WO2021114037A1 - Cyclic adenosine monophosphate fluorescent probe based on fluorescent brightness width variation - Google Patents

Abstract

Disclosed is a cyclic adenosine monophosphate fluorescent probe based on fluorescent brightness width variation, having the structure as shown in formula I: MlotiK1 CNBD-N-linker1-cpEGFP-linker2-MlotiK1 CNBD-C, wherein MlotiK1 CNBD-N is an N-terminal of MlotiK1 CNBD, and has an amino acid sequence as shown in SEQ ID NO: 3; cpEGFP has an amino acid sequence as shown in SEQ ID NO: 4; MlotiK1 CNBD-C is a C-terminal of MlotiK1 CNBD, and has an amino acid sequence as shown in SEQ ID NO: 5; linker1 is WG, and linker2 is RV.

Description

Cyclic Adenosine Phosphate Fluorescent Probe with Wide Variation of Fluorescence Brightness Technical field

The invention belongs to the field of biological detection, and particularly relates to a cAMP fluorescent probe and its application.

Background technique

Cyclic Adenosine Phosphate (cAMP) 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. . 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]. In practical applications, dynamic range (variation amplitude of fluorescence brightness, ΔF/F ₀ ) is a very important parameter, which is directly related to detection sensitivity. In cells cultured at a physiological temperature of 37°C, the dynamic range of the above-mentioned probes is relatively small. In summary, 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.

references:

1.Odaka H, Arai S, Inoue T, Kitaguchi T (2014) Genetically-encoded yellow fluorescent cAMP indicator with an expanded dynamic range for dual-color imaging.PLoS One 9:e100252.

2.Tewson PH, Martinka S, Shaner NC, Hughes TE, Quinn AM (2016) New DAG and cAMP Sensors Optimized for Live-Cell Assays in Automated Laboratories. J Biomol Screen 21:298-305.

3. Hackley CR, Mazzoni EO, Blau J (2018) cAMPr: A single-wavelength fluorescent sensor for cyclic AMP. Sci Signal 11.

4. Harada K, Ito M, Wang X, Tanaka M, Wongso D, et al. (2017) Red fluorescent protein-based cAMP indicator applicable to optogenetics and in vivo imaging. Sci Rep 7: 7351.

5.https://montanamolecular.com/live-cell-camp-assay-caddis/red-caddis-camp-protocol/

6.Ohta Y, Furuta T, Nagai T, Horikawa K (2018) Red fluorescent cAMP indicator with increased affinity and expanded dynamic range. Sci Rep 8: 1866.

7.CN109627344A

Summary of the invention

In order to solve the problem of the small change in the fluorescence brightness of the cAMP probe, 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.

Compared with the existing fluorescent probes, the probe created by the present invention has a larger dynamic range (ΔF/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-linker1-cpEGFP-linker2-MlotiK1 CNBD-C Formula I

Wherein, 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.

In the technical solution of the present invention, 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.

The present invention will be described in detail below with reference to the accompanying drawings.

(1) First construct mICNBD-N-linker1-cpEGFP-linker2-mICNBD-C (Cyclic Nucleotide-binding domain, CNBD, cyclic nucleotide binding domain; mICNBD-N, the N-terminus of mICNBD; mICNBD-C, mICNBD C-terminus; cpEGFP, circularly rearranged green fluorescent protein; linker, connecting peptide). Screening linker1 and linker2 to get #252 probe, its linker1 and linker2 are WG and RV respectively (Figure 1). The amino acid sequence of #252 is also given (Figure 2).

(2) Mutation of several amino acids of #252, to obtain the G-Flamp1 probe, the sequence is shown in SEQ ID No.2.

(3) The G-Flamp1 probe was expressed in bacteria, cultured at room temperature for 2 days to collect the bacteria, and sonicated in HEPES buffer (containing 150mM KCl and 50mM HEPES) at pH=7.3, using HisPur Cobalt Resin (purchased from Pierce) Company) Purify the probe, and dissolve the probe in HEPES buffer pH=7.3 through Econo-Pac 10DG desalting column (purchased from Bio-Rad, USA), and use BCA kit (purchased from Thermo Scientific, USA) Determine the probe concentration. Take a 2mM probe solution, use the multifunctional microplate reader Infinite M1000PRO to detect the probe's response to the saturated concentration of cAMP (~500μM), and it can be seen that the signal increases by ~8 times (Figure 3).

(4) 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. Using the IX83 fluorescence microscope to detect the brightness of the probe, it can be seen that G-Flamp1 has the largest signal change amplitude (ΔF/F ₀ ) after the cells are stimulated by 60 μM 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.

Description of the drawings

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.

Figure 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 μM. 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 60μM Forskolin was used. Fluorescence brightness changes after stimulation. (B) Response of the R-FlincA probe.

Detailed ways

In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings, but they should not be understood as limiting the scope of implementation of the present invention.

Example 1

First construct 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

Mutation of several amino acids of probe #252, see SEQ ID No. 2 in bold, to obtain G-Flamp1 probe, and its sequence see SEQ ID No. 2.

G-Flamp1 probe: SEQ ID No. 2

Between WG and RV is the circularly rearranged green fluorescent protein sequence. Before WG is the sequence of mICNBD-N, after RV is the sequence of mICNBD-C. WG and RV are connectors

Example 2

The G-Flamp1 probe was expressed in bacteria, cultured at room temperature for 2 days to collect the bacteria, then sonicated in HEPES buffer (containing 150mM KCl and 50mM HEPES) at pH=7.3, and purified by HisPur Cobalt Resin (purchased from Pierce) The probe is dissolved in HEPES buffer at pH=7.3 through Econo-Pac 10DG desalting column (purchased from Bio-Rad, USA), and the probe is measured with BCA kit (purchased from Thermo Scientific, USA) concentration. Take a 2mM probe solution, use the multi-function microplate reader Infinite M1000PRO to detect the probe's response to the saturated concentration of cAMP (~500μM), and it can be seen that its fluorescence signal has increased by ~8 times (Figure 2).

Example 3

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. Using the IX83 fluorescence microscope built by our laboratory to detect the brightness of the probe, it can be seen that after the cells are stimulated by 60μM Forskolin (purchased from Biyuntian Biotechnology), G-Flamp1 has the largest signal change amplitude (ΔF/F ₀ ) reaching 2.2 Times, see attached figure 3. So far, the fluorescence imaging procedure of the changes in the concentration of cAMP in mammalian cells is completed.

Claims (8)

1. A cAMP fluorescent probe, the cAMP fluorescent probe has a structure as shown in formula I:

   MlotiK1 CNBD-N-linker1-cpEGFP-linker2-MlotiK1 CNBD-C Formula I

   Wherein, MlotiK1 CNBD-N is the N-terminal of MlotiK1 CNBD, and has an amino acid sequence as shown in SEQ ID NO: 3;

   MRGSHHHHHHHGMASMTGGQQMGRDLYDDDDKDPMGFYQEVRRGDFVRNWQLVAAVPLFQKLGPAVLVEIVRALRARTVPAGAVICRIGEPGDRMFFVVEGSVSVATN SEQID 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;

   NVYITADKQKNGIKANFKIRHNVEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPWPTLVTTLHMKTRAKN

   cpEGFP has an amino acid sequence as shown in SEQ ID NO: 5

   NPVELGPGAFFGEMALISGEPRVATVSAATTVSLLSLHSADFQMLCSSSPEIAEIFRKTALERRGAAASA SEQ ID No. 5;

   Linker1 is WG, and linker2 is RV.
2. A nucleic acid encoding the nucleotide sequence of the cAMP fluorescent probe according to claim 1.
3. An expression vector comprising a nucleic acid encoding the cAMP fluorescent probe according to claim 1.
4. A host cell transformed or transfected with the expression vector of claim 3.
5. The method for preparing the cAMP fluorescent probe according to claim 1, comprising: culturing the host cell according to claim 4 and inducing the expression of the cAMP fluorescent probe.
6. The use of the cAMP fluorescent probe of claim 1 in the detection of cAMP.
7. The use of the cAMP fluorescent probe of claim 1 in the detection of cAMP in living cells at 37°C.
8. A kit containing the cAMP fluorescent probe according to claim 1.

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