CAMP fluorescent probe r-flamp2m, its applications and kits

Abstract

The application relates to the field of biomedical technology, and particularly relates to a cAMP fluorescent probe R-Flamp2m, application and kit thereof.A cAMP fluorescent probe R-Flamp2m, the amino acid sequence of the R-Flamp2m is shown as SEQ ID NO:1.The single-photon excitation wavelength peak of the fluorescent probe R-Flamp2m is 570 nm, and the emission wavelength peak is about 600 nm.The application has the following advantages: (1) a larger dynamic range; (2) high detection sensitivity; (3) can be used in cAMP signal detection of active objects; (4) wide application range, and can be combined with a short-wavelength (400-500 nm) probe or a photosensitive protein.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a cAMP fluorescent probe R-Flamp2m, its applications, and a kit. Background Technology

[0002] Cyclic adenosine monophosphate (cAMP) is a downstream messenger molecule of the G protein-coupled receptor (GPCR) family, currently the largest drug target. Cellular and in vivo cAMP fluorescence imaging is an important direction for basic research and drug development of the GPCR signaling pathway. cAMP fluorescent probes are mainly divided into fluorescence resonance energy transfer (FRET) probes based on fluorescent proteins and probes based on single fluorescent proteins. The latter has a larger dynamic range and is simpler to use. Currently, cAMP probes based on single fluorescent proteins are divided into two subclasses: green and red. The former mainly includes Flamindo2, cADDis, and cAMPr, while the latter mainly includes R-Flamp1m, Pink Flamindo, Red cADDis, and R-FlincA.

[0003] Live-cell cAMP fluorescence imaging refers to the expression of cAMP fluorescent probes in cells and then detecting changes in probe fluorescence intensity using fluorescence microscopy. Fluorescent probes are crucial for cAMP fluorescence imaging analysis. In recent years, internationally renowned laboratories have developed cAMP single-fluorescent protein probes with varying performance and spectra using different fluorescent proteins, different cAMP sensing modules, and different fusion sites. These include the Flamindo / Flamindo2 / PinkFlamindo probe developed by Dr. Tetsuya Kitaguchi, the cADDis / Red cADDis probe developed by Dr. Anne Marie Quinn, the R-FlincA probe developed by Dr. Kazuki Horikawa, and the cAMPr probe developed by Dr. Justin Blau. For green cAMP probes, the applicant has developed a higher-performance probe, G-Flamp1 (CN201911251920.X and CN202010354936.X).

[0004] Compared with short-wavelength green probes, long-wavelength red probes have many advantages: (1) Red probes can be used in conjunction with a large number of existing green probes or blue light-sensitive tool proteins; (2) Red probes have lower fluorescence background interference, less phototoxicity and deeper tissue penetration.

[0005] However, existing red probes for cAMP have the following shortcomings: (1) They have very low brightness in mammalian cells cultured at 37°C, which is not conducive to cell localization and observation of small cellular structures such as neuronal dendrites. (2) The dynamic range (fluid intensity variation ΔF / F0) in mammalian cells cultured at 37°C is very small, which seriously affects the detection sensitivity.

[0006] Therefore, developing high-performance red probes and improving their brightness and dynamic range in practical applications is of great significance for enhancing detection sensitivity. Summary of the Invention

[0007] The purpose of this invention is to overcome the above-mentioned technical problems and provide a cAMP fluorescent probe R-Flamp2m with a large dynamic range, high detection sensitivity, ability to detect changes in cAMP concentration after cell stimulation, and the ability to be used in conjunction with a large number of existing green probes or blue light-sensitive tool proteins for simultaneous dual-color imaging or optical control / fluorescence imaging, as well as its applications and kits.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A cAMP fluorescent probe R-Flamp2m, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0009] Furthermore, the peak excitation wavelength of the single photon from the fluorescent probe R-Flamp2m is 570 nm.

[0010] Furthermore, the fluorescent probe R-Flamp2m can be used at a single-photon excitation wavelength of 550-570 nm.

[0011] The present invention also provides a method for preparing the cAMP fluorescent probe R-Flamp2m.

[0012] A method for preparing a cAMP fluorescent probe R-Flamp2m, comprising: using mEpac1, which can bind cAMP, as the sensing domain, linking it to mApple fluorescent protein, and obtaining the R-Flamp2m probe through several amino acid mutations; The amino acid sequence of R-Flamp2m is shown in SEQ ID NO: 1.

[0013] The present invention also provides a method for detecting cAMP fluorescence imaging in live cells for purposes other than disease diagnosis or treatment.

[0014] A method for detecting cAMP fluorescence imaging in live cells for purposes other than disease diagnosis or treatment, the method comprising the following steps: (1) Live cell culture, when the cell density is 60±5%, transfect R-Flamp2m plasmid; (2) After transfecting, the cells were cultured overnight and starved for 2-4 hours. The culture medium was then replaced with a colorless and transparent buffer solution. (3) Fluorescence microscopy imaging analysis at excitation wavelengths of 550-570 nm for single photons.

[0015] The present invention also provides a method for detecting cAMP fluorescence imaging in mammalian cells for purposes other than disease diagnosis or treatment.

[0016] A method for detecting cAMP fluorescence imaging in mammalian cells for purposes other than disease diagnosis or treatment, the method comprising the following steps: (1) Mammalian cell culture: The culture medium was DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, the culture temperature was 37℃, and the CO2 content was 5%; When the cell density was 60±5%, the R-Flamp2m plasmid was transfected using the Lipofectamine 2000 kit; (2) After transfecting mammalian cells, the cells were cultured overnight and starved for 2-4 hours in a medium without serum and phenol red. Then the medium was replaced with a colorless and transparent fluorescent imaging buffer. (3) Fluorescence microscopy imaging analysis, in which the excitation wavelength of single photons is 550~570nm.

[0017] The present invention also provides another method for detecting cAMP fluorescence imaging in mammalian cells for purposes other than disease diagnosis or treatment.

[0018] A method for detecting cAMP fluorescence imaging in mammalian cells for purposes other than disease diagnosis or treatment, the method comprising the following steps: (1) Mammalian cell culture: The culture medium was DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, the culture temperature was 37℃, and the CO2 content was 5%; When the cell density was 60±5%, the R-Flamp2m plasmid was transfected using the Lipofectamine 2000 kit; (2) After transfecting mammalian cells, the cells were cultured overnight and starved for 2-4 hours in a medium without serum and phenol red. Then the medium was replaced with a colorless and transparent fluorescent imaging buffer. (3) The excitation wavelength of single photons is 550-570nm. Fluorescence microscopy imaging analysis is performed at a frequency of one frame every 15 seconds. Forskolin is added in the tenth frame until the final concentration of Forskolin is 60μM. The intensity change of the fluorescence of the R-Flamp2m probe is detected.

[0019] This invention also provides an application of the cAMP fluorescent probe R-Flamp2m.

[0020] Application of a cAMP fluorescent probe R-Flamp2m in the detection of cAMP for non-disease diagnosis or treatment purposes.

[0021] This invention also provides an application of another cAMP fluorescent probe, R-Flamp2m.

[0022] Application of a cAMP fluorescent probe R-Flamp2m in the detection of cAMP in live cells and / or live animals for non-disease diagnosis or treatment purposes.

[0023] The present invention also provides a kit for the cAMP fluorescent probe R-Flamp2m.

[0024] A kit containing the fluorescent probe R-Flamp2m.

[0025] The fluorescent probe R-Flamp2m provided by this invention uses mEpac1, which can bind cAMP, as its sensing domain, and interacts with the mApple fluorescent protein via... Figure 1 The probe is obtained by connecting the components as shown in the diagram and performing several amino acid mutations.

[0026] The fluorescent probe R-Flamp2m provided by this invention is a red cAMP probe with a peak excitation wavelength of 570 nm and a peak emission wavelength of approximately 600 nm. In cells cultured at a physiological temperature of 37°C, R-Flamp2m exhibits a dynamic range (ΔF / F0) of approximately 10 under single-photon excitation at 550-570 nm, which is about 66% higher than that of R-Flamp1m (ΔF / F0 is approximately 6). This results in a larger dynamic range and higher detection sensitivity in cell imaging. The amino acid sequence of R-Flamp1m is shown in SEQ ID NO:2.

[0027] The fluorescent probe R-Flamp2m provided by this invention is currently the red cAMP probe with the largest dynamic range.

[0028] Compared to green cAMP probes, the fluorescent probe R-Flamp2m provided by this invention is a red probe. The red probe can be used in conjunction with a large number of existing green probes or blue-light-sensitive protein tools, enabling simultaneous two-color imaging or optical control / fluorescence imaging.

[0029] The fluorescent probe R-Flamp2m provided by this invention is expressed in mammalian cells, and changes in cAMP concentration after cells are stimulated by a common fluorescence microscope can be detected.

[0030] Compared with existing technologies, the advantages of the cAMP fluorescent probe R-Flamp2m, its applications, and kits provided by this invention are as follows: (1) Larger dynamic range.

[0031] (2) High detection sensitivity.

[0032] (3) It can be used in the detection of cAMP signals of moving objects.

[0033] (4) It has a wide range of applications and can be used in conjunction with short wavelength (400-500nm) probes or photosensitive proteins. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the composition of R-Flamp1m and R-Flamp2m provided by the present invention, wherein His is a protein purification tag, mApple-N and mApple-C are the amino-terminal and carboxyl-terminal portions of the red fluorescent protein mApple, respectively (the two portions constitute the fluorescent module of the probe), mEpac1 (205~353) is a cAMP-binding domain (sensing module), and linker peptide 1 and linker peptide 2 are the connection portions between the sensing module and different portions of the fluorescent protein; Figure 2 This invention provides the R-Flamp1m and R-Flamp2m sequence alignments. Figure 3 This is a comparison of the affinity curves of R-Flamp1m and the R-Flamp2m probe provided in this invention, including the fluorescence responses of R-Flamp1m and R-Flamp2m probes at different cAMP concentrations. Bacteria expressing the probe were suspended in HEPES buffer at pH 7.2, sonicated, and the supernatant was used to measure the probe's response to different concentrations of cAMP. Figure 4 This is a comparison of the brightness of R-Flamp1m and R-Flamp2m provided in this invention in HEK293T cells; Figure 5The responses of Flamp1m and the R-Flamp2m probe provided in this invention in HEK293T cells under single-photon excitation are shown in the figures. (A) are representative fluorescence images before and after Fsk stimulation; scale bar: 20 μm; excitation wavelength: 568 nm ± 10 nm, fluorescence reception wavelength: 630 nm ± 25 nm; (B) are fluorescence change curves; curve data are expressed as mean ± standard error of mean (SEM). Detailed Implementation

[0035] To enable those skilled in the art to better understand the technical solutions of the present invention, the following embodiments provide a more detailed description of the present invention. These embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0036] A cAMP fluorescent probe R-Flamp2m, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0037] Furthermore, the excitation wavelength of the single photon of the fluorescent probe R-Flamp2m is 570 nm.

[0038] Furthermore, the fluorescent probe R-Flamp2m can be used at a single-photon excitation wavelength of 550-570 nm.

[0039] The present invention also provides a method for preparing the cAMP fluorescent probe R-Flamp2m.

[0040] A method for preparing a cAMP fluorescent probe R-Flamp2m, comprising: using mEpac1, which can bind cAMP, as the sensing domain, linking it to mApple fluorescent protein, and obtaining the R-Flamp2m probe through several amino acid mutations; The amino acid sequence of R-Flamp2m is shown in SEQ ID NO: 1.

[0041] The present invention also provides a method for detecting cAMP fluorescence imaging in live cells for purposes other than disease diagnosis or treatment.

[0042] A method for detecting cAMP fluorescence in live cells, the method comprising the following steps: (1) Live cell culture, when the cell density is 60±5%, transfect R-Flamp2m plasmid; (2) After transfecting, the cells were cultured overnight and starved for 2-4 hours. The culture medium was then replaced with a colorless and transparent buffer solution. (3) Fluorescence microscopy imaging analysis at excitation wavelengths of 550-570 nm for single photons.

[0043] The present invention also provides a method for detecting cAMP fluorescence imaging in mammalian cells for purposes other than disease diagnosis or treatment.

[0044] A method for detecting cAMP fluorescence imaging in mammalian cells for purposes other than disease diagnosis or treatment, the method comprising the following steps: (1) Mammalian cell culture: The culture medium was DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, the culture temperature was 37℃, and the CO2 content was 5%; When the cell density was 60±5%, the R-Flamp2m plasmid was transfected using the Lipofectamine 2000 kit; (2) After transfecting mammalian cells, the cells were cultured overnight and starved for 2-4 hours in a medium without serum and phenol red. Then the medium was replaced with colorless and transparent live cell imaging buffer. (3) Fluorescence microscopy imaging analysis, in which the excitation wavelength of single photons is 550-570nm.

[0045] The present invention also provides another method for detecting cAMP fluorescence imaging in mammalian cells for purposes other than disease diagnosis or treatment.

[0046] A method for detecting cAMP fluorescence imaging in mammalian cells for purposes other than disease diagnosis or treatment, the method comprising the following steps: (1) Mammalian cell culture: The culture medium was DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, the culture temperature was 37℃, and the CO2 content was 5%; When the cell density was 60±5%, the R-Flamp2m plasmid was transfected using the Lipofectamine 2000 kit; (2) After transfecting mammalian cells, the cells were cultured overnight and starved for 2-4 hours in a medium without serum and phenol red. Then the medium was replaced with colorless and transparent live cell imaging buffer. (3) The excitation wavelength of single photons is 550-570nm. Fluorescence microscopy imaging analysis is performed at a frequency of one frame every 15 seconds. Forskolin is added in the tenth frame until the final concentration of Forskolin is 60μM. The intensity change of the fluorescence of the R-Flamp2m probe is detected.

[0047] This invention also provides an application of the cAMP fluorescent probe R-Flamp2m.

[0048] Application of a cAMP fluorescent probe R-Flamp2m in the detection of cAMP for non-disease diagnosis or treatment purposes.

[0049] This invention also provides an application of another cAMP fluorescent probe, R-Flamp2m.

[0050] Application of a cAMP fluorescent probe R-Flamp2m in the detection of cAMP in live cells and / or live animals for non-disease diagnosis or treatment purposes.

[0051] The present invention also provides a kit for the cAMP fluorescent probe R-Flamp2m.

[0052] A kit containing the fluorescent probe R-Flamp2m.

[0053] Example 1 Construction of R-Flamp2m Combination Figure 1 As shown, mEpac1, which can bind cAMP, serves as the sensing domain, and interacts with mApple fluorescent protein via... Figure 1 The probes were connected as shown, and after several amino acid mutations, the R-Flamp2m probe was obtained. The amino acid sequence of R-Flamp2m is shown in SEQ ID NO:1. The amino acid sequence of R-Flamp1m is shown in SEQ ID NO:2.

[0054] His is the protein purification tag, mApple-N and mApple-C are the N-terminal and C-terminal portions of the red fluorescent protein mApple, respectively (the two portions constitute the fluorescent module of the probe), mEpac1 (205~353) is the cAMP-binding domain (sensing module), and linker peptide 1 and linker peptide 2 are the connection parts between the sensing module and different portions of the fluorescent protein.

[0055] Example 2 Excitation and emission spectra of the R-Flamp2m probe The R-Flamp2m probe was expressed in bacteria, and the cells were collected after culturing at room temperature for 3 days. The cells were then sonicated in HEPES buffer (containing 150 mM KCl and 50 mM HEPES) at pH 7.2, and the supernatant (containing the probe) was obtained by centrifugation.

[0056] 120 μL of probe solution was taken, and the response of the probe to different cAMP concentrations was detected using an Infinite M1000 PRO multi-functional microplate reader. The excitation wavelength of fluorescence was 560 ± 5 nm, and the receiving wavelength was 610 ± 10 nm. The curve of ΔF / F0 versus cAMP concentration was obtained to compare the dynamic range of R-Flamp1m and R-Flamp2m at different cAMP concentrations. The results are shown below. Figure 3 As shown.

[0057] As can be seen from the figure, the R-Flamp2m provided by the present invention has a larger dynamic range ΔF / F0 and higher detection sensitivity.

[0058] Example 3 Comparison of the brightness of R-Flamp1m and R-Flamp2m in HEK293T cells HEK293T cells were cultured in 12-well plates in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C with 5% CO2. The same mass of plasmids expressing R-Flamp1m or R-Flamp2m were transfected into cells in different wells using a Lipofectamine 2000 kit. After approximately 40 hours, the cells were washed once with colorless, clear live cell imaging buffer, then resuspended in 200 μl of live cell imaging buffer and transferred to 96-well microplates. After incubation at 37°C for 10 minutes, the fluorescence intensity of the cells was detected using a microplate reader. The results are shown below. Figure 4 As shown.

[0059] Example 4 Response of Flamp1m and R-Flamp2m probes in HEK293T cells under single-photon excitation HEK293T cells were cultured in glass-bottomed culture dishes in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin at a temperature of 37°C and a CO2 content of 5%.

[0060] When the cell density is around 60%, transfect the R-Flamp1m or R-Flamp2m plasmids using the Lipofectamine 2000 kit.

[0061] After overnight culture, cells were starved for 2 to 4 hours using serum- and phenol red-free medium (purchased from GIBCO). The medium was then replaced with colorless, transparent live cell imaging buffer. Imaging analysis was performed using an IX83 single-photon fluorescence microscope (excitation wavelength 568nm ± 10nm, emission wavelength 630nm ± 25nm) built in our laboratory. The imaging frequency was one frame every 15 seconds. Forskolin (purchased from Beyotime Biotechnology) was added at the tenth frame, with a final concentration of 60 μM (the culture dish contained 500 μL of live cell imaging buffer; 4 μL of 12 mM stock solution was dissolved in 300 μL of live cell imaging buffer, and at the tenth frame, the diluted 300 μL of Forskolin solution was added dropwise to the cell dish being imaged).

[0062] The changes in fluorescence intensity of R-Flamp1m and R-Flamp2m in cells after Forskolin stimulation are shown in the following results. Figure 5 As shown. This completes the fluorescence imaging step for changes in cAMP concentration within mammalian cells.

[0063] Figure 5 The curve data in the figure are represented as: mean ± standard deviation; ΔF / F0 is the ratio of the change in fluorescence intensity to the initial fluorescence intensity.

[0064] from Figure 5 As can be seen, after Forskolin stimulation, HEK293T cells showed that R-Flamp2m had a larger signal change amplitude (ΔF / F0) compared to Flamp1m, and the dynamic range and sensitivity were greatly improved.

[0065] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0066] It should also be noted that the various specific technical features and steps described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0067] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A cAMP fluorescent probe R-Flamp2m, characterized in that: The amino acid sequence of the R-Flamp2m is shown in SEQ ID NO:

1.

2. A method for preparing a cAMP fluorescent probe R-Flamp2m, characterized in that, The method is as follows: using mEpac1, which can bind cAMP, as the sensing domain, it is linked to mApple fluorescent protein, and after several amino acid mutations, the R-Flamp2m probe is obtained. The amino acid sequence of R-Flamp2m is shown in SEQ ID NO:

1.

3. A method for detecting cAMP fluorescence imaging in live cells for purposes other than disease diagnosis or treatment, characterized in that, The method includes the following steps: (1) Live cell culture, at a cell density of 60±5%, transfected with a plasmid containing the cAMP fluorescent probe R-Flamp2m as described in claim 1; (2) After transfecting, the cells were cultured overnight and starved for 2-4 hours. The culture medium was then replaced with a colorless and transparent buffer solution. (3) Fluorescence microscopy imaging analysis at excitation wavelengths of 550-570 nm for single photons.

4. A method for detecting cAMP fluorescence imaging in mammalian cells for purposes other than disease diagnosis or treatment, characterized in that, The method includes the following steps: (1) Mammalian cell culture: The culture medium was DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, the culture temperature was 37℃, and the CO2 content was 5%; At a cell density of 60±5%, the plasmid containing the cAMP fluorescent probe R-Flamp2m as described in claim 1 was transfected using the Lipofectamine 2000 kit. (2) After transfecting mammalian cells, culture them overnight, starve them for 2-4 hours with serum-free and phenol red-free medium, and then replace the medium with colorless and transparent fluorescent imaging buffer. (3) Fluorescence microscopy imaging analysis, in which the excitation wavelength of single photons is 550-570nm.

5. A method for detecting cAMP fluorescence imaging in mammalian cells for purposes other than disease diagnosis or treatment, characterized in that, The method includes the following steps: (1) Mammalian cell culture: The culture medium was DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, the culture temperature was 37℃, and the CO2 content was 5%; At a cell density of 60±5%, the plasmid containing the cAMP fluorescent probe R-Flamp2m as described in claim 1 was transfected using the Lipofectamine 2000 kit. (2) After transfecting mammalian cells, culture them overnight, starve them for 2-4 hours with serum-free and phenol red-free medium, and then replace the medium with colorless and transparent fluorescent imaging buffer. (3) The excitation wavelength of single photons is 550-570nm. Fluorescence microscopy imaging analysis is performed at a frequency of one frame every 15 seconds. Forskolin is added in the tenth frame until the final concentration of Forskolin is 60μM. The intensity change of the fluorescence of the R-Flamp2m probe is detected.

6. The application of the cAMP fluorescent probe R-Flamp2m as described in claim 1 in the detection of cAMP for purposes other than disease diagnosis or treatment.

7. The use of the cAMP fluorescent probe R-Flamp2m as described in claim 1 in the detection of cAMP in live cells and / or live animals for non-disease diagnosis or treatment purposes.

8. A kit comprising the fluorescent probe R-Flamp2m according to any one of claims 1 to 7.

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