughput method.

[0009] Preferably, the step of screening using a high-throughput method comprises saturating the amino acid sites within 6Å near the S205 site of the cyclically rearranged fluorescent protein, and then imaging and screening using a high-content Opera Phenix instrument.

[0010] Furthermore, the sequence of the connecting peptide includes the N-terminal connecting peptide of the cyclically rearranged fluorescent protein and the C-terminal connecting peptide of the cyclically rearranged fluorescent protein.

[0011] Furthermore, the cyclically rearranged fluorescent protein is cpEGFP.

[0012] Furthermore, the N-terminal connecting peptide sequence of the cpEGFP is LEEGG.

[0013] Furthermore, the C-terminal connecting peptide sequence of the cpEGFP is TGATR.

[0014] Furthermore, the sequence of the cpEGFP is shown as SEQ ID NO.1.

[0015] Furthermore, the sequence of the cpEGFP loop region is shown as SEQ ID NO.2.

[0016] Further, the GRAB probe is an NTS probe.

[0017] Furthermore, the sequence of the NTS probe is shown in SEQ ID NO.3.

[0018] Furthermore, there are 16 amino acid sites within 6 Å near the cpEGFP S205 site, including the connecting peptide group N1 (L), N2 (E), N3 (E), N4 (G), and C1 (T), the S205 site group V203, Q204, H206, and L207, the E222 site group L220, L221, E222, and F223, and the chromophore group V52 and Y57.

[0019] Preferably, the key amino acid site for improving the excitation ratio property of the GRAB probe is the connecting peptide group N3(E), and the N3(E) is the third amino acid of the connecting peptide at the N-terminus of cpEGFP.

[0020] The second aspect of the present invention provides a screening system for key mutation sites that improves the ratiometric properties of GRAB probe excitation.

[0021] Further, the system comprises: Structure prediction module: using Alphafold to predict the structure of the loop region of the cyclically rearranged fluorescent protein in the GRAB probe; the loop region of the cyclically rearranged fluorescent protein is the cyclically rearranged fluorescent protein in the GRAB probe and the connecting peptide region with a length of 5 amino acids at both ends; Amino acid site analysis module: Based on the structural prediction results, the amino acid sites within 6Å around the cyclically rearranged fluorescent protein S205 site were analyzed in the structure; Saturation mutagenesis module: saturation mutation of amino acid sites within 6Å around the cyclically rearranged fluorescent protein S205 site; Screening module for key mutation sites: The amino acid sites that significantly improve the response amplitude of the fluorescent probe under Ex405 and Ex488 / 405 are selected as key mutation sites for improving the excitation ratio properties of the GRAB probe.

[0022] Furthermore, the module for screening key mutation sites includes a high-throughput screening unit.

[0023] Preferably, the high-throughput screening unit comprises saturation mutagenesis of amino acid sites within 6Å of the S205 site of the cyclically rearranged fluorescent protein, followed by imaging and screening using a high-content Opera Phenix instrument.

[0024] Furthermore, the sequence of the connecting peptide includes the N-terminal connecting peptide of the cyclically rearranged fluorescent protein and the C-terminal connecting peptide of the cyclically rearranged fluorescent protein.

[0025] Furthermore, the cyclically rearranged fluorescent protein is cpEGFP.

[0026] Furthermore, the N-terminal connecting peptide sequence of the cpEGFP is LEEGG.

[0027] Furthermore, the C-terminal connecting peptide sequence of the cpEGFP is TGATR.

[0028] Furthermore, the sequence of the cpEGFP is shown as SEQ ID NO.1.

[0029] Furthermore, the sequence of the cpEGFP loop region is shown as SEQ ID NO.2.

[0030] Further, the GRAB probe is an NTS probe.

[0031] Further, the sequence of the NTS probe is shown in SEQ ID NO.3. Furthermore, there are 15 amino acid sites within 6 Å near the cpEGFP S205 site, including the connecting peptide group N1 (L), N2 (E), N3 (E), N4 (G), and C1 (T), the S205 site group V203, Q204, H206, and L207, the E222 site group L220, L221, E222, and F223, and the chromophore group V52 and Y57.

[0032] Preferably, the key amino acid site for improving the excitation ratio property of the GRAB probe is the connecting peptide group N3(E), and the N3(E) is the third amino acid of the connecting peptide at the N-terminus of cpEGFP.

[0033] A third aspect of the present invention provides a computer device and a computer-readable storage medium.

[0034] Further, the device comprises: A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the screening method described in the first aspect of the present invention is implemented; The computer-readable storage medium stores a computer program, and when the computer program is executed by the processor, the screening method described in the first aspect of the present invention is implemented.

[0035] The fourth aspect of the present invention provides the use of the screening method described in the first aspect of the present invention in screening key mutation sites that improve the excitation ratio properties of GRAB probes.

[0036] Furthermore, the GRAB probe is an NTS probe.

[0037] Advantages and beneficial effects of the present invention: The present invention combines the newly developed Alphafold to simulate the probe structure, and through structural and amino acid network analysis, it digs out the potential amino acid sites that may enhance the excitation ratio properties. Combined with high-throughput screening methods, efficient screening of candidate probes is achieved, providing ideas for the preparation of ratio GRAB probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A schematic diagram of the process of screening key mutation sites for improving the excitation ratio properties of GRAB probes provided by the present invention; Figure 2 A schematic diagram of the structure of a screening system for key mutation sites for improving the ratiometric properties of GRAB probe excitation provided by the present invention; Figure 3 A schematic diagram of the structure of a computer device provided by the present invention; Figure 4 Shows the spectra of wild-type GFP and EGFP fluorescent proteins; A is the spectrum of wild-type GFP, B is the spectrum of EGFP; the solid line is the excitation spectrum, and the dotted line is the emission spectrum; Figure 5 Schematic diagram of the excitation spectrum model of the new generation ratiometric neuropeptide probe; left: schematic diagram of the excitation spectrum of the non-ratiometric probe, right: excitation spectrum of the ratiometric neuropeptide probe; the dotted line represents the probe without neuropeptide binding, and the solid line represents the probe with neuropeptide binding; Figure 6 GFP spectrum and schematic diagram of GFP undergoing ESPT; A is the excitation spectrum (solid line) and emission spectrum (dashed line) of GFP; B is the schematic diagram of GFP undergoing ESPT, and the structures from left to right are Neutral form, State I and Ionized form respectively; Figure 7 Schematic diagram of the structure-guided ratiometric probe screening process; Figure 8 Figure 1 shows the predicted structure analysis of the NTS1.3 probe cpEGFP; Figure A shows the structure of the NTS1.3 probe cpEGFP predicted by Alphafold; Figure B shows the spatial distribution of 15 sites within 6Å around the S205 site. These 15 sites can be divided into four groups according to their spatial positions, namely the site in the connecting peptide region (dark blue), the site next to S205 (pink), the site next to E222 (bright blue), and the site close to the chromophore (green); Figure C shows the frequency of occurrence of each group of sites in the spatial distance from near to far, with the S205 site as the center; Fig. 9The results of ratiometric NTS1.3 probe screening are shown; Figure A shows the spatial distribution of 15 sites within 6Å around the S205 site in the NTS1.3 probe cpEGFP. These 15 sites can be divided into four groups according to their spatial positions, namely the site in the connecting peptide region (dark blue), the site next to S205 (pink), the site next to E222 (bright blue), and the site close to the chromophore (green); Figure B shows the screening results of four groups of amino acid sites; Figure C shows the saturation mutation screening results of the N3 (E) site of the N-terminal connecting peptide of the NTS1.3 probe cpEGFP; Fig.10 Displays the prediction and analysis of the NTS probe structure; Figure A shows the structural comparison of the NTS1.3 and NTS1.3-N3(E)L candidate probes, with the gray color representing the NTS1.3 probe cpEGFP structure predicted by Alphafold and the green color representing the NTS1.3-N3(E)L candidate probe cpEGFP structure predicted by Alphafold; Figure B shows the relative spatial positions of the N3(E), S205 and E222 sites in the NTS1.3 probe cpEGFP (top) and the N3(E)L, S205 and E222 sites in the NTS1.3-N3(E)L candidate probe cpEGFP (bottom); Fig.11 The spectrum and pH stability test of the probe after N3(E)L mutation are shown; A is the single-photon excitation (left) and emission (right) fluorescence spectra of the probe after N3(E)L mutation, the dotted line indicates the absence of NTS, and the solid line indicates the presence of 1μM NTS; B is the single-photon excitation spectrum of the probe after N3(E)L mutation at different pH values; C is a statistical graph of the probe fluorescence brightness changing with pH; Fig.12 It shows that the ratio brightness of the probe after N3(E)L mutation is not affected by the changes in probe expression level and laser power; among them, A is a bar graph of Ex488 / 405, Ex488 and Ex405 at different expression levels of the probe after N3(E)L mutation; B is a statistical graph of Ex488 / 405, Ex488 and Ex405 at different expression levels of the probe after N3(E)L mutation; C is a bar graph of Ex488 / 405, Ex488 and Ex405 at different expression levels of the probe after N3(E)L mutation; D is a statistical graph of Ex488 / 405, Ex488 and Ex405 at different expression levels of the probe after N3(E)L mutation. DETAILED DESCRIPTION

[0039] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0040] In some of the processes described in the specification and claims of the present invention and the above-mentioned figures, multiple operations that appear in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this article or executed in parallel. The serial numbers of the operations, such as 101, 102, etc., are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel. It should be noted that the descriptions of "first", "second", etc. in this article are used to distinguish different messages, devices, modules, etc., do not represent the order of precedence, and do not limit the "first" and "second" to be different types.

[0041] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0042] Figure 1 A schematic flow chart of a method for screening key mutation sites for improving the ratiometric properties of GRAB probe excitation provided by the present invention; specifically, the method comprises: 101: structure prediction, using Alphafold to perform structure prediction on the loop region of the cyclically rearranged fluorescent protein in the GRAB probe; the loop region of the cyclically rearranged fluorescent protein is the cyclically rearranged fluorescent protein in the GRAB probe and its connecting peptide regions with a length of 5 amino acids at both ends; The principle of the GRAB probe (GPCR Activation Based Sensor) of the present invention is: inserting a cyclically rearranged fluorescent protein into the third intracellular loop of the GPCR, the binding of the ligand to the GPCR will induce a conformational change of the GPCR, thereby causing a conformational change of the cyclically rearranged fluorescent protein, causing a change in the intensity of the fluorescent signal, thereby converting the conformational change of the GPCR induced by the ligand into a change in the optical signal.

[0043] In the present invention, the GRAB probe refers to a fusion protein constructed by inserting a cyclic rearranged fluorescent protein at an appropriate position in the third intracellular loop between the fifth and sixth transmembrane regions of a GPCR, and connecting the cyclic rearranged fluorescent protein to the third intracellular loop with a connecting peptide at the N-terminus and C-terminus of the cyclic rearranged fluorescent protein, which can be used as a probe for detecting the ligand of the GPCR. In a specific embodiment of the present invention, the GRAB probe is an NTS probe, that is, a probe for detecting NTS, and its sequence is (SEQ ID NO.3).

[0044] In the present invention, the circular permutated fluorescent protein (cpFP) is well known to those skilled in the art, and refers to a fluorescent protein formed by connecting the molecular ends of the original fluorescent protein and then disconnecting the protein from any site to form a new carbon end and nitrogen end. The fluorescent protein itself has its own chromophore center composed of three amino acids, and the chemical reaction that occurs determines the spectral properties and fluorescence intensity of the fluorescent protein. Most of the chromophores of the fluorescent protein are located inside the protein and are protected by the barrel structure of the surrounding β folding. When the fluorescent protein is fused with the target protein, the involvement of the end of the fluorescent protein is difficult to cause environmental changes around the chromophore, and it is difficult to change the fluorescence intensity of the fluorescent protein. The chromophore of the circularly rearranged 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 involve the end of the circularly rearranged fluorescent protein, resulting in changes in the environment around the chromophore, thereby increasing or decreasing the fluorescence intensity of the fluorescent protein, thereby converting the conformational change of the target protein into a change in its fluorescence intensity, so that real-time detection can be performed by an optical imaging method. In a specific embodiment of the present invention, the circularly rearranged fluorescent protein refers to cpEGFP, and its sequence is NVYIKADKQKNGIKAAFNIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITWGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVGGEGEGDATEGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN (SEQ ID NO.1).

[0045] In one embodiment of the present invention, the applicant found that the wild-type GFP has an excitation ratio property based on the spectrum of the wild-type GFP, that is, it has two excitation peaks of Ex405 nm and Ex488 nm ( Figure 4A), the fluorescent protein states correspond to the neutral state (Neutral form) and the ionized state (Ionized form). Among them, the Neutral form undergoes proton transfer in the excited state, which is called the excited state proton transfer (ESPT) process, and transforms into the intermediate state State I, which then emits light and the fluorescence brightness is detected by the outside world. Unfortunately, EGFP introduces three mutations on and near the chromophore based on GFP to increase the brightness, but it also loses the Ex405 nm excitation peak and only retains an Ex488 nm excitation peak ( Figure 4 B).

[0046] Based on this, the applicant of the present invention proposes a hypothesis, whether it is possible to predict and analyze the structure of the probe cpEGFP using Alphafold, find the key sites that affect ESPT, restore the ESPT network of the probe, and promote the occurrence of ESPT, thereby preparing an excitation ratio fluorescent probe. This excitation ratio probe has two excitation peaks, Ex405 nm and Ex488 nm, and after adding neuropeptides, it will cause a negative response (off response) of Ex405 and a positive response (on response) of Ex488 ( Figure 5 In this case, using the Ex488 / 405 ratio to calculate the reaction will have a larger response amplitude, and the ratio can also be used to correct for signal fluctuations caused by pH, expression level, laser power, etc.

[0047] 102: Analyze the amino acid sites. Based on the structure prediction results, analyze the amino acid sites within 6Å around the S205 site of the cyclically rearranged fluorescent protein in the structure; In one embodiment of the present invention, the applicant found that for wild-type GFP, the fluorescent protein states under the two excitation peaks Ex405 and Ex488 of its excitation spectrum correspond to the neutral form and the ionized form, respectively. Figure 6 Å). In the excited state, the neutral form undergoes proton transfer, i.e., the ESPT process, and is converted into the intermediate state State I, which then emits light and is detected by the outside world. In this network, the transfer of protons depends on the integrity of the ESPT network, which is composed of the chromophore, the main chain carbon hydroxyl group at the S205 site, the main chain carbon carboxyl group at the E222 site, and water molecules ( Figure 6B). In order to make the GRAB probe have the property of excitation ratio, it is necessary to increase the response amplitude of the probe under 405 nm excitation, especially to increase the brightness of Ex405 in the inactive state. According to the experience of previous studies, when the S205 and E222 sites in GFPs are mutated to other amino acids, the occurrence of fluorescent protein ESPT will be affected to a certain extent. For example, the E222Q mutation will cause the ESPT of GFP to be completely blocked, because the main chain carbon carboxyl group at the 222 site is missing to participate in the ESPT network. In contrast, the S205V mutation will reduce the brightness of GFP, but GFP can still undergo ESPT. This suggests that the E222 site is essential for the occurrence of ESPT. If the amino acid composition of the E222 site and its adjacent positions is directly changed, it is likely to inhibit the occurrence of ESPT, which is not conducive to the development of ratio neuropeptide probes.

[0048] Therefore, the applicant chose to optimize the composition of amino acids near the S205 site in order to screen candidate probes with better ratio properties, promote the occurrence of ESPT, and have a larger response amplitude under Ex405. Specifically, cpEGFP and its connecting peptide regions with a length of 5 amino acids at both ends were first named and extracted as the cpEGFP loop, and then predicted using Alphafold. Finally, based on the prediction results, the amino acid sites within 6Å near the S205 site were analyzed in the structure and saturation mutations were screened, and their response amplitudes under Ex488, Ex405 and Ex488 / 405 were tested, respectively, which are referred to as ΔF / F0(488), ΔF / F0(405) and ΔR / R0( Figure 7 ). In one embodiment of the present invention, taking the NTS1.3 probe as an example, the cpEGFP loop region sequence thereof is LEEGGNVYIKADKQKNGIKAAFNIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITWGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVGGEGEGDATEGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNTGATR (SEQ ID NO.2).

[0049] In the present invention, Alphafold is a deep learning algorithm developed by DeepMind for predicting the 3D structure of proteins. By utilizing a large amount of known protein sequence and structure data, as well as the powerful learning ability of deep learning algorithms, Alphafold can predict the 3D structure of a protein based solely on its amino acid sequence. Therefore, it is easy for a person skilled in the art to use Alphafold to determine the position of an amino acid site within 6Å near the cpEGFP S205 site.

[0050] In one embodiment of the present invention, the applicant uses the NTS1.3 probe as an example and finds that there are 15 amino acid sites within 6Å of S205 ( Figure 8 A). These amino acid sites are divided into four groups according to their spatial positions, namely, linker group, sites near S205 group, sites near E222 group, and sites near chromophore group. Figure 8 B). Among them, the amino acids in the sites near S205 and sites near E222 are closer to S205, appearing at 3.0Å from S205. Further expanding the distance screening range, it can be found that the amino acids in the connecting peptide group (N-linker) gradually appear ( Figure 8 C), and all of these amino acids are located near the carboxyl and amine groups on the Cα of S205, that is, on the opposite side of the hydroxyl group of S205. It is worth mentioning that glutamic acid, the third amino acid of the N-linker, is numbered N3(E), which corresponds to the number 146 of the wild-type GFP. In the ESPT network of GFP, it can be found that the 146 site is close to the ESPT network in the spatial structure, suggesting that its conformational changes are likely to affect the occurrence of ESPT.

[0051] 103: Saturation mutagenesis, saturation mutagenesis of amino acid sites within 6Å near the S205 site of the cyclically rearranged fluorescent protein; In one embodiment of the present invention, the applicant found that the 15 amino acid sites mentioned above are spatially close to the S205 site ( Fig. 9 A), it is very likely that the occurrence of probe ESPT can be promoted by changing its amino acid composition. Therefore, based on the NTS1.3 probe, these 15 sites were screened for saturation mutations, and the probes were excited with light at wavelengths of 488 nm and 405 nm, respectively, and their response amplitudes after the addition of 1 μM NTS were detected.

[0052] 104: Screen key mutation sites, and select the amino acid sites that can significantly improve the response amplitude of the fluorescent probe under Ex405 and Ex488 / 405 after mutation as the key mutation sites for improving the excitation ratio properties of the GRAB probe.

[0053] In one embodiment of the present invention, the saturation mutagenesis results showed that different mutations in the amino acids adjacent to the E222 site and the amino acid sites in the connecting peptide region increased the ΔF / F0 (405) of the mutant to a certain extent. In contrast, the ΔF / F0 (405) of the mutant in the amino acid site in the connecting peptide region was significantly higher than that of the control probe NTS1.3 ( Fig. 9 B). These mutants were further tested repeatedly on cells to ensure the credibility of the signal enhancement. The results showed that among the five sites in the linker peptide region, only when the glutamic acid at the N3(E) site mutated to other amino acids, the brightness and response amplitude of the candidate probe at 488 nm and 405 nm could be improved to a certain extent. In particular, when the glutamic acid at the N3(E) site mutated to L (leucine), the brightness and response amplitude of the candidate probe at 488 nm and 405 nm were the largest ( Fig. 9 C).

[0054] In order to analyze why the mutation of E (glutamic acid) to L (leucine) at the N3 site of the NTS1.3 probe can significantly improve the ESPT of the probe, the sequences of cpEGFP and its left and right connecting peptides of the NTS1.3 probe and the NTS1.3-N3(E) candidate probe were extracted and the structures were predicted using Alphafold. The predicted structures of the two probes are very similar, but there are significant conformational differences between the two probes in the N-terminal connecting peptide region of cpEGFP ( Fig.10 A). The N-terminal connecting peptide of cpEGFP of the NTS1.3-N3(E) candidate probe is closer to the chromophore cpEGFP side than the N-terminal connecting peptide of cpEGFP of the NTS1.3 probe, and the farthest end is displaced inward by about 7.4Å. Specifically, after NTS1.3 carries the N3(E)L mutation, the N-terminal connecting peptide undergoes a large displacement toward the cpEGFP side. It is speculated that this may be caused by the chemical bond formed by N3(E)L and certain amino acids on cpEGFP, resulting in an interaction. By searching within 3Å of the N3(E)L site, it was found that N3(E)L can form two hydrogen bonds with the S205 site, while this interaction was not found in NTS1.3 ( Fig.10B). In addition, the hydrogen bond between S205 and E222 can be found in the predicted structure of NTS1.3-N3(E)L, which is also the structural basis for proton transfer in the ESPT network. The above results show that N3(E)L forms a hydrogen bond with the S205 site, stabilizes the amino acid orientation of S205, and makes it more prone to ESPT. Since the NTS1.3-N3(E)L candidate probe has a large response amplitude at 405 and a large ratio response amplitude, it is named NTS1.4.

[0055] Ratio-type neuropeptide probes have the ability to show opposite reactions triggered by binding neuropeptides under 488 and 405 excitation light, so using the Ex488 / 405 ratio for calculation will significantly increase the probe's response amplitude, providing the possibility of sensitively detecting dynamic changes in neuropeptides. However, the advantage of ratio-type neuropeptide probes is not only that they have a higher response amplitude, but they can also be used to correct the fluctuations in fluorescence values ​​caused by certain environmental factors (such as pH and excitation power in the environment). Because the probe itself has only one fluorescent protein and contains one chromophore, when external factors (pH, excitation power, etc.) change, the fluorescence of the probe will change accordingly. However, since the fluorescence of the ratio-type probe under 488 nm and 405 nm excitation comes from the same chromophore, the effects of external factors on Ex488 and Ex405 are in the same direction. Therefore, using the Ex488 / 405 ratio may be beneficial to correct the changes in fluorescence intensity caused by changes in pH or excitation power, thereby improving the stability of the probe and ensuring the specificity of the probe detection signal.

[0056] Furthermore, the applicant characterized the spectral properties of the probe after the NTS1.3 N(3) site mutation, and the results showed that the probe after the mutation had two excitation peaks at 400 nm and 505 nm, respectively, and the corresponding emission peaks were both at 525 nm ( Fig.11 A). In order to verify the pH stability of the probe after mutation, the pH in the cell environment and in the cell was changed. The results showed that as the pH decreased, the fluorescence brightness of the probe at 488 and 405 also gradually decreased ( Fig.11 B). In contrast, the ratio of Ex488 / 405 is less affected by different pH values. For example, at pH = 5, Ex488 is only 30% of the maximum brightness, but Ex488 / 405 is 80% of the maximum brightness, and its fluorescence brightness is significantly higher than Ex488 ( Fig.11 C). These results suggest that the mutated probes are less affected by pH changes when detecting dynamic changes in neuropeptide release in vivo.

[0057] In addition, the brightness of fluorescent proteins will be greatly affected by the expression level of the probe and the laser power. For example, the higher the expression level of the probe, the higher the brightness of Ex488 and Ex405 of cpEGFP; the higher the laser power used in the experiment, the higher the Ex488 and Ex405 of cpEGFP. Because the effects of the probe expression level and the laser power on Ex488 and Ex405 of cpEGFP are in the same direction, it is speculated that Ex488 / 405 may remain stable. In order to detect whether the fluorescence brightness of the probe after the N(3) site mutation is affected by the probe expression level, 50 ng, 100 ng, 200 ng and 300 ng of the probe after the N(3) site mutation were expressed in HEK293T cells, and Ex488, Ex405 and Ex488 / 405 at different expression levels of the probe were detected. The experimental results showed that with the increase in the expression level of the probe, Ex488 and Ex405 also gradually increased, but Ex488 / 405 remained stable ( Fig.12 AB).

[0058] To further verify whether the fluorescence brightness of the probe after N(3) site mutation is affected by the excitation power, 300 ng of the probe after N(3) site mutation was expressed in HEK293T cells, and the probe was excited with 10%, 30%, 50%, 70% and 90% excitation power, and the brightness of Ex488, Ex405 and Ex488 / 405 under different excitation power intensities was detected. The experimental results showed that as the excitation power of the probe increased, Ex488 and Ex405 also increased proportionally, but Ex488 / 405 remained relatively stable. For example, when the excitation power was 10%, Ex488 was 10% of the maximum brightness, but Ex488 / 405 was 70% of the maximum brightness, and its fluorescence brightness was significantly higher than Ex488 ( Fig.12 CD).

[0059] Figure 2 Schematic diagram of the structure of the screening system for key mutation sites for improving the excitation ratio properties of GRAB probes provided by the present invention.

[0060] The system is programmed or otherwise configured to include a structure prediction module 201, an amino acid site analysis module 202, a saturation mutation module 203, and a key mutation site screening module 204: Structure prediction module: using Alphafold to predict the structure of the loop region of the cyclically rearranged fluorescent protein in the GRAB probe; the loop region of the cyclically rearranged fluorescent protein is the cyclically rearranged fluorescent protein in the GRAB probe and the connecting peptide region with a length of 5 amino acids at both ends; Amino acid site analysis module: Based on the structural prediction results, the amino acid sites within 6Å around the cyclically rearranged fluorescent protein S205 site were analyzed in the structure; Saturation mutagenesis module: saturation mutation of amino acid sites within 6Å around the cyclically rearranged fluorescent protein S205 site; Screening module for key mutation sites: The amino acid sites that can significantly improve the response amplitude of the fluorescent probe under Ex405 and Ex488 / 405 after mutation are selected as key mutation sites for improving the excitation ratio properties of the GRAB probe.

[0061] The system may be the user's electronic device or a computer system located remotely from the electronic device.

[0062] Figure 3 A schematic diagram of the structure of a computer device provided by the present invention.

[0063] The computer device 300 includes a processor 301 and a memory 302 coupled to the processor 301 , wherein the memory 302 stores program instructions, and when the program instructions are executed by the processor 301 , the processor 301 executes the above-mentioned method for screening key mutation sites for improving the excitation ratio property of GRAB probes.

[0064] The processor 301 may also be referred to as a CPU (Central Processing Unit). The processor 301 may be an integrated circuit chip having signal processing capabilities. The processor 301 may also be a general-purpose processor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components. The general-purpose processor may be a microprocessor or the processor may also be any conventional processor, etc.

[0065] The computer device 300 may be a mobile electronic device.

[0066] It should be understood that the systems, devices and methods described in the present invention can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the modules is only a logical function division. There may be other division methods in actual implementation, such as multiple modules or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or modules, which can be electrical, mechanical or other forms.

[0067] The modules described as separate components may or may not be physically separated, and the components shown as modules may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0068] In addition, each functional module in each embodiment of the present invention may be integrated into one processing module, or each module may exist physically separately, or two or more modules may be integrated into one module. The above-mentioned integrated module may be implemented in the form of hardware or in the form of software functional modules.

[0069] The above are only implementation methods of the present application, and are not intended to limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the contents of the present application specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A method for screening key mutation sites for improving the ratiometric properties of GRAB probe excitation, characterized in that: The method comprises: Structure prediction: Alphafold was used to predict the structure of the loop region of the cyclically rearranged fluorescent protein in the GRAB probe; the loop region of the cyclically rearranged fluorescent protein is the cyclically rearranged fluorescent protein in the GRAB probe and its connecting peptide region with a length of 5 amino acids at both ends; Analyze amino acid sites: Based on the structural prediction results, analyze the amino acid sites within 6Å around the cyclically rearranged fluorescent protein S205 site in the structure; Saturation mutagenesis: Saturation mutagenesis of amino acid sites within 6Å of the cyclically rearranged fluorescent protein S205 site; Screening of key mutation sites: The amino acid sites that can significantly improve the response amplitude of the fluorescent probe under Ex405 and Ex488 / 405 after mutation are selected as the key mutation sites for improving the excitation ratio properties of the GRAB probe.

2. The screening method according to claim 1, characterized in that The step of screening key mutation sites includes screening using a high-throughput method; Preferably, the step of screening using a high-throughput method comprises saturating the amino acid sites within 6Å near the S205 site of the cyclically rearranged fluorescent protein, and then imaging and screening using a high-content Opera Phenix instrument.

3. The screening method according to claim 1, characterized in that The sequence of the connecting peptide includes the N-terminal connecting peptide of the cyclically rearranged fluorescent protein and the C-terminal connecting peptide of the cyclically rearranged fluorescent protein; Preferably, the cyclically rearranged fluorescent protein is cpEGFP; Preferably, the N-terminal connecting peptide sequence of the cpEGFP is LEEGG; Preferably, the C-terminal connecting peptide sequence of the cpEGFP is TGATR; Preferably, the sequence of the cpEGFP is shown as SEQ ID NO.1; Preferably, the sequence of the cpEGFP loop region is shown as SEQ ID NO.

2.

4. The screening method according to claim 1, characterized in that The GRAB probe is an NTS probe; Preferably, the sequence of the NTS probe is shown as SEQ ID NO.

3.

5. The screening method according to any one of claims 1 to 4, characterized in that There are 15 amino acid sites within 6Å near the cpEGFP S205 site, including the connecting peptide group N1 (L), N2 (E), N3 (E), N4 (G), C1 (T), the S205 site group V203, Q204, H206, L207, the E222 site group L220, L221, E222, F223, and the chromophore group V52 and Y57; Preferably, the key amino acid site for improving the excitation ratio property of the GRAB probe is the connecting peptide group N3(E), and the N3(E) is the third amino acid of the connecting peptide at the N-terminus of cpEGFP.

6. A screening system for key mutation sites that improves the ratiometric properties of GRAB probe excitation, characterized in that: The system comprises: Structure prediction module: using Alphafold to predict the structure of the loop region of the cyclically rearranged fluorescent protein in the GRAB probe; the loop region of the cyclically rearranged fluorescent protein is the cyclically rearranged fluorescent protein in the GRAB probe and the connecting peptide region with a length of 5 amino acids at both ends; Amino acid site analysis module: Based on the structural prediction results, the amino acid sites within 6Å around the cyclically rearranged fluorescent protein S205 site were analyzed in the structure; Saturation mutagenesis module: saturation mutation of amino acid sites within 6Å around the cyclically rearranged fluorescent protein S205 site; Screening module for key mutation sites: The amino acid sites that can significantly improve the response amplitude of the fluorescent probe under Ex405 and Ex488 / 405 after mutation are selected as key mutation sites for improving the excitation ratio properties of the GRAB probe.

7. The screening system according to claim 6, characterized in that The module for screening key mutation sites includes a high-throughput screening unit; Preferably, the high-throughput screening unit comprises saturation mutagenesis of amino acid sites within 6Å of the S205 site of the cyclically rearranged fluorescent protein, followed by imaging and screening using a high-content Opera Phenix instrument.

8. The screening system according to claim 6, characterized in that: The sequence of the connecting peptide includes the N-terminal connecting peptide of the cyclically rearranged fluorescent protein and the C-terminal connecting peptide of the cyclically rearranged fluorescent protein; Preferably, the cyclically rearranged fluorescent protein is cpEGFP; Preferably, the N-terminal connecting peptide sequence of the cpEGFP is LEEGG; Preferably, the C-terminal connecting peptide sequence of the cpEGFP is TGATR; Preferably, the sequence of the cpEGFP is shown as SEQ ID NO.1; Preferably, the sequence of the cpEGFP loop region is shown as SEQ ID NO.2; Preferably, the sequence of the NTS probe is shown as SEQ ID NO.3; Preferably, there are 15 amino acid sites within 6Å near the cpEGFP S205 site, including the connecting peptide group N1 (L), N2 (E), N3 (E), N4 (G), C1 (T), the S205 site group V203, Q204, H206, L207, the E222 site group L220, L221, E222, F223, and the chromophore group V52, Y57; Preferably, the key amino acid site for improving the excitation ratio property of the GRAB probe is the connecting peptide group N3(E), and the N3(E) is the third amino acid of the connecting peptide at the N-terminus of cpEGFP.

9. A computer device and a computer readable storage medium, characterized in that: The device comprises: A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the screening method according to any one of claims 1 to 5 is implemented; The computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the screening method according to any one of claims 1 to 5 is implemented.

10. Use of the screening method according to any one of claims 1 to 5 in screening key mutation sites for improving the excitation ratio properties of GRAB probes; Preferably, the GRAB probe is an NTS probe.

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