High-sensitivity low-cytotoxicity red calcium ion probe based on cpFP and application
Through the design of fluorescent protein, the red calcium ion probe sRCaMP1 has solved the problems of low brightness, insufficient sensitivity and high cytotoxicity of existing red calcium ion probes, and achieved the effects of large dynamic range, high sensitivity and low cytotoxicity. It is suitable for rapid imaging of neuronal activities and two-color collaborative imaging.
Patent Information
- Application Number
- CN202411939522.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-26
- Publication Date
- 2025-04-25
AI Technical Summary
The existing red calcium ion probes have low brightness, insufficient sensitivity, easy aggregation and high cytotoxicity in living organs, which limit their application in living cells and live imaging.
Through fluorescent protein engineering, a new red calcium ion probe sRCaMP1 was designed, and the mutation screened out probes with large dynamic range, high sensitivity, extremely fast response and low cytotoxicity.
sRCaMP1 achieves a larger dynamic range and high sensitivity, is suitable for high-sensitive rapid imaging of neuronal activity, and can be combined with signal molecular probes of other colors to achieve dual-color collaborative imaging of cell activity.
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Figure CN119874935A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a high-sensitivity, low-cytotoxicity red calcium ion probe based on cpFP and an application thereof. Background Art
[0002] Calcium ions (Ca 2 +) is an important second messenger in cells and is involved in regulating a variety of biological processes, including nerve signal transduction, muscle contraction, and cell cycle regulation. Therefore, the development of efficient and sensitive calcium ion probes is of great significance for studying calcium signal transduction in cells and tissues.
[0003] Genetically encoded neural activity probes based on fluorescent proteins combined with modern in vivo microscopy techniques have achieved non-invasive and high-throughput recording of specific populations of neurons, allowing high-resolution, high-throughput functional imaging. Among them, calcium ion probes are mainly divided into three categories: fluorescence resonance energy transfer (FRET) probes, bioluminescence resonance energy transfer (BRET) probes, and cyclized permuted fluorescent protein (cpFP) probes. FRET and BRET probes have the following three main disadvantages: 1) The distance between luminescent proteins changes slowly, resulting in a slow response speed (about 100ms), which cannot resolve high-frequency calcium ion changes; 2) It occupies two fluorescence channels and is difficult to use simultaneously with other spectral molecular probes; 3) The luminescence efficiency of BRET is low, making it difficult to use for fast optical imaging. The cpFP probe overcomes the above shortcomings by inducing rapid changes in the fluorescence of a single cpFP through conformational changes in the calcium ion sensing module, and can achieve millisecond-level rapid responses and optical imaging with a high signal-to-noise ratio.
[0004] The design principle of the cpFP calcium ion probe is to cyclize and rearrange the barrel structure of the fluorescent protein, open the position near the luminescent group in the middle of its outer side, and connect calmodulin (CaM) and calmodulin binding peptides (such as M13 and ckkap, etc.) respectively. When the calcium ion concentration is low, CaM and M13 / ckkap do not bind, the gap is large, resulting in darker fluorescence; when the calcium ion concentration increases, CaM and M13 / ckkap bind, the gap becomes smaller, and the fluorescence is significantly enhanced.
[0005] Although the cpFP probe, first proposed in 1999, appeared relatively late, its GCaMP series of calcium ion probes based on cyclized permuted green fluorescent protein (cpGFP) has made significant progress since 2010. With its ultra-high sensitivity and signal-to-noise ratio, the GCaMP probe has become the most widely used genetically encoded calcium ion probe. However, compared with the green probe, the development of the red cpFP calcium ion probe is relatively slow. Red probes generally have problems such as low brightness, insufficient sensitivity, easy aggregation and high cytotoxicity, which limits their practical application in living cells and in vivo imaging. These technical bottlenecks urgently need to be solved through design optimization. Summary of the invention
[0006] In order to solve the problems of low brightness of red calcium ion probes in the prior art, inability to show a good signal-to-noise ratio in living bodies, low sensitivity, and many easy aggregation in living cells and high cytotoxicity, the present invention aims to provide a red calcium ion probe based on cyclized permuted fluorescent protein (cpFP), which has a large dynamic range, high sensitivity and low cytotoxicity, can be used for long-term monitoring of neuronal activity at the cellular and subcellular scales, and can be used in combination with signal molecule probes of other colors (such as green) to achieve dual-color collaborative imaging of cell activity.
[0007] In one aspect, the present invention provides a red calcium ion probe based on cpFP, the amino acid sequence of the red calcium ion probe is shown in SEQ ID NO.1.
[0008] Another aspect of the present invention provides a polynucleotide sequence encoding the above-mentioned red calcium ion probe.
[0009] Another aspect of the present invention provides an expression vector, wherein the expression vector comprises the polynucleotide sequence encoding the red calcium ion probe.
[0010] Furthermore, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.
[0011] In another aspect, the present invention provides use of the red calcium ion probe, the polynucleotide sequence and the expression vector in preparing a reagent for detecting calcium concentration in a sample.
[0012] In another aspect, the present invention provides the use of the red calcium ion probe, the polynucleotide sequence and the expression vector in preparing a reagent for monitoring the dynamic changes of calcium signals in neurons or cells.
[0013] Furthermore, the cells are mammalian cells.
[0014] In yet another aspect, the present invention provides a kit, which comprises any one or more of the above-mentioned red calcium ion probe, the above-mentioned polynucleotide sequence and the above-mentioned expression vector.
[0015] Furthermore, the kit also contains signal molecule probes of colors other than red.
[0016] In another aspect, the present invention provides a method for rapid imaging of neuronal activity, the method comprising the following steps:
[0017] S1) transfecting the above expression vector into isolated neurons;
[0018] S2) Use imaging equipment to perform imaging analysis and observe rapid imaging of neuronal activity.
[0019] Furthermore, the imaging device is a fluorescence microscope or a confocal microscope.
[0020] In another aspect, the present invention provides a method for monitoring the change process of calcium signal, the method comprising the following steps:
[0021] S1) transfecting the above expression vector into living cells or mixing the above cpFP-based red calcium ion probe with a sample to be detected and incubating;
[0022] S2) Use imaging equipment to perform imaging analysis to observe the fluctuation of intracellular calcium ion concentration under specific stimulation or environmental conditions.
[0023] Furthermore, the calcium signal changes in milliseconds.
[0024] Beneficial Effects
[0025] The present invention provides a red calcium ion probe sRCaMP1, which has a larger dynamic range, which is more than three times that of K-GeCO1. sRCaMP1 is suitable for high-sensitivity rapid imaging of neuronal activity and other biological detection scenarios where calcium ions change rapidly on a millisecond scale. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 The protein crystal structure model of K-GeCO1. The red one is cpRFP, the cyan one is the calcium ion sensing module, the yellow one is the sensing module binding peptide, the black one is the connecting peptide, and the green one is the amino acid site screened by some libraries.
[0027] Figure 2 Comparison of the amino acid sequences of K-GeCO1 and sRCaMP1. The mutant amino acids are marked with different color backgrounds.
[0028] Figure 3 The brightness of sRCaMP1 changes with calcium ion concentration (Kd value detection).
[0029] Figure 4 The left figure shows the expression of sRCaMP1 probe in HEK293T cells and CHO cells, bar=10μm; the right figure shows the expression of sRCaMP1 probe in CHO cells, bar=5μm.
[0030] Figure 5 The brightness of sRCaMP1 in HEK293T and CHO cells is compared (normalized to the brightness of K-GeCO1).
[0031] Figure 6 The expression of sRCaMP1 in primary cultured neurons isolated from rat hippocampus.
[0032] Figure 7 The red arrows indicate the response of different red calcium ion probes to ionomycin stimulation in HEK293T cells. Each curve shows the change of probe brightness over time in a single cell. DETAILED DESCRIPTION
[0033] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention are described in detail below, but it should not be understood as limiting the applicable scope of the present invention.
[0034] Through fluorescent protein engineering, a new red calcium ion probe sRCaMP1 was screened and identified through mutation. sRCaMP1f has a large dynamic range, high sensitivity, extremely fast response and low cytotoxicity. It can be used for long-term monitoring of neuronal activity at the cellular and subcellular scales, and can be used in combination with signal molecule probes of other colors (such as green) to achieve dual-color collaborative imaging of cell activity.
[0035] The present invention specifically provides a red calcium ion probe based on cpFP, and the amino acid sequence of the red calcium ion probe is shown in SEQ ID NO.1.
[0036] Some specific embodiments of the present invention also provide a polynucleotide sequence encoding the above-mentioned red calcium ion probe, and an expression vector comprising the above-mentioned polynucleotide sequence.
[0037] In some embodiments, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.
[0038] Some specific embodiments of the present invention also provide the use of the above-mentioned red calcium ion probe, the above-mentioned polynucleotide sequence and the above-mentioned expression vector in preparing a reagent for detecting the calcium concentration in a sample.
[0039] Some specific embodiments of the present invention also provide the use of the above-mentioned red calcium ion probe, the above-mentioned polynucleotide sequence and the above-mentioned expression vector in the preparation of a reagent for monitoring the dynamic changes of calcium signals in neurons or cells.
[0040] In some embodiments, the cell is a mammalian cell.
[0041] In some specific embodiments of the present invention, a kit is also provided, which contains any one or more of the above-mentioned red calcium ion probe, the above-mentioned polynucleotide sequence and the above-mentioned expression vector.
[0042] Some specific embodiments of the present invention also provide a method for rapid imaging of neuronal activity, the method comprising the following steps:
[0043] S1) transfecting the above expression vector into isolated neurons;
[0044] S2) Use imaging equipment to perform imaging analysis and observe rapid imaging of neuronal activity.
[0045] The imaging device is a fluorescence microscope or a confocal microscope.
[0046] Some specific embodiments of the present invention also provide a method for monitoring the change process of calcium signal in living cells, the method comprising the following steps:
[0047] S1) transfecting the above expression vector into living cells;
[0048] S2) Use imaging equipment to perform imaging analysis to observe the fluctuation of intracellular calcium ion concentration under specific stimulation or environmental conditions.
[0049] Example 1 Screening for mutants of the red calcium ion probe K-GeCO1
[0050] The crystal structure model of the existing red calcium ion probe K-GeCO1 with low cytotoxicity was analyzed. The structure is shown in Figure 1 , where red is cpRFP; cyan is the calcium ion sensing module, yellow is the sensing module binding peptide; black is the connecting peptide. The inventors of the present invention rationally designed and site-directed mutated the key sites that affect the probe response and the amino acids that interact with it, and then expressed and screened the mutants on the constitutive expression vector pNCS, and the expression strain used was Stellar. To ensure the integrity of the library, 10 clones were set for each mutant. Finally, the mutants with higher expression brightness were screened out by visual distinction and the fluorescent properties of the mutants were detected through an orange acrylic filter using blue LED excitation light. The dynamic range of mutants expressed and detected in bacteria, the mutants with the largest dynamic range were selected to construct into mammalian cell expression plasmids, the brightness, cell aggregation, response speed and dynamic range were evaluated in cells, and the mutants with the best comprehensive performance were selected for the next round of library screening. The mutant with low cytotoxicity, rapid response and large dynamic range finally screened out was named sRCaMP1. The amino acid sequence comparison of K-GeCO1 and sRCaMP1 is shown in Figure 2 .
[0051] The amino acid sequence of sRCaMP1 is shown in SEQ ID NO.1;
[0052] SEQ ID NO.1:
[0053] MGSVKLIPSLTTVILVKSMLRKRSFGNPFKYNTETLYPADGGLEGRCDMALKLVGGGHLNCSLVTTYRSKKPATNLKMPGVYNVDHMLERIKEADDETYVELHEVAVARHIGVGGGGGTGGSVSELIKENMPTKLYME GTVNNHHFKCTHEGEGKPYEGTQTARIKVVEGGPLPFAFDILATSFMYGSRTFIKHPPDIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKVRGMNFPANGPVMQKKTLGWEASNGQLTEEQIAEF KEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGDGTFFDFPEFLTMMARKMSYRVTEEEIREAFRVFDKDGNGYIGAAELRHVMTDLGEKLTDEEVDEMIRVADIDGDGQVNYEEFVQMMTAK
[0054] sRCaMP1 mutated 10 sites based on K-GeCO1, namely A46R, E64V, R87M, Y110H, V111I, L113V, M133T, S150H, M163A, and G197D.
[0055] Example 2 Evaluation of sRCaMP1 dynamic range
[0056] Bacteria expressing sRCaMP1 were lysed using B-PER II (purchased from Pierce), and the lysate supernatant was used to detect the dynamic range of the probe in a buffer environment without calcium ions and in a buffer environment saturated with calcium ions (used to measure the difference in brightness changes in the absence of calcium ions and in the absence of calcium ions, ΔF / F0).
[0057] The results showed that the dynamic range of sRCaMP1 bacterial lysis supernatant was 26-fold.
[0058] Example 3 Evaluation of the extinction coefficient, quantum yield and molecular brightness of sRCaMP1
[0059] The bacteria expressing the probe were lysed using B-PER II (purchased from Pierce), and then the protein was purified using HisPurCobalt Resin (purchased from Pierce), followed by desalting through an Econo-Pac 10DG gravity flow column (purchased from Bio-Rad, USA). After completing the above protein purification steps, the extinction coefficient and quantum yield of sRCaMP1 at the peak were detected in a buffer environment without calcium ions and saturated with calcium ions.
[0060] The results showed that when calcium ions were saturated, the extinction coefficient was 62mM. -1 cm -1 , the quantum yield is 0.51 and the molecular brightness is 32; the quantum yield is 0.12 when there is no calcium ion.
[0061] Example 4 Evaluation of sRCaMP1 calcium ion affinity
[0062] Purified sRCaMP1 proteins were used to detect their affinity for calcium ions using Lambda35 UV / VIS and LS-55 fluorescence spectrometers (purchased from Perkin Elmer).
[0063] Results Figure 3 The results showed that sRCaMP1 has a high affinity for calcium ions, with a Kd value of 98nM.
[0064] Example 5 Expression and response of sRCaMP1 in cells
[0065] 1) The nucleotide sequence encoding sRCaMP1 was constructed into the eukaryotic expression vector pC DNA3.1-CAG, and the mammalian cell lines HEK293T and CHO were transfected using lipo2000 (Thermo Fisher), and imaging was performed after 48 hours of expression.
[0066] The experimental results are shown in Figure 4 The results showed that sRCaMP1 was uniformly expressed in both cell lines without aggregation in the cells.
[0067] 2) Using the brightness of mTurqueise2 as a reference, the brightness of the new probe sRCaMP1 and the parent probe K-GeCO1 were compared in different cell lines HEK293T and CHO.
[0068] The experimental results are shown in Figure 5 The results showed that sRCaMP1 was brighter than K-GeCO1 in different cells, with brightness increases of 12% (HEK293T) and 28% (CHO), respectively.
[0069] 3) sRCaMP1 was transfected into primary cultured rat hippocampal neurons.
[0070] The experimental results are shown in Figure 6 The results showed that sRCaMP1 was also uniformly expressed in isolated neurons in the hippocampus without aggregation, so sRCaMP1 can be used for rapid imaging of neuronal activity.
[0071] 4) sRCaMP1 and other red calcium ion probes were expressed in HEK293T cells and imaged after 24-48 hours.
[0072] Imaging procedure: Imaging over time, frequency of 1 Hz, ionomycin and CaCl2 were added after collecting 1 min baseline, and the total imaging time was 250 s.
[0073] The experimental results are shown in Figure 7 The results showed that the dynamic ranges of the other three probes in response to ionomycin and CaCl2 were between 2-4 times, while the dynamic range of sRCaMP1 response was 5-12 times.
Claims
1. A red calcium ion probe based on cpFP, characterized in that: The amino acid sequence of the red calcium ion probe is shown in SEQ ID NO.
1.
2. A polynucleotide sequence encoding the red calcium ion probe according to claim 1.
3. An expression vector, characterized in that: The expression vector comprises the polynucleotide sequence according to claim 2.
4. The expression vector according to claim 3, characterized in that The expression vector is a eukaryotic expression vector or a prokaryotic expression vector.
5. Use of the red calcium ion probe according to claim 1, the polynucleotide sequence according to claim 2, and the expression vector according to claim 3 or 4 in preparing a detection reagent for detecting calcium concentration in a sample.
6. Use of the red calcium ion probe according to claim 1, the polynucleotide sequence according to claim 2, and the expression vector according to claim 3 or 4 in preparing a detection reagent for monitoring the dynamic changes of calcium signals in neurons or cells.
7. The use according to claim 6, characterized in that The cells are mammalian cells.
8. A kit, comprising any one or more of the red calcium ion probe according to claim 1, the polynucleotide sequence according to claim 2, and the expression vector according to claim 3 or 4; Preferably, the kit further comprises signal molecule probes of colors other than red.
9. A method for rapid imaging of neuronal activity, the method comprising the following steps: S1) transfecting the above expression vector into isolated neurons; S2) Use imaging equipment to perform imaging analysis and observe rapid imaging of neuronal activity; Preferably, the imaging device is a fluorescence microscope or a confocal microscope.
10. A method for monitoring calcium signal changes, characterized in that: The method comprises the following steps: S1) transfecting the above expression vector into living cells or mixing the above cpFP-based red calcium ion probe with a sample to be detected and incubating; S2) using imaging equipment to perform imaging analysis to observe the fluctuation of intracellular calcium ion concentration under specific stimulation or environmental conditions; Preferably, the calcium signal changes in milliseconds.