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 PDF

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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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camp
cnbd
probe
mlotik1
fluorescent
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PCT/CN2019/124070
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English (en)
Chinese (zh)
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储军
王亮
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中国科学院深圳先进技术研究院
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Priority to PCT/CN2019/124070 priority Critical patent/WO2021114037A1/fr
Publication of WO2021114037A1 publication Critical patent/WO2021114037A1/fr

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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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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
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  • Medicinal Chemistry (AREA)
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  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une sonde fluorescente de monophosphate d'adénosine cyclique basée sur une variation de largeur de luminosité fluorescente, ayant la structure telle que représentée dans la formule I : MlotiK1 CNBD-N-lieur1-cpEGFP-lieur2-MlotiK1 CNBD-C, dans laquelle MlotiK1 CNBD-N représente une extrémité N-terminale de MlotiK1 CNBD, et comprend une séquence d'acides aminés telle que représentée dans la SEQ ID NO : 3 ; cpEGFP comprend une séquence d'acides aminés telle que représentée dans la SEQ ID NO : 4 ; MlotiK1 CNBD-C représente une extrémité C-terminale de MlotiK1 CNBD, et comprend une séquence d'acides aminés telle que représentée dans la SEQ ID NO : 5 ; lieur1 représente un WG, et lieur2 représente un RV.
PCT/CN2019/124070 2019-12-09 2019-12-09 Sonde fluorescente de monophosphate d'adénosine cyclique basée sur une variation de largeur de luminosité fluorescente WO2021114037A1 (fr)

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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)

* Cited by examiner, † Cited by third party
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
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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