WO2021114037A1 - Sonde fluorescente de monophosphate d'adénosine cyclique basée sur une variation de largeur de luminosité fluorescente - Google Patents
Sonde fluorescente de monophosphate d'adénosine cyclique basée sur une variation de largeur de luminosité fluorescente Download PDFInfo
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- WO2021114037A1 WO2021114037A1 PCT/CN2019/124070 CN2019124070W WO2021114037A1 WO 2021114037 A1 WO2021114037 A1 WO 2021114037A1 CN 2019124070 W CN2019124070 W CN 2019124070W WO 2021114037 A1 WO2021114037 A1 WO 2021114037A1
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 26
- IVOMOUWHDPKRLL-KQYNXXCUSA-N Cyclic adenosine monophosphate Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-KQYNXXCUSA-N 0.000 title abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 11
- 210000004899 c-terminal region Anatomy 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 21
- 238000001514 detection method Methods 0.000 claims description 7
- 239000013604 expression vector Substances 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims description 2
- 150000007523 nucleic acids Chemical class 0.000 claims 2
- 102000039446 nucleic acids Human genes 0.000 claims 2
- 108020004707 nucleic acids Proteins 0.000 claims 2
- 239000000523 sample Substances 0.000 description 46
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 8
- 239000007995 HEPES buffer Substances 0.000 description 8
- OHCQJHSOBUTRHG-KGGHGJDLSA-N FORSKOLIN Chemical compound O=C([C@@]12O)C[C@](C)(C=C)O[C@]1(C)[C@@H](OC(=O)C)[C@@H](O)[C@@H]1[C@]2(C)[C@@H](O)CCC1(C)C OHCQJHSOBUTRHG-KGGHGJDLSA-N 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 125000004122 cyclic group Chemical group 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000000799 fluorescence microscopy Methods 0.000 description 4
- 108091006047 fluorescent proteins Proteins 0.000 description 4
- 102000034287 fluorescent proteins Human genes 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 108010075254 C-Peptide Proteins 0.000 description 3
- SUZLHDUTVMZSEV-UHFFFAOYSA-N Deoxycoleonol Natural products C12C(=O)CC(C)(C=C)OC2(C)C(OC(=O)C)C(O)C2C1(C)C(O)CCC2(C)C SUZLHDUTVMZSEV-UHFFFAOYSA-N 0.000 description 3
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 3
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 3
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- OHCQJHSOBUTRHG-UHFFFAOYSA-N colforsin Natural products OC12C(=O)CC(C)(C=C)OC1(C)C(OC(=O)C)C(O)C1C2(C)C(O)CCC1(C)C OHCQJHSOBUTRHG-UHFFFAOYSA-N 0.000 description 3
- 239000005090 green fluorescent protein Substances 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000012097 Lipofectamine 2000 Substances 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 229910017052 cobalt Inorganic materials 0.000 description 2
- 239000010941 cobalt Substances 0.000 description 2
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 238000000695 excitation spectrum Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- IVOMOUWHDPKRLL-UHFFFAOYSA-N UNPD107823 Natural products O1C2COP(O)(=O)OC2C(O)C1N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-UHFFFAOYSA-N 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- 229950006790 adenosine phosphate Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 229940095074 cyclic amp Drugs 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- 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
Definitions
- the invention belongs to the field of biological detection, and particularly relates to a cAMP fluorescent probe and its application.
- Cyclic Adenosine Phosphate is the downstream messenger molecule of the G-protein coupled receptor (GPCR) family, the largest drug target at present, and cAMP fluorescent probes and microscopic imaging research are the basic research of GPCR signaling pathway and an important research direction for drug development. .
- GPCR G-protein coupled receptor
- cAMP fluorescent probes are mainly divided into fluorescent resonance energy transfer probes based on fluorescent proteins and probes based on single fluorescent proteins. The latter has a larger dynamic range and is simpler to use. At present, cAMP probes based on a single fluorescent protein are divided into two categories: green and red.
- the former mainly includes Flamindo2[1], cADDis[2] and cAMPr[3], and the latter mainly includes Pink Flamindo[4] and Red cADDis[5] ] And R-FlincA[6].
- dynamic range (variation amplitude of fluorescence brightness, ⁇ F/F 0 ) is a very important parameter, which is directly related to detection sensitivity.
- the dynamic range of the above-mentioned probes is relatively small.
- improving the dynamic range of the probe in practical applications is of great significance for improving the detection sensitivity.
- Fluorescence imaging of cAMP in living cells refers to expressing cAMP fluorescent probes in the cells, and then using a fluorescence microscope to detect changes in the fluorescence intensity of the probes. Fluorescent probes are the key to cAMP fluorescence imaging analysis.
- the existing cAMP probes and dynamic ranges based on a single fluorescent protein are shown in the following table. #252 is the probe designed by the inventor in the 2018 patent application. It can be seen from this table that the cells were cultured at a physiological temperature of 37°C.
- the present invention optimizes the probe part of the cAMP imaging technology to obtain a green probe, which has the largest dynamic range in cells cultured at a physiological temperature of 37°C. In actual use, it can be expressed in mammalian cells by using a common fluorescence microscope to detect whether the cAMP concentration changes after the cells are stimulated.
- the probe created by the present invention has a larger dynamic range ( ⁇ F/F 0 ) in cells cultured at 37° C., that is, a higher detection sensitivity.
- One aspect of the present invention provides a cAMP fluorescent probe, which has a structure as shown in formula I:
- MlotiK1 CNBD-N is the N-terminal of MlotiK1 CNBD, and has an amino acid sequence as shown in SEQ ID NO: 3;
- the MlotiK1 CNBD-C is the C-terminal of MlotiK1 CNBD, and has an amino acid sequence as shown in SEQ ID NO: 4;
- cpEGFP has an amino acid sequence as shown in SEQ ID NO: 5
- Linker1 is WG, and linker2 is RV.
- the cAMP fluorescent probe has a sequence as SEQ ID No.2.
- Another aspect of the present invention provides nucleotides encoding cAMP fluorescent probes as described above.
- Another aspect of the present invention provides an expression vector, which includes a nucleotide encoding a cAMP fluorescent probe as described above.
- Yet another aspect of the present invention provides a host cell transformed or transfected with an expression vector as described above.
- Another aspect of the present invention provides a method for preparing the cAMP fluorescent probe as described above, which includes: culturing the host cell as described above and inducing the expression of the cAMP fluorescent probe.
- Another aspect of the present invention provides the use of the cAMP fluorescent probe described above in the detection of cAMP.
- Another aspect of the present invention provides the use of the cAMP fluorescent probe described above in detecting cAMP in living cells at 37°C.
- Another aspect of the present invention provides a kit containing the cAMP fluorescent probe described above.
- Probes such as cAMPr, Flamindo2, G-Flamp1, Pink-Flamindo, and R-FlincA were constructed on the eukaryotic expression vector (CAG promoter), and transfected with Lipofectamine 2000 kit and cultured in a glass bottom petri dish HEK293T cells (purchased from GE Healthcare Dharmacon) in the middle, after overnight culture, the cells were starved with serum-free and phenol red-free medium (purchased from GIBCO) for 6 hours.
- G-Flamp1 has the largest signal change amplitude ( ⁇ F/F 0 ) after the cells are stimulated by 60 ⁇ M Forskolin (purchased from Biyuntian Biotechnology Company), as shown in Figure 4. So far, the fluorescence imaging procedure of the changes in the concentration of cAMP in mammalian cells is completed.
- Figure 1 shows the design of the #252 probe and the G-Flamp1 probe of the present invention. Insert the mutant cpEGFP into the cAMP affinity domain.
- the connecting peptides on the left and right are WG and RV, respectively.
- the sequence of mlCNBD-N before WG and the sequence of mlCNBD-C after RV are used to obtain the G-Flamp1 probe probe.
- RSET is the leader sequence on the plasmid vector and can be used for protein purification.
- FIG. 2 shows the dynamic range measurement of the purified G-Flamp1 probe.
- the G-Flamp1 probe purified from bacteria was diluted in a HEPES solution of pH 7.3 to a final concentration of 2 ⁇ M.
- the graph shows the fluorescence excitation spectrum of the probe concentration in HEPES solution and saturated concentration cAMP.
- the bottom line is the spectrum without cAMP probe, and the top line is the spectrum with cAMP probe.
- the dotted line is the excitation spectrum, and the solid line is the emission spectrum.
- Figure 3 shows the brightness and response of the probe in HEK293T cells.
- A Transfect HEK cells with a plasmid containing cAMPr, Flamindo2, G-Flamp1, and Pink-Flamindo probes with Lipofectamine. After overnight culture, they were starved with DMEM cell culture medium without phenol red and serum for 6 hours, and then 60 ⁇ M Forskolin was used. Fluorescence brightness changes after stimulation.
- B Response of the R-FlincA probe.
- mICNBD-N-linker1-cpEGFP-linker2-mICNBD–C Cyclic nucleotide-binding domain, CNBD, cyclic nucleotide binding domain; mICNBD-N, the N-terminal of mICNBD; mICNBD-C, the C-terminal of mICNBD; cpEGFP, circularly rearranged green fluorescent protein; linker, connecting peptide).
- the linker1 and linker2 were screened and probe #252 was obtained, and the linker1 and linker2 were WG and RV respectively ( Figure 1).
- the amino acid sequence of #252 is shown in SEQ ID No. 1.
- Probe 252 SEQ ID No. 1
- G-Flamp1 probe SEQ ID No. 2
- WG and RV are connectors
- Probes such as cAMPr, Flamindo2, G-Flamp1, Pink-Flamindo and R-FlincA were constructed on the eukaryotic expression vector (CAG promoter), and the HEK293T cultured in a glass bottom culture dish was transfected by Lipofectamine 2000 kit The cells (purchased from GE Healthcare Dharmacon) were cultured overnight, and the cells were starved with serum-free and phenol red-free medium (purchased from GIBCO) for 6 hours.
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- Medicinal Chemistry (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
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PCT/CN2019/124070 WO2021114037A1 (fr) | 2019-12-09 | 2019-12-09 | Sonde fluorescente de monophosphate d'adénosine cyclique basée sur une variation de largeur de luminosité fluorescente |
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PCT/CN2019/124070 WO2021114037A1 (fr) | 2019-12-09 | 2019-12-09 | Sonde fluorescente de monophosphate d'adénosine cyclique basée sur une variation de largeur de luminosité fluorescente |
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105646716A (zh) * | 2014-11-14 | 2016-06-08 | 华东理工大学 | 一种基因编码的环腺苷酸荧光探针及其制备方法和应用 |
CN109627344A (zh) * | 2018-12-28 | 2019-04-16 | 深圳先进技术研究院 | cAMP荧光探针及其应用 |
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- 2019-12-09 WO PCT/CN2019/124070 patent/WO2021114037A1/fr active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN105646716A (zh) * | 2014-11-14 | 2016-06-08 | 华东理工大学 | 一种基因编码的环腺苷酸荧光探针及其制备方法和应用 |
CN109627344A (zh) * | 2018-12-28 | 2019-04-16 | 深圳先进技术研究院 | cAMP荧光探针及其应用 |
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