CN112480271A - High-performance red cAMP fluorescent probe and application thereof - Google Patents
High-performance red cAMP fluorescent probe and application thereof Download PDFInfo
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- CN112480271A CN112480271A CN202011473325.3A CN202011473325A CN112480271A CN 112480271 A CN112480271 A CN 112480271A CN 202011473325 A CN202011473325 A CN 202011473325A CN 112480271 A CN112480271 A CN 112480271A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Abstract
The invention belongs to the field of biological detection. In particular to a high-performance red cAMP (cyclic adenosine monophosphate) fluorescent probe and application thereof in detecting cAMP concentration in living cells. Compared with a green cAMP probe, the R-flash 1m is a red probe, and the red probe can be used together with a large amount of existing green probes or blue light sensitive tool proteins, and can be used for simultaneously carrying out two-color imaging or optical control/fluorescence imaging; in cells cultured at 37 ℃, the fluorescence brightness of R-Flamp1m is more than 10 times of that of the existing red cAMP probes Pink Flamiddo and R-FlincA, and the red cAMP probes have a larger dynamic range (delta F/F0-5), namely, the red cAMP probes have higher detection sensitivity.
Description
Technical Field
The invention belongs to the field of biological detection. In particular to a high-performance red cAMP (cyclic adenosine monophosphate) fluorescent probe and application thereof in detecting cAMP concentration in living cells.
Background
Cyclic adenosine monophosphate (cAMP), is a downstream messenger molecule of the largest drug target G protein-coupled receptor (GPCR) family at present, and cAMP fluorescence imaging at cellular and living levels is an important direction for fundamental research and drug development of GPCR signaling pathways. cAMP fluorescence imaging in living cells is carried out by expressing a cAMP fluorescent probe in a cell and detecting the change in the intensity of probe fluorescence using a fluorescence microscope. Fluorescent probes are key to the cAMP fluorescence imaging assay. cAMP fluorescent probes are mainly classified into fluorescent protein-based fluorescence resonance energy transfer probes and single fluorescent protein-based probes, the latter having a larger dynamic range than the former and being simple to use.
In recent years, cAMP single fluorescent protein probes of different properties and spectra have been developed in the internationally known laboratory using different fluorescent proteins, different cAMP sensing modules and different fusion sites (see Table 1), including the Flumido/Flumido 2/Pink Flumido probe developed by doctor Tetsuya Kitaguchi, the cADDis/Red cADDis probe developed by doctor Anne Marie Quinn, the R-FlucA probe developed by doctor Kazuki Horikawa and the cAMPr probe developed by doctor Justin Blau.
Table 1 summary of cAMP monofluorescent protein probes for different properties and spectra.
Currently, cAMP probes based on a single fluorescent protein are classified into subclasses of green and Red 2, the former mainly including Flamido 2, cADDis and cAMPr, and the latter mainly including Pink Flamido, Red cADDis and R-FlincA. For green cAMP probes, we have developed a higher performance probe G-flash 1 (201911251920.X and 202010354936. X). The long wavelength red probe has many advantages over the short wavelength green probe: (1) the red probe can be used in combination with a large number of existing green probes or blue light sensitive tool proteins; (2) the red probe has lower fluorescence background interference, less phototoxicity and deeper tissue penetration ability. However, the conventional red probe has the following disadvantages: (1) the brightness is very low (such as Pink Flamido/R-FlincA) when mammalian cells are cultured at 37 ℃, and the positioning of the cells and the observation of the structures of tiny cells such as neuron dendrites and the like are not facilitated; (2) the dynamic range (the change amplitude of fluorescence brightness is delta F/F0) is very small (such as Pink Fluamino/R-FlincA) in the mammalian cell cultured at 37 ℃, and the detection sensitivity is seriously influenced; (3) some probes are poorly expressed, e.g., R-FlincA is expressed in mammalian cells for a longer period of time to form spots.
In conclusion, the invention develops the high-performance red cAMP fluorescent probe, improves the brightness and dynamic range of the probe in practical application, and has important significance for improving the detection sensitivity.
Disclosure of Invention
In order to solve the technical problems, the invention provides a high-performance red cAMP (cyclic adenosine monophosphate) fluorescent probe R-flash 1m and application thereof in detecting cAMP concentration in living cells.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the invention provides a cAMP fluorescent probe R-Flamp1m with an amino acid sequence SEQ ID NO 1.
The R-flash 1m has the structure shown in the formula I:
His-mApple-N-linker peptide 1-mEpacl-linker peptide 2-mApple-C (formula I).
Wherein the content of the first and second substances,
the His is His tag (i.e. Histag), and the amino acid sequence of the His is SEQ ID NO. 2.
The amino acid sequence of mApple-N is SEQ ID NO. 3.
The amino acid sequence of the connecting peptide 1 (linker 1) is SEQ ID NO. 4.
The amino acid sequence of mEpacl is SEQ ID NO. 5.
The amino acid sequence of the connecting peptide 2 (linker 2) is SEQ ID NO. 6.
The amino acid sequence of mApple-C is SEQ ID NO. 7.
In a preferred embodiment of the present invention, the amino acid sequence further includes an amino acid sequence obtained by substituting, deleting or adding one or more amino acids, and the amino acid sequence has the same or similar function as the amino acid sequence; the amino acid sequence is an amino acid sequence with at least 80 percent of homology.
The cAMP fluorescent probe R-Flamp1m is applied to living cells and animal living bodies, and is particularly applied to an imaging detection method, especially a two-photon imaging method.
Furthermore, the cAMP fluorescent probe R-Flamp1m can be used for cAMP signal detection of a moving object.
Furthermore, the cAMP fluorescent probe R-Flamp1m can be used for detecting the cAMP concentration in HEK293T cells.
Furthermore, the cAMP fluorescent probe R-Flamp1m can be used for detecting the concentration of cAMP in a solution.
The invention has the beneficial effects.
The probe of the invention is a red cAMP probe R-Flamp1m, which is optimized on the basis of ping Flamiddo. The R-Flamp1m probe developed by the inventor is cultured in cells at 37 ℃ and physiological temperature, the dynamic range (delta F/F0) of R-Flamp1m under 560nm single photon excitation is about 5, and the dynamic range (delta F/F0) under 1000 nm two-photon excitation is about 8; the optimal excitation wavelength of R-Flamp1m is about 560nm, the maximum emission wavelength is about 600 nm, and as can be seen from the data in Table 1, R-Flamp1m is the red cAMP probe with the largest dynamic range at present.
Compared with the existing fluorescent probe, the R-flash 1m of the invention has the following advantages: (1) compared with a green cAMP probe, R-Flamp1m is a red probe which can be used in combination with a large amount of existing green probes or blue light-sensitive tool proteins, and can be used for simultaneous two-color imaging or optical control/fluorescence imaging; (2) in cells cultured at 37 ℃, the fluorescence brightness of R-Flamp1m is more than 10 times of that of the existing red cAMP probes Pink Flamiddo and R-FlincA, and the red cAMP probes have a larger dynamic range (delta F/F0-5), namely, the red cAMP probes have higher detection sensitivity.
Drawings
The invention is further described below with reference to the accompanying drawings.
FIG. 1 is a schematic structural design diagram of R-Flamp1 m.
FIG. 2 fluorescence spectra and affinity of purified R-Flamp1m probe.
FIG. 3 comparison of the intensity of Pink-Flamiddo, R-FlincA and R-Flamp1m in Hek293T cells at the same transfection concentration and under the same imaging conditions.
FIG. 4 response of different probes in HEK293T cells under single photon excitation.
FIG. 5 response of R-Flamp1m probe in HEK293T cells under two-photon excitation.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The following describes the implementation of the present invention in detail with reference to specific embodiments.
The same or similar reference numerals in the drawings of the present embodiment correspond to the same or similar components; in the description of the present invention, it should be understood that if there is an orientation or positional relationship indicated by the terms "upper", "lower", "left", "right", etc. based on the orientation or positional relationship shown in the drawings, it is only for convenience of describing the present invention and simplifying the description, but it is not intended to indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes and are not to be construed as limiting the present patent, and the specific meaning of the terms may be understood by those skilled in the art according to specific circumstances.
Referring to fig. 1-5, preferred embodiments of the present invention are shown.
The present invention will be described in detail with reference to the following embodiments.
The cAMP fluorescent probe R-Flamp1m disclosed by the invention and the application thereof as a red fluorescent probe in living cells and animal living bodies can be realized by appropriately modifying process parameters by taking the contents in the text for reference. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be included in the invention. While the method and application of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the method and application described herein may be made and equivalents employed without departing from the spirit and scope of the invention.
The term "fluorescent probe" used in the present invention refers to a polypeptide sensitive to cAMP in the environment, which is fused with a fluorescent protein, specifically mEpac1 protein, wherein the conformational change of the fluorescent protein caused by the binding of cAMP binding domain specific to mEpac1 with cAMP causes the change of the conformation of the fluorescent protein, thereby causing the change of the generated fluorescence, and the fluorescence of the fluorescent protein measured at different cAMP concentrations is used to draw a standard curve, thereby detecting and analyzing the existence level of cAMP in cells.
The invention provides a cAMP fluorescent probe R-flash 1m, which comprises: protein domains sensitive to cAMP and red fluorescent protein; the amino acid sequence of R-Flamp1m is shown in SEQ ID NO. 1, the structural design schematic diagram of R-Flamp1m is shown in FIG. 1, wherein the amino acid sequence of 1-37 is His tag (His tag) part, 38-188 is the amino terminal part of fluorescent protein mApplel, 189-191 is connecting peptide 1, 192-340 is sensing module part mEpac1(205-353), 341-344 is connecting peptide 2, 345-428 is the carboxyl terminal part of red fluorescent protein mApplel.
The detection method of the cAMP fluorescent probe comprises the following steps:
(1) mEpac1 capable of binding cAMP is taken as a sensing structural domain, is connected with mApplel fluorescent protein in a mode shown in figure 1, and is subjected to mutation by a plurality of amino acids to obtain the R-Flamp1m probe.
(2) Expressing the R-Flamp1m probe in bacteria, culturing at room temperature for 3 days to collect the cells, sonicating in HEPES buffer (containing 150 mM KCl and 50 mM HEPES) at pH =7.2, purifying the probe with HisPur Cobalt Resin (available from Pierce), dissolving the probe in HEPES buffer at pH =7.2 through Econo-Pac 10DG desalting column (available from Bio-Rad, USA), and determining the probe concentration with BCA kit (available from Thermo scientific, USA); taking 2 μ M probe solution, detecting the response of probe to saturated concentration cAMP (500 μ M) by using multifunctional microplate reader Infinite M1000 PRO, and observing the signal increase 19 times, and the specific result is shown in FIG. 2, wherein (A) R-Flamp1M probe purified from bacteria is diluted in HEPES solution with pH 7.2 (final concentration is 2 μ M), and then saturated concentration cAMP (500 μ M) is added; the fluorescence excitation and emission spectra are shown as the figure, the dotted line is the excitation spectrum, and the solid line is the emission spectrum; (B) the fluorescence change of 2. mu.M purified probe mixed with different concentrations of cAMP or cGMP is shown in the graph, and it can be seen from the graph that R-Flamp1M has an affinity of about 2. mu.M for cAMP and an affinity of about 35. mu.M for cGMP. However, the affinity of Pink Flamindo for cAMP/cGMP was 3.2. mu.M/22. mu.M, respectively. Therefore, the fluorescent probe disclosed by the invention has improved cAMP affinity and is more suitable for detecting low-concentration cAMP; on the other hand, the affinity for cGMP is reduced, and the specificity of the fluorescent probe is better.
(3) The R-Flamp1m HEK293T cells need to be cultured in a culture dish with a glass bottom, the culture medium is DMEM containing 10% fetal calf serum and 1% penicillin-streptomycin, the culture temperature is 37 ℃, and the content of CO2 is 5%. At a cell density of about 60%, plasmids of the same mass of the red probes Pink-Flamdo, R-FlincA and R-Flamp1m were transfected with the Lipofectamine 2000 kit. After about 24 hours, under the same imaging conditions (the excitation wavelength, the emission wavelength, the excitation light intensity, the imaging time and the like are the same), the brightness of the Pink-Flamdo, the R-FlincA and the R-Flamp1m in Hek293T cells is shown in the specific result of FIG. 3, wherein (A) is the cell imaging graph of R-Flamp1 m; (B) is a cell image of Pink-Flamindo; (C) is a cytogram of R-FlincA; (D) normalized bar graph of mean value of cell brightness in A/B/C plot.
(4) The R-Flamp1m HEK293T cells need to be cultured in a culture dish with a glass bottom, the culture medium is DMEM containing 10% fetal calf serum and 1% penicillin-streptomycin, the culture temperature is 37 ℃, and the content of CO2 is 5%. At a cell density of about 60%, cAMPr, Flamido 2, G-Flamp1, Pink-Flamido, R-FlancA and R-Flamp1m were transfected with Lipofectamine 2000 kit. After overnight culture, starving the cells for 2 to 4 hours with a serum-and phenol red-free medium (purchased from GIBCO), changing the medium to a colorless and transparent live cell imaging buffer, and performing imaging analysis by using an IX83 single photon fluorescence microscope (R-flash 1m excitation wavelength of about 560nm and maximum emission wavelength of about 600 nm) or a commercial two-photon microscope (R-flash 1m excitation wavelength of about 1000 nm and maximum emission wavelength of about 600 nm) which is self-constructed in the laboratory. Forskolin (from Biyunnan Biotech) was added at 15 s/scan to the tenth frame at a final concentration of 60. mu.M (e.g.: 500. mu.l live cell imaging buffer in a petri dish, 4. mu.l of 12 mM stock solution was dissolved in 300. mu.l live cell imaging buffer, and 300. mu.l of the diluted Forskolin solution was dropped into the cell dish being imaged at the tenth frame). Changes in fluorescence intensity of cAMPr, Flamido 2, G-Flamp1, Pink-Flamido, R-FlancA and R-Flamp1M in the cells after Forskolin stimulation, as shown in FIG. 4, wherein (A) and (B) are plasmids transfected by Lipofectamine into HEK293T cells containing cAMPr, Flamido 2, Pink-Flamido, R-FlancA probes, after overnight culture, after starvation for 4 hours with DMEM cell culture medium without phenol red and serum, the fluorescence intensity changes after stimulation with 60 μ M Forskolin; (C) is the response of R-flash 1m and the negative control probe R-flash 1m R279E; the excitation wavelength is 560nm +/-20 nm; the curve data represents: mean ± standard deviation, scale: 100 μm.
Lipofectamine is used for transfecting plasmids containing R-Flamp1M probes of HEK cells, after overnight culture, after starvation for 4 hours by DMEM cell culture solution without phenol red and serum, under the condition that the two-photon excitation wavelength is 1000 nm, the fluorescence brightness change is caused after 60 mu M Forskolin stimulation, and the result is shown in figure 5, and curve data show that: mean ± standard deviation, scale: 100 μm.
Thus, the fluorescence imaging procedure for changes in cAMP concentration in mammalian cells is completed.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> Shenzhen advanced technology research institute of Chinese academy of sciences
<120> high-performance red cAMP fluorescent probe and application thereof
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Claims (10)
1. A cAMP fluorescent probe R-flash 1m is characterized in that the fluorescent probe R-flash 1m has an amino acid sequence SEQ ID NO of 1;
the amino acid sequence can also be an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO. 1, and the amino acid sequence has the same or similar function with the amino acid sequence; the amino acid sequence is an amino acid sequence with at least 80 percent of homology.
2. The cAMP fluorescent probe R-flash 1m according to claim 1, wherein the fluorescent probe R-flash 1m comprises His, mApple-N, linker peptide 1, mEpacl, linker peptide 2 and mApple-C.
3. The cAMP fluorescent probe R-Flamp1m according to claim 2, wherein His is His tag (Histag) and its amino acid sequence is SEQ ID NO 2;
the amino acid sequence can also be an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO. 2, and the amino acid sequence has the same or similar function with the amino acid sequence; the amino acid sequence is an amino acid sequence with at least 80 percent of homology.
4. The cAMP fluorescent probe R-flash 1m according to claim 2, wherein the mApple-N amino acid sequence is SEQ ID NO 3;
3 is obtained by substituting, deleting or adding one or more amino acids, and has the same or similar function with the amino acid sequence; the amino acid sequence is an amino acid sequence with at least 80 percent of homology.
5. The cAMP fluorescent probe R-Flamp1m according to claim 2, wherein the amino acid sequence of the linker peptide 1 (linker 1) is SEQ ID NO 4;
the amino acid sequence can also be an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO. 4, and the amino acid sequence has the same or similar function with the amino acid sequence; the amino acid sequence is an amino acid sequence with at least 80 percent of homology.
6. The cAMP fluorescent probe R-Flamp1m according to claim 2, wherein the mEpacl amino acid sequence is SEQ ID NO 5;
the amino acid sequence can also be an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO. 5, and the amino acid sequence has the same or similar function with the amino acid sequence; the amino acid sequence is an amino acid sequence with at least 80 percent of homology.
7. The cAMP fluorescent probe R-Flamp1m according to claim 2, wherein the amino acid sequence of the linker2 (i.e. linker 2) is SEQ ID NO 6;
the amino acid sequence can also be an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO. 6, and the amino acid sequence has the same or similar function with the amino acid sequence; the amino acid sequence is an amino acid sequence with at least 80 percent of homology.
8. The cAMP fluorescent probe R-flash 1m according to claim 2, wherein the mApple-C amino acid sequence is SEQ ID NO. 7;
the amino acid sequence can also be an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO. 7, and the amino acid sequence has the same or similar function with the amino acid sequence; the amino acid sequence is an amino acid sequence with at least 80 percent of homology.
9. Use of the cAMP fluorescent probe R-Flamp1m according to claim 2 in living cells and animal living bodies, in particular for imaging detection methods, especially two-photon imaging methods.
10. The use of the cAMP fluorescent probe R-Flamp1m according to claim 1, wherein the cAMP fluorescent probe is used for cAMP signal detection of a living body, for cAMP concentration detection in HEK293T cells or for cAMP concentration detection in a solution.
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