ATP fluorescent probe and application thereof

Abstract

This invention relates to an ATP fluorescent probe and its applications. The invention provides an ATP-binding protein whose sequence, from the N-terminus to the C-terminus, includes: amino acid sequences from positions 1 to 107 of the ATP-binding domain, an N-terminal linker sequence, a dye-binding domain sequence, a C-terminal linker sequence, and amino acid sequences from positions 108 to 131 of the ATP-binding domain. The ATP-binding protein of this invention, when labeled with a fluorescent dye, forms a far-red fluorescent ATP probe, which specifically responds to dynamic changes in ATP levels, enabling precise and sensitive measurement of ATP content and localization of ATP. The far-red fluorescent ATP probe of this invention can be used in conjunction with green and red fluorescent probes, offering the advantage of simultaneous detection of multiple probe molecules when investigating issues related to energy metabolism reprogramming. Furthermore, the far-red fluorescent ATP probe of this invention can also be used in conjunction with an ADP probe in the same cell, enabling simultaneous detection of dynamic changes in ATP and ADP molecules.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe technology, specifically to an ATP fluorescent probe and its applications. Background Technology

[0002] Adenosine triphosphate (ATP) is a nucleotide that functions in cells to store and transfer chemical energy, and plays a crucial role in nucleic acid synthesis. Detecting ATP helps in understanding metabolic processes in life, and developing novel ATP probes is of great significance and has broad application prospects.

[0003] Currently, ATP fluorescent probes used for ATP detection mainly fall into two categories: one is gene-encoded ATP fluorescent probes based on the fluorescence resonance energy transfer (FRET) principle and consisting of two fluorescent proteins; the other is probes based on a single fluorescent protein. The basic structure of FRET-based ATP fluorescent probes includes an ATP-binding module and a fluorescent reporter module. Their working principle is that the fluorescent reporter module detects ATP binding in response to conformational changes in the ATP-binding module, thereby reporting the ATP concentration level. The fluorescent protein in the reporter module is gene-encoded, enabling cell type or subcellular specific labeling. Unlike the FRET principle (which relies on two fluorescent proteins in the fluorescent module), single-fluorescent protein probes require only one fluorescent protein. In 2018, Tetsuya Kitaguchi's research group first reported the MaLion series of ATP probes based on a single fluorescent protein (Arai et al., 2018). In 2019, Loren Looger and Baljit Khakh's research group reported iATPSnFR, an ATP probe based on a single green fluorescent protein with superior detection performance. This probe was subsequently optimized over five years by Timothy Ryan's research group (optimizing the amino acid residues connecting the fluorescent module and the ATP-binding module, and introducing mutations in the binding module to adjust affinity), resulting in a version, iATPSnFR2, that performs better in cells (Marvin et al., 2024). Furthermore, Li Yulong's research group developed a high-performance GRAB-ATP probe for in vivo detection of extracellular ATP, utilizing the principle that conformational changes in the hP2Y1 receptor during ATP binding can modulate the optical signal of a single green fluorescent protein (Wu et al., 2022). Currently, high-performance gene-encoded ATP probes such as iATPSnFR2 and GRAB-ATP are mainly in the green wavelength range, with only the MaLionR series of ATP probes operating in the red wavelength range. In addition, existing high-performance probes based on single fluorescent proteins are susceptible to interference from pH and temperature changes (Wu et al., 2022, Marvin et al., 2024). When detecting ATP molecules, changes in intracellular pH (such as cytoplasmic acidification induced by neuronal electrical activity or the accumulation of acidic metabolites) (Rossano et al., 2013, Chesler, 2003) or ambient temperature (such as fluctuations in body temperature) (Miyazaki et al., 2002, Gaskill et al., 2024) can interfere with the signal, leading to decreased detection precision and accuracy. Currently, the spectra of ATP fluorescent probes are mainly limited to the green and red wavelength ranges.

[0004] In the field of fluorescent probes, in addition to gene-encoded probes based on fluorescent proteins, there are also chemically encoded probes based on fluorescent dyes. Unlike fluorescent proteins, fluorescent dyes themselves do not possess gene-encoded properties, making it difficult to achieve cell type or subcellular specific labeling. However, due to their good membrane permeability, this problem can be effectively solved by combining them with gene-encoded self-labeling protein tags. Self-labeling proteins are a class of protein tags that, after being fused with and expressed with a target protein, can undergo a specific covalent reaction with a specific fluorescent dye. They are widely used in live-cell imaging, protein localization tracking, and interaction studies (Wilhelm et al., 2021), and mainly include HaloTag, SNAP-Tag, and CLIP-Tag (Gautier et al., 2008, Los et al., 2008, Wilhelm et al., 2021, Kühn et al., 2025). In 2021, Luke Lavis and Eric Schreiter's research group combined the cyclically permuted HaloTag (cpHaloTag) with Ca... 2+ By combining protein fusion, far-red Ca2+ was successfully developed. 2+ The fluorescent probe Halo-GCaMP (Deo et al., 2021). Further research is needed targeting K... + Probes for ATP (Cheng et al., 2024) and dopamine (HaloDA1.0, Zheng et al., 2025) have been developed. However, no far-red ATP fluorescent probes based on cpHaloTag have been reported to date.

[0005] cpGFP is a circularly arranged green fluorescent protein, including cpsfGFP and cpEGFP; cpsfGFP is a circularly arranged superfolded green fluorescent protein, which introduces superfolding properties on the basis of cpGFP; cpEGFP is a circularly arranged enhanced green fluorescent protein, which is modified from enhanced green fluorescent protein (EGFP) by changing its circular arrangement.

[0006] F0F1 of bacteria _The ε subunit of ATP synthase (hereinafter referred to as the ε subunit) or its variants possess stable and hydrolysis-independent ATP-binding capacity, high selectivity for ATP, and significant conformational changes triggered by ATP binding. These characteristics make it an ideal choice for ATP probe binding modules (Imamura et al., 2009, Lobas et al., 2019b, Arai et al., 2018, Hellweg et al., 2023). However, whether the ε subunit can be used for the development of chemically encoded probes remains unknown.

[0007] HaloTag is a protein capable of irreversible, covalent binding to specific chemical groups (such as chloroalkanes). HaloTag has undergone long-term optimization, including improvements to its stability and labeling efficiency (e.g., HaloTag7, HaloTag9, HaloTag10, and HaloTag11) (Frei et al., 2022). The research group of Luke Lavis and Eric Schreiter cyclically rearranged HaloTag to obtain cpHaloTag (circularly permuted HaloTag), and then combined it with Ca... 2+ By combining protein fusion, far-red Ca2+ was successfully developed. 2+ Fluorescent probes (Deo et al., 2021). No cpHaloTag-based ATP fluorescent probes have been reported to date. However, it remains unclear whether conformational changes in the ε subunit can affect the conformational changes of cpHaloTag and whether cpHaloTag binding to fluorescent dyes elicits a fluorescent response. Summary of the Invention

[0008] This invention provides an ATP probe, which is itself an ATP-binding protein comprising an ATP-binding domain and a dye-binding domain. The basic principle of this probe is as follows (…). Figure 1 A) As follows: During use, a far-red fluorescent dye is added to fluorescently label the ATP-binding protein. The far-red fluorescent dye covalently binds to the dye-binding domain of the ATP-binding protein, or the ATP-binding protein is directly labeled with the far-red fluorescent dye. In this case, the fluorescent dye is in a dark state. When the ATP concentration increases, ATP binds to the ATP-binding domain of the ATP-binding protein, triggering a conformational change in the ATP-binding domain, which is then transferred to the dye-binding domain, causing it to also undergo a conformational change. This results in the fluorescent dye becoming bright. The amount of ATP can be determined based on the generated fluorescence intensity. Figure 1 ).

[0009] This invention utilizes the ε subunit of ATP synthase as the ATP-binding domain and cpHaloTag as the dye-binding domain to develop and optimize a novel far-red chemogenetic ATP probe. cpHaloTag is a circularly arranged HaloTag, the sequence of which can be found in the PDB protein database (www.rcsb.org), PDB ID: 6U2M (Deo et al., 2021).

[0010] In some embodiments, the ε subunit of the ATP synthase is F0F1 from the thermophilic Bacillus PS3 strain. _ The ε subunit of ATP synthase (abbreviated as PS3 ε subunit). The sequence of PS3 ε subunit can be found in the PDB protein database, PDB ID: 2E5Y (Lobas et al., 2019a, Marvin et al., 2024, Yagi et al., 2007).

[0011] The specific construction strategy of the ATP probe of the present invention is as follows: Figure 1 As shown in B, cpHaloTag is inserted into the 107th amino acid residue of the ATP-binding domain (PS3 ε subunit) via linker peptides (N-Linker and C-Linker).

[0012] The conformation of the HL-ATP probe before and after ATP binding was predicted using Alphafold3 software, such as... Figure 1 As shown in Figure C, the spatial conformation of the ATP-binding protein undergoes significant changes before and after binding to ATP.

[0013] In the ATP probe of this invention, the ATP-binding domain and the dye-binding domain (cpHaloTag) are linked by linker peptides (including N-Linker and C-Linker). Figure 1 B).

[0014] This invention provides the following technical solutions:

[0015] 1. An ATP-binding protein, the sequence of which, from the N-terminus to the C-terminus, comprises: amino acid sequence 1 to 107 of the ATP-binding domain, an N-terminal linker sequence, a dye-binding domain sequence, a C-terminal linker sequence, and amino acid sequence 108 to 131 of the ATP-binding domain;

[0016] The ATP-binding domain sequence is as follows:

[0017] (1) The sequence shown in SEQ ID NO:2;

[0018] (2) The sequence shown in SEQ ID NO:4;

[0019] (3) The sequence shown in SEQ ID NO:6;

[0020] (4) The sequence shown in SEQ ID NO:8; or

[0021] (5) The sequence shown in SEQ ID NO:10;

[0022] The dye-binding domain sequence is as follows:

[0023] (A) The sequence shown in SEQ ID NO:11; or

[0024] (B) The sequence shown in SEQ ID NO:12;

[0025] The sequences of the N-terminal connector and the C-terminal connector are as follows:

[0026] (a) The sequences shown in SEQ ID NO:27 and SEQ ID NO:28;

[0027] (b) The sequences shown in SEQ ID NO:30 and SEQ ID NO:31;

[0028] (c) The sequences shown in SEQ ID NO:33 and SEQ ID NO:34; or

[0029] (d) The sequences shown in SEQ ID NO:36 and SEQ ID NO:37.

[0030] 2. The ATP-binding protein described in Project 1, wherein:

[0031] (I) Having the sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35 or SEQ ID NO:38;

[0032] (II) A sequence having mutations at one, two, three, four, five or more sites in the sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35 or SEQ ID NO:38 and having the ability to bind ATP, said mutations including amino acid modifications, substitutions or deletions; or

[0033] (III) A sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with (I) or (II) and has the ability to bind to ATP.

[0034] 3. The ATP-binding protein described in Project 1 or 2, further comprising a localization sequence, a purification tag, or an immunoblot tag.

[0035] In some embodiments, the localization sequence is a sequence that guides the ATP-binding protein to a specific site.

[0036] 4. The ATP-binding protein described in Project 3, wherein the localization sequence is a nuclear export signal sequence, a mitochondrial localization sequence, an endoplasmic reticulum localization sequence, a neuronal localization sequence, an astrocyte localization sequence, a calreticulin signal sequence, and / or an endoplasmic reticulum residency sequence;

[0037] Preferably, the nuclear output signal sequence is shown in SEQ ID NO:15, the mitochondrial localization sequence is shown in SEQ ID NO:16, the calreticulin signal sequence is shown in SEQ ID NO:21, the endoplasmic reticulum resident sequence is shown in SEQ ID NO:22, the neuron localization sequence is shown in SEQ ID NO:39, and the astrocyte localization sequence is shown in SEQ ID NO:40.

[0038] 5. The ATP-binding protein described in Project 3, wherein the purification tag includes a histidine tag, a Strep tag, or a FLAG tag.

[0039] In some embodiments, the histidine tag is 3×His, 4×His, 5×His, 6×His, 7×His, 8×His, 9×His, or 10×His, which are composed of 3, 4, 5, 6, 7, 8, 9, or 10 histidine (H) residues, respectively.

[0040] In some embodiments, the sequence of the Strep tag is preferably WSHPQFEK (SEQ ID NO.44).

[0041] In some embodiments, the preferred sequence of the FLAG tag is DYKDDDDDK (SEQ ID NO.45).

[0042] 6. The ATP-binding protein described in Project 3, wherein the immunoblot tag includes an HA tag, a Myc tag, or a V5 tag.

[0043] In some implementations, the sequence of the HA tag is preferably YPYDVPDYA (SEQ ID NO.46).

[0044] In some implementations, the sequence of the Myc tag is preferably EQKLISEEDL (SEQ ID NO.47).

[0045] In some implementations, the sequence of the V5 tag is preferably GKPIPNPLLGLDST (SEQ ID NO.48).

[0046] In some embodiments, the localization sequence, purification tag, or immunoblotting tag is located at the C-terminus of the amino acid sequence of the ADP-binding protein.

[0047] In some implementations, a linker may be present between the ADP-binding protein and the localization sequence, purification tag, or immunoblotting tag.

[0048] In some embodiments, the connector is preferably GGGGS (SEQ ID NO:49) or QSGS (SEQ ID NO:50).

[0049] 7. A far-red fluorescent ATP probe comprising an ATP-binding protein labeled with any one of items 1-6 using a far-red fluorescent dye.

[0050] 8. The far-red fluorescent ATP probe described in Project 7, wherein the far-red fluorescent dye is a dye with a chloroalkane group, preferably, the dye includes JF635, JF639, JF646, SiR650, BD626 or BD666.

[0051] The far-red fluorescent ATP probe of the present invention is an ATP-binding protein labeled with a far-red fluorescent dye, wherein the far-red fluorescent dye and the dye-binding domain of the ATP-binding protein are covalently bound.

[0052] 9. A nucleic acid molecule comprising:

[0053] (1) The coding sequence of the ATP-binding protein as described in any one of items 1-6;

[0054] (2) A sequence complementary to (1).

[0055] 10. Nucleic acid constructs, including the nucleic acid molecules described in item 9, preferably, said nucleic acid constructs are cloning vectors, expression vectors or recombinant vectors.

[0056] 11. A host cell comprising, expressing, or secreting: the ATP-binding protein of any one of items 1-6, the nucleic acid molecule of item 9, and / or the nucleic acid construct of item 10.

[0057] 12. A kit for detecting ATP, comprising: an ATP-binding protein as described in any one of items 1-6, a nucleic acid molecule as described in item 9, a nucleic acid construct as described in item 10, and / or a host cell as described in item 11, and a far-red fluorescent dye.

[0058] 13. The kit described in Item 12, wherein the far-red fluorescent dye is a dye with a chloroalkane group, preferably, the dye includes JF635, JF639, JF646, SiR650, BD626 or BD666.

[0059] 14. A method for preparing the ATP-binding protein according to any one of items 1-6, comprising: culturing the host cells described in item 11, and isolating the ATP-binding protein from the culture.

[0060] In some embodiments, the method includes the steps of: 1) incorporating a nucleic acid molecule encoding the ATP-binding protein into an expression vector; 2) transferring the expression vector into a host cell; 3) culturing the host cell under conditions suitable for expression of the expression vector; and 4) isolating the ATP-binding protein.

[0061] 15. The use of the far-red fluorescent ATP probe described in item 7 or 8 in the detection of ATP.

[0062] 16. Use of the far-red fluorescent ATP probe described in item 7 or 8 in the intracellular and / or extracellular localization of ATP.

[0063] In some embodiments, the method includes: contacting the sample with the ADP-binding protein or the host cell, and detecting fluorescence changes, and locating ADP based on the location of the fluorescence change.

[0064] In some embodiments, the sample is a solution, cells, subcellular components, or tissue.

[0065] 17. A method for detecting ATP in a sample, the method comprising:

[0066] The ATP-binding protein described in any one of Projects 1-6, the nucleic acid molecule described in Project 9, or the nucleic acid construct described in Project 10 is introduced into the sample and labeled with a far-red fluorescent dye. The amount of ATP is determined based on the generated fluorescence intensity; or

[0067] The far-red fluorescent ATP probe described in Project 7 or 8 is introduced into the sample, and the amount of ATP is determined based on the generated fluorescence intensity.

[0068] 18. A method for localizing ATP intracellularly and / or extracellularly, the method comprising:

[0069] The ATP-binding protein described in any one of Projects 1-6, the nucleic acid molecule described in Project 9, or the nucleic acid construct described in Project 10 is introduced into the cells and / or extracellularly, and labeled with a far-red fluorescent dye. The location of ATP is determined based on the position of fluorescence.

[0070] The far-red fluorescent ATP probe described in Project 7 or 8 is introduced into the sample, and the location of ATP is determined based on the location where fluorescence is generated.

[0071] 19. A method for simultaneously detecting ATP and ADP in a sample, the method comprising:

[0072] The ATP-binding protein described in any one of Projects 1-6, the nucleic acid molecule described in Project 9, or the nucleic acid construct described in Project 10 is introduced into the sample and labeled with a far-red fluorescent dye. An ADP-binding protein, whose sequence includes the sequence shown in SEQ ID NO. 25, is then introduced into the sample. The amounts of ATP and ADP are determined based on the generated fluorescence intensity.

[0073] The far-red fluorescent ATP probe described in Project 7 or 8 is introduced into the sample, and the ADP-binding protein is introduced into the sample. The ADP-binding protein sequence includes the sequence shown in SEQ ID NO. 25. The amount of ATP and ADP is determined based on the generated fluorescence intensity.

[0074] 20. The method of Item 19, wherein the ADP-binding protein further comprises a localization sequence, a purification tag, or an immunoblot tag.

[0075] In some embodiments, the localization sequence, purification tag, or immunoblotting tag is located at the C-terminus of the amino acid sequence of the ADP-binding protein. Preferably, the purification tag includes a histidine tag, a Strep tag, or a FLAG tag; the immunoblotting tag includes an HA tag, a Myc tag, or a V5 tag.

[0076] In some embodiments, the histidine tag is 3×His, 4×His, 5×His, 6×His, 7×His, 8×His, 9×His, or 10×His, which are composed of 3, 4, 5, 6, 7, 8, 9, or 10 histidine (H) residues, respectively.

[0077] In some embodiments, the sequence of the Strep tag is preferably WSHPQFEK (SEQ ID NO.44).

[0078] In some embodiments, the preferred sequence of the FLAG tag is DYKDDDDDK (SEQ ID NO.45).

[0079] In some implementations, the immunoblot label is preferably an HA label, a Myc label, or a V5 label.

[0080] In some implementations, the sequence of the HA tag is preferably YPYDVPDYA (SEQ ID NO.46).

[0081] In some implementations, the sequence of the Myc tag is preferably EQKLISEEDL (SEQ ID NO.47).

[0082] In some implementations, the sequence of the V5 tag is preferably GKPIPNPLLGLDST (SEQ ID NO.48).

[0083] In some implementations, a linker may be present between the ADP-binding protein and the localization sequence, purification tag, or immunoblotting tag.

[0084] In some embodiments, the connector is preferably GGGGS (SEQ ID NO:49) or QSGS (SEQ ID NO:50).

[0085] 21. A method for simultaneously detecting ATP and glucose and / or lactate in a sample, the method comprising:

[0086] The ATP-binding protein described in any one of Items 1-6, the nucleic acid molecule described in Item 9, or the nucleic acid construct described in Item 10 is introduced into the sample and labeled with a far-red fluorescent dye. The glucose probe iGlucoSnFR and / or the lactate probe R-iLACCO1.2 are then introduced into the sample, and the amounts of ATP, glucose, and / or lactate are determined based on the generated fluorescence intensity; or

[0087] The far-red fluorescent ATP probe described in Project 7 or 8 is introduced into the sample, and the glucose probe iGlucoSnFR and / or the lactate probe R-iLACCO1.2 is introduced into the sample. The amount of ATP, glucose and / or lactate is determined based on the fluorescence intensity produced.

[0088] 22. The method of any one of items 17-21, wherein the far-red fluorescent dye is a dye with a chloroalkane group, preferably, the dye includes JF635, JF639, JF646, SiR650, BD626 or BD666.

[0089] The beneficial effects of this invention are:

[0090] The far-red fluorescent ATP probe of this invention specifically responds to dynamic changes in ATP levels, enabling precise and sensitive measurement of ATP content and localization of ATP. It can be used for ATP detection during energy metabolism homeostasis imbalances both intracellularly and intercellularly. This far-red fluorescent ATP probe can be used in conjunction with green and red fluorescent probes, offering the advantage of simultaneous multi-probe molecular detection when investigating energy metabolism reprogramming. Furthermore, this far-red fluorescent ATP probe can be used in conjunction with an ADP probe in the same cell, enabling simultaneous detection of dynamic changes in ATP and ADP molecules. Attached Figure Description

[0091] Figure 1 Figure A shows the design schematic of the far-red fluorescent ATP probe; Figure B shows the specific construction strategy of the HL-ATP probe, with the upper figure showing the connection relationship of each part in the ATP-binding protein and the lower figure showing the spatial conformation of cpHaloTag inserted into the PS3 ε subunit; Figure C shows the spatial conformation of the HL-ATP probe before and after ATP binding as predicted by Alphafold3, with the left figure showing the spatial conformation of the HL-ATP probe without ATP binding and the right figure showing the spatial conformation of the HL-ATP probe after ATP binding. The positions of the luminescent fluorescent dyes are marked in magenta.

[0092] Figure 2 Figure A is a schematic diagram of the binding of fluorescent dye and cpHaloTag, where the red and orange parts are fluorescent dyes, the red parts indicate the dyes, the orange parts indicate chloroalkane groups, and the blue parts are cpHaloTags; Figure B is a schematic diagram of the structure of an exemplary fluorescent dye.

[0093] Figure 3 Figure A shows the statistical graph of the maximum fluorescence response amplitude of HL-ATP0.3, HL-ATP0.5, HL-ATP0.7 and HL-ATP0.9 probes labeled with fluorescent dye JF635; Figure B shows the ATP concentration-dependent response curves (n=3) of HL-ATP0.9 probe labeled with fluorescent dyes JF635, JF639, JF646 and SiR650 respectively.

[0094] Figure 4The optimization of the ε subunit of the HL-ATP probe is shown in Figure A, which is a schematic diagram of the optimization screening of the HL-ATP 0.9 probe at amino acid residues 60 and 95 of the binding protein ε subunit; Figure B shows the results of high-throughput screening, with the affinity index under 200 μM ATP and 1 mM ATP conditions and 1 mM ATP. The fluorescence response amplitude under ATP was used to evaluate the probe's responsiveness; Figure C shows the ATP concentration-dependent response curves (n=3) of the HL-ATP1.0 probe labeled with fluorescent dyes JF635, JF639, JF646, and SiR650, respectively; Figure D shows a schematic diagram of the construction of the HL-ATPmut probe, introducing mutations at amino acid residues 89 and 92 of the binding protein ε subunit; Figure E shows a schematic diagram of the ATP-binding active pocket of the ε subunit; Figure F shows the ATP concentration-dependent curves (n=3) of the HL-ATPmut probe labeled with fluorescent dyes JF635, JF639, JF646, and SiR650, respectively, compared with HL-ATP1.0; Figure G shows the optimization diagram of mutation screening at amino acid residues 113 and 115 of the binding protein ε subunit for the HL-ATP0.9 probe; Figure H shows the results of the high-throughput screening shown in Figure G, with the affinity index under 400 μM ATP and 2 mM ATP conditions and 2 mM ATP. The fluorescence response amplitude under ATP conditions was used to evaluate the probe’s responsiveness to ATP. Figure I shows the ATP concentration-dependent response curve of the HL-ATP1.0 (R103A) probe labeled with the fluorescent dye SiR650, compared with that of the HL-ATP1.0 probe (n=3).

[0095] Figure 5 The optimization of the HL-ATP probe cpHaloTag is shown in Figure A, which is a schematic diagram of the key mutation sites of the HL-ATP1.0 probe cpHaloTag (cpHT); Figure B shows the results of high-throughput screening, using 1 mM ATP to test the fluorescence response amplitude to evaluate the probe's ATP responsiveness; Figure C shows the ATP concentration-dependent response curves (n=3) when the HL-ATP1.0 probe and the HL-ATP1h probe are labeled with the fluorescent dye SiR650, respectively; Figure D is a schematic diagram of the construction of the key mutation sites of the HL-ATP1l probe, which is constructed by combining the mutation R103A on the ATP-binding protein that reduces the apparent affinity with the mutations (D18T, Q19T) on cpHaloTag that increase the response amplitude; Figure E shows the ATP concentration-dependent response curves (n=3) when the HL-ATP1.0 probe, the HL-ATP1h probe, and the HL-ATP1.0l probe are labeled with the fluorescent dye SiR650, respectively; cpHT is an abbreviation for cpHaloTag in all figures.

[0096] Figure 6 The spectral characteristics of HL-ATP series probes labeled with different dyes are shown. Figures A, B, and C show the excitation and emission spectra of HL-ATP1.0, HL-ATP1h, and HL-ATP1l probes labeled with fluorescent dye BD626, respectively; Figures D, E, and F show the excitation and emission spectra of HL-ATP1.0, HL-ATP1h, and HL-ATP1l probes labeled with fluorescent dye JF635, respectively; Figures G, H, and I show the excitation and emission spectra of HL-ATP1.0, HL-ATP1h, and HL-ATP1l probes labeled with fluorescent dye JF639, respectively; Figures J, K, and L show the excitation and emission spectra of HL-ATP1.0, HL-ATP1h, and HL-ATP1l probes labeled with fluorescent dye JF639, respectively; and Figures J, K, and L show the excitation and emission spectra of HL-ATP1.0, HL-ATP1h, and HL-ATP1l probes labeled with fluorescent dye JF639, respectively. The excitation and emission spectra of HL-ATP1.0, HL-ATP1h, and HL-ATP1l probes labeled with fluorescent dye JF646 are shown. The M, N, and O spectra are the excitation and emission spectra of HL-ATP1.0, HL-ATP1h, and HL-ATP1l probes labeled with fluorescent dye SiR650, respectively. The P, Q, and R spectra are the excitation and emission spectra of HL-ATP1.0, HL-ATP1h, and HL-ATP1l probes labeled with fluorescent dye BD666, respectively. "+ATP" indicates the presence of ATP, and "-ATP" indicates the absence of ATP. In the excitation spectroscopy test, the emission wavelength was fixed at 710 nm, and the fluorescence intensity was tested from 500 nm to 690 nm. In the emission spectroscopy test, the excitation wavelength was fixed at 600 nm, and the fluorescence intensity was tested from 620 nm to 800 nm. The ATP concentration was 5 mM saturation concentration, and the fluorescence intensity was normalized using the maximum fluorescence intensity after adding ATP.

[0097] Figure 7The diagram shows a comparison of the response characteristics of HL-ATP series probes labeled with different dyes. Figures A, B, C, and D show the ATP concentration-dependent response curves when HL-ATP 1.0, HL-ATP 1h, HL-ATP 1l, and HL-ATP mut probes are labeled with fluorescent dyes BD626, JF635, JF639, JF646, SiR650, or BD666, respectively. Figures E, F, G, and H are statistical graphs showing the maximum fluorescence response amplitudes based on figures A, B, C, and D. Figures I, J, K, and L show the ATP concentration-dependent response curves when HL-ATP 1.0, HL-ATP 1h, HL-ATP 1l, and HL-ATP mut probes are labeled with fluorescent dyes BD626, JF635, JF639, JF646, SiR650, or BD666, respectively. The graphs show the relative fluorescence intensity of HL-ATP1h, HL-ATP1l, and HL-ATPmut probes when they are not bound to saturated ATP concentrations. The M, N, O, and P graphs show the relative fluorescence intensity of HL-ATP1.0, HL-ATP1h, HL-ATP1l, and HL-ATPmut probes after binding to saturated ATP concentrations when they are labeled with fluorescent dyes BD626, JF635, JF639, JF646, SiR650, or BD666, respectively. "+ATP" indicates the presence of ATP, and "-ATP" indicates the absence of ATP. The relative fluorescence intensity is defined as the ratio of the fluorescence intensity when using the fluorescent dye to the fluorescence intensity when using fluorescent dye JF635.

[0098] Figure 8 Figure A shows a schematic diagram of the purification process of the HL-ATP probe, using Ni 2+ -NTA affinity chromatography was used to purify the His-tagged HL-ATP probes; Figure B shows the SDS-PAGE gel electrophoresis images of each probe, with 1 mg / mL BSA solution used as the loading volume control, where 1.0 represents the HL-ATP 1.0 probe, 1h represents the HL-ATP 1h probe, 1l represents the HL-ATP 1l probe, and mut represents the HL-ATP mut probe.

[0099] Figure 9The substrate selectivity of the HL-ATP1.0, HL-ATP1h, and HL-ATP1l probes is shown. Figure A is a comparative statistical graph of the response of the HL-ATP1.0 probe (labeled with JF635) to ATP and its analogues; Figure B is the concentration-dependent response curve of the HL-ATP1.0 probe (labeled with JF635) to ATP, ADP, and AMP (n = 3); Figure C is a comparative statistical graph of the response of the HL-ATP1h probe (labeled with SiR650) to ATP and its analogues; Figure D is the concentration-dependent response curve of the HL-ATP1h probe (labeled with SiR650) to ATP, ADP, and AMP (n = 3); Figure E is a comparative statistical graph of the response of the HL-ATP1l probe (labeled with SiR650) to ATP and its analogues; Figure F is the concentration-dependent response curve of the HL-ATP1h probe (labeled with SiR650) to ATP, ADP, and AMP (n = 3).

[0100] Figure 10 The pH sensitivity comparison of different HL-ATP series probes and the iATPSnFR2l probe is shown. Figure A shows the relative fluorescence intensity of the iATPSnFR2l probe at different pH values; Figure B shows the relative fluorescence intensity of the HL-ATP1.0 probe (labeled with JF635) at different pH values; Figure C shows the relative fluorescence intensity of the HL-ATP1h probe (labeled with SiR650) at different pH values; Figure D shows the relative fluorescence intensity of the HL-ATP1L probe (labeled with SiR650) at different pH values. Relative fluorescence intensity is defined as the fluorescence intensity relative to 25℃, pH=7, and without added ATP. The final ATP concentration is 4 mM (n = 6–8, from individual test wells). "+ATP" indicates the presence of ATP, and "-ATP" indicates the absence of ATP.

[0101] Figure 11 The temperature sensitivity of different HL-ATP series probes and the iATPSnFR2l probe is compared. Figure A shows the relative fluorescence intensity of the iATPSnFR2l probe at different temperatures; Figure B shows the relative fluorescence intensity of the HL-ATP1.0 probe (labeled with JF635) at different temperatures; Figure C shows the relative fluorescence intensity of the HL-ATP1h probe (labeled with SiR650) at different temperatures; Figure D shows the relative fluorescence intensity of the HL-ATP1L probe (labeled with SiR650) at different temperatures. Relative fluorescence intensity is defined as fluorescence intensity relative to 25°C, pH=7, and without added ATP. The final ATP concentration is 4 mM (n = 6–8, from individual test wells). "+ATP" indicates the presence of ATP, and "-ATP" indicates the absence of ATP.

[0102] Figure 12 The images are from confocal microscopy. The left image shows the expression localization of the HL-ATP1.0 probe in HeLa cells, and the right image shows the expression localization of the NES-HL-ATP1.0 probe in HeLa cells. The scale bar is 20 μm in both images.

[0103] Figure 13 The dynamic detection of ATP in live cells by HL-ATP1.0, HL-ATP1h, HL-ATP1L, and HL-ATPmut probes carrying nuclear export sequences (NES) is shown. Figures A and D are confocal images of the expression and response of these probes in the cytoplasm under different treatments (scale bar: 10 μm). Figures B and E show the changes in fluorescence response amplitude of these probes under different treatments (n = 9 fields of view, from 3 independent wells). Figure C is a statistical graph of the maximum fluorescence response amplitude corresponding to Figure B; Figure F is a statistical graph of the maximum fluorescence response amplitude corresponding to Figure E. In each figure, Basal represents the baseline after probe expression, 2-DG represents 2-deoxy-D-glucose, Oligo represents oligomycin, and Glc represents glucose.

[0104] Figure 14 The dynamic detection of ATP in live cells when the HL-ATP1.0 probe is labeled with different fluorescent dyes is shown. Figures A and C are confocal images of the expression and response of the HL-ATP1.0 probe labeled with fluorescent dyes JF635 and BD666 in the cytoplasm under different treatments, with a scale bar of 10 μm. Figures B and D are graphs showing the changes in fluorescence response amplitude of the above probes under different treatments (n = 9 fields of view, from 3 independent test wells). Figure E is a statistical graph of the maximum fluorescence response amplitude corresponding to different treatments according to Figures B and D. Figure F is a comparison of the baseline fluorescence intensity of the HL-ATP1.0 probe labeled with fluorescent dyes JF635 and BD666 according to Figures A and C. In each figure, Basal represents the baseline after probe expression, 2-DG represents 2-deoxy-D-glucose, Oligo represents oligomycin, and Glc represents glucose.

[0105] Figure 15The detection capabilities of the HL-ATP probe and the iATPSnFR2l probe are compared. Figures A and D are confocal images of the expression and response of the above probes in the cytoplasm under different treatments, scale bar: 10 μm. Figures B and E are graphs showing the changes in fluorescence response amplitude of the above probes under different treatments (n = 9 fields of view, from 3 independent wells). Figure C is a statistical graph of the maximum fluorescence response amplitude corresponding to Figure B. Figure F is a statistical graph of the maximum fluorescence response amplitude corresponding to Figure E. Figures G, H, and I are graphs showing the changes in fluorescence response amplitude of the iATPSnFR2l probe and the HL-ATP1.0, HL-ATP1h, and HL-ATP1mut probes under different treatments, respectively. Figure J is a statistical graph of the relative fluorescence response amplitudes of the HL-ATP1.0, HL-ATP1h, and HL-ATP1mut probes based on Figures G, H, and I. Figures K, L, and M are graphs showing the relative fluorescence response amplitudes of the iATPSnFR2l probe and the HL-ATP1.0 probe, respectively. The graph shows the changes in fluorescence response amplitude of the probe, HL-ATP1h probe, and HL-ATP1mut probe under different treatments; the N graph is a statistical graph of the relative fluorescence response amplitude of the HL-ATP1.0 probe, HL-ATP1h probe, and HL-ATP1mut probe based on the K, L, and M graphs; in each graph, Basal represents the baseline after probe expression, 2-DG represents 2-deoxy-D-glucose, Oligo represents oligomycin, and Glc represents glucose.

[0106] Figure 16 The expression and detection of the HL-ATP probe in neurons and astrocytes are shown. Figure A is a schematic diagram and confocal image of the expression of the HL-ATP1.0 probe in primary cultured neurons, with a scale bar of 10 μm. Figure B shows the fluorescence response amplitude of the HL-ATP1.0 probe in neurons to treatment with 10 mM 2-DG or 10 mM 2-DG and 1 μM oligomycin (n = 9 fields of view, from 3 independent test wells). Figure C is a schematic diagram and confocal image of the expression of the HL-ATP1.0 probe in primary cultured astrocytes, with a scale bar of 10 μm. Figure D shows the fluorescence response amplitude of the HL-ATP1.0 probe in astrocytes to treatment with 10 mM 2-DG or 10 mM 2-DG and 1 μM oligomycin (n = 9 fields of view, from 3 independent test wells).

[0107] Figure 17The implementation of the Mito-HL-ATP probe and its localization in HeLa cell mitochondria are shown. Figure A is a schematic diagram of the structure and expression of the Mito-HL-ATP probe, showing that the probe is expressed in mitochondria by adding four tandem COX8 sequences upstream. Figure B is a confocal imaging image using different Mito-HL-ATP probes, where mitochondrial localization uses green fluorescent protein EGFP expressed on the inner mitochondrial membrane (shown in green), the Mito-HL-ATP probe (shown in red), and the co-localization of the probe and mitochondria is shown in yellow. The scale bar is 10 μm.

[0108] Figure 18 The expression and detection of different Mito-HL-ATP probes in HeLa cell mitochondria are shown. Figure A shows confocal imaging of the expression and detection of Mito-HL-ATP1.0, Mito-HL-ATP1h, Mito-HL-ATP1l, and Mito-HL-ATPmut probes in HeLa cell mitochondria, with a scale bar of 10 μm. Figure B shows the fluorescence response amplitude of the above probes to 10 mM 2-DG treatment (n = 9 fields of view, from 3 independent test wells). Figure C is a statistical graph of the maximum fluorescence response amplitude corresponding to Figure B.

[0109] Figure 19 The expression and detection of different ER-HL-ATP probes in the endoplasmic reticulum of HeLa cells are shown. Figure A shows confocal imaging of the expression and detection of ER-HL-ATP1.0, ER-HL-ATP1h, ER-HL-ATP1l, and ER-HL-ATPmut probes in the endoplasmic reticulum of HeLa cells, with a scale bar of 10 μm. Figure B shows the changes in fluorescence response amplitude of the above probes under different treatments (n = 9 fields of view, from 3 independent test wells). Figure C is a statistical graph of the maximum fluorescence response amplitude corresponding to Figure B.

[0110] Figure 20 The multicolor imaging capability of the HL-ATP probes is shown. Figures A and C are confocal images of the expression of the HL-ATP1l probe, iGlucoSnFR probe, and R-iLACCO1.2 probe in HeLa cells under different treatments, with a scale bar of 10 μm. Figures B and D are graphs showing the changes in fluorescence response amplitude of the above probes under different treatments (n = 9 fields of view, from 3 independent test wells). In each figure, Basal represents the baseline after probe expression, 2-DG represents 2-deoxy-D-glucose, Oligo represents oligomycin, and Glc represents glucose.

[0111] Figure 21The combined use of the iADP0.7 and HL-ATP1l probes in HEK293T cells is illustrated. Figure A shows confocal imaging of the expression and detection of the iADP0.7 and HL-ATP1l probes in the HEK293T cell line under different treatments, with a scale bar of 10 μm. Figure B shows the changes in fluorescence response amplitude of the above probes under different treatments (n = 9 fields of view, from 3 independent test wells). In each figure, Basal represents the baseline after probe expression, 2-DG represents 2-deoxy-D-glucose, Oligo represents oligomycin, and Glc represents glucose. Detailed Implementation

[0112] The ATP-binding protein of the present invention can be prepared by the following method:

[0113] First, after obtaining the nucleotide sequence encoding the ATP-binding protein (ATP probe), the nucleotide sequence encoding the ATP probe is inserted into an expression vector to obtain a recombinant expression vector. The terms "expression vector" and "recombinant vector" used herein are interchangeable and refer to prokaryotic or eukaryotic vectors well-known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors that can replicate and be stably expressed in the host.

[0114] Methods for constructing expression vectors containing the ATP probe coding sequence of this invention and suitable transcription / translation control signals are well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques.

[0115] In one embodiment, the expression vector may be a commercially available pET28a vector. Exemplarily, the nucleotide sequence encoding the ATP probe and the expression vector are double-digested with BamHI and XhoI, respectively, and then the digestion products are ligated to obtain the recombinant expression vector. This invention does not specifically limit the specific steps and parameters of the digestion and ligation; conventional steps and parameters in the art can be used.

[0116] Next, after obtaining the recombinant expression vector, the vector is transferred to a host cell to produce a protein or peptide including the ATP probe. This transfer process can be performed using conventional techniques well known to those skilled in the art, such as transformation or transfection. The host cell described in this invention refers to a cell capable of receiving and accommodating recombinant DNA molecules, serving as the site for recombinant gene amplification. Ideally, the recipient cell should meet the conditions of easy acquisition and proliferation.

[0117] The "host cell" in this invention may include prokaryotic and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells, and mammalian cells. The host cell is preferably a cell type well-suited for gene product expression or fermentation production, and such cells are well-known and commonly used in the art. Specifically, it may include bacterial cells such as *Escherichia coli*, *Streptomyces*, and *Salmonella typhimurium*; fungal cells such as yeast; plant cells; insect cells such as *Drosophila S2* or *Sf9*; animal cells such as CHO, COS, HEK293, HeLa cells, or Bowes melanoma cells.

[0118] The method for transferring DNA to host cells described in this invention is a conventional method in the art, including calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipid transfection, native competent cells, chemically mediated transfer, or electroporation. When the host is a prokaryote such as *Escherichia coli*, the method preferably uses the CaCl2 or MgCl2 method, and the steps used are well known in the art. When the host cell is a eukaryotic cell, the following DNA transfection methods can be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0119] This invention involves transferring an expression vector into host cells, followed by amplification and expression culture of the host cells to isolate the ATP probe. The host cell amplification and expression culture can be performed using conventional methods. Depending on the type of host cells used, the culture medium can be any conventional medium. Culture is carried out under conditions suitable for host cell growth.

[0120] The ATP probe of this invention is expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated or purified using various separation methods based on its physical, chemical, and other properties. This invention does not specifically limit the method for separating the ADP probe and ADP-binding protein; conventional protein separation methods in the art can be used. These methods are well known to those skilled in the art and include, but are not limited to: conventional refolding treatment, salting out, centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof. In one embodiment, the ATP probe is separated using His-tagged affinity chromatography.

[0121] This invention also provides the application of the ATP probe in the real-time localization and quantitative detection of ATP. In one aspect, the ATP probe is preferably linked to a signal peptide at different sites within the cell, transferred into the cell, and its real-time localization is achieved by detecting the intensity of fluorescence signals within the cell; the corresponding ATP is then quantitatively detected using a standard ATP titration curve. The standard ATP titration curve described in this invention is plotted based on the fluorescence signals of the ATP probe at different ATP concentrations. Because the ATP probe of this invention is directly transferred into the cell, the real-time localization and quantitative detection of ATP does not require a time-consuming sample processing step, resulting in greater accuracy.

[0122] In ATP-binding proteins or ATP probes, the N-Linker and C-Linker are located at the N-terminus and C-terminus of the dye-binding domain, respectively, and are used to connect the PS3 ε subunit and cpHaloTag or its variants. Here, N-Linker refers to the N-terminal linker, and C-Linker refers to the C-terminal linker. The same applies below.

[0123] The ATP-binding domain (PS3 ε subunit or a variant thereof) of this invention is a functional domain for recognizing and binding ATP, specifically binding to ATP and undergoing a conformational change upon ATP binding. The far-red fluorescent dye does not directly participate in ATP recognition or binding; it specifically binds to the dye-binding domain (cpHaloTag or a variant thereof) covalently, rather than directly to the ATP-binding domain. The ATP-binding domain and the dye-binding domain are connected by linkers (N-Linker and C-Linker). When ATP binds to the ATP-binding domain, the ATP-binding domain undergoes a conformational change, which is transmitted to the dye-binding domain via the linkers, causing a corresponding spatial conformational change or local microenvironmental change in the dye-binding domain. The conformational change of the dye-binding domain further alters the molecular microenvironment and / or the electron cloud distribution, intramolecular conformation, and photophysical properties of the far-red fluorescent dye covalently bound to it, thereby causing changes in the fluorescence intensity, fluorescence lifetime, and / or other fluorescence optical parameters of the far-red fluorescent dye, ultimately producing a detectable fluorescence change. The ATP-binding domain primarily functions as an ATP-recognizing, binding, and conformational transduction unit; the linker peptide primarily functions as a spatial coupling unit for conformational changes and a signal transduction unit; the dye-binding domain primarily functions as a unit for converting conformational changes into fluorescence output; and the far-red fluorescent dye primarily functions as an optical signal output unit. Through the interactions between these different functional modules of the ATP-binding protein, the far-red fluorescent dye emits light, achieving the conversion of ATP molecule recognition into a far-red fluorescent signal.

[0124] In this invention, the terms fluorescence change amplitude and fluorescence response amplitude are used interchangeably.

[0125] In this application, ATP synthase, F0F1 _ ATP synthase can be used interchangeably.

[0126] The fluorescent dye for the ATP probe in this application is preferably a dye with a chloroalkane group, and the dye is selected from: JF635, JF639, JF646, SiR650, BD626, and BD666. The structural composition of the fluorescent dye for the ATP probe is described in [reference needed]. Figure 2 B, The fluorescent dyes used in this application include:

[0127] The structural formula of fluorescent dye JF635 is: For preparation methods, please refer to (Grimm et al., 2017 and Zhang et al., 2025).

[0128] The structural formula of fluorescent dye JF639 is: For preparation methods, please refer to (Deo et al., 2021 and Zhang et al., 2025).

[0129] The structural formula of the fluorescent dye JF646 is: For preparation methods, please refer to (Grimm et al., 2017 and Zhang et al., 2025).

[0130] The structural formula of the fluorescent dye SiR650 is: For preparation methods, please refer to (Lukinavičius et al., 2013 and Zhang et al., 2025).

[0131] The structural formula of the fluorescent dye BD626 is: For the preparation method, please refer to (Zhang et al., 2025);

[0132] The structural formula of the fluorescent dye BD666 is: For the preparation method, please refer to (Zhang et al., 2025).

[0133] The test buffer described in this application consists of 100 mM NaCl, 10 mM HEPES buffer (pH 7.2-7.4), and 1 mM DTT.

[0134] Example

[0135] The reagents used in the following embodiments of the present invention:

[0136] Test Buffer: 100 mM NaCl, 10 mM HEPES buffer (pH 7.2-7.4), 1 mM dithiothreitol (DTT). The ATP or ADP probe protein used in this invention is diluted with the test buffer. The ATP or ADP solution used in this invention is diluted with the test buffer.

[0137] The lysis buffer used to resuspend the bacterial cells during protein purification consisted of 50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole, with the pH adjusted to 7.35.

[0138] The washing buffer used to clean impurity proteins during protein purification consists of 50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole, with the pH adjusted to 7.35.

[0139] The elution buffer used to elute the target protein during protein purification consists of 50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole, with the pH adjusted to 7.35.

[0140] The cell imaging solution consisted of 150 mM NaCl, 4 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM glucose, with the pH adjusted to 7.3-7.4.

[0141] The testing and analysis methods used in the following examples are as follows:

[0142] Preparation method of ATP probe:

[0143] The probe sequence was amplified by PCR, and the PCR product was recovered after gel electrophoresis. The gene fragment was constructed into the pET28a plasmid (purchased from Addgene, catalog number 69864-3) using the Gibson Assembly method (Gibson et al., 2009) or the BsaI Golden Gate Assembly method (Chen et al., 2017) to obtain the recombinant expression vector. The correctly sequenced recombinant expression vector was transformed into DH5α cells to induce expression, and positive clones were screened in LB medium containing antibiotics. Next, positive colonies were picked and inoculated into LB medium, cultured at 37°C with shaking until the OD600 value reached 0.6-0.8. Expression of the target protein was induced using 0.5 mM IPTG at 30°C. After lysis, the supernatant was collected, the cell pellet was collected by centrifugation, and the cells were further lysed using sonication to obtain a soluble protein solution. Insoluble impurities were removed by centrifugation to obtain the probe protein supernatant. Next, protein purification was performed using affinity chromatography. The target protein containing the 6×His tag was purified using a Ni2+-NTA affinity column to obtain the HL-ATP probe.

[0144] Preparation method of ADP probe:

[0145] ParM protein, green fluorescent protein, and linker peptides were amplified by PCR. The PCR products were recovered after gel electrophoresis. The gene fragments were then constructed into the pET28a plasmid (purchased from Addgene, catalog number 69864-3) using either the Gibson Assembly method (Gibson et al., 2009) or the BsaI Golden Gate Assembly method (Chen et al., 2017) to obtain a recombinant expression vector. The correctly sequenced recombinant expression vector was transformed into *E. coli* BL21 competent cells (purchased from Novizan, catalog number C504-02) to induce expression, and positive clones were screened in LB medium containing antibiotics. Positive colonies were then picked and inoculated into LB medium, cultured at 37°C with shaking until the OD600 value reached 0.6-0.8. Expression of the target protein was induced using 0.5 mM IPTG at 30°C. After lysis, the supernatant was collected, the cell pellet was collected by centrifugation, and the cells were further lysed using sonication to obtain a soluble protein solution. Insoluble impurities were removed by centrifugation to obtain the probe protein supernatant. Next, protein purification was performed using affinity chromatography with Ni. 2+ The ADP probe was purified using an NTA affinity column.

[0146] The concentration of the purified probe protein was determined by BCA method or ultraviolet spectrophotometry, and the protein purity was analyzed by SDS-PAGE electrophoresis to ensure that the expected purity requirements were met.

[0147] In ADP-binding proteins or ADP probes, N-Linker and C-Linker are located at the N-terminus and C-terminus of the fluorescent protein, respectively, and are used to connect the fluorescent protein and the ADP-binding protein.

[0148] ATP concentration-dependent fluorescence response assay method:

[0149] 1. Prepare probe solution: Dilute the purified ATP probe to 16 μM using test buffer, add 1 μM fluorescent dye and incubate at room temperature for 1 hour.

[0150] 2. Fluorescence assay: ATP solutions of different concentrations (using test buffer as solvent) were added to the purified probe solution to be tested, and the fluorescence intensity was measured using a microplate reader. When screening ATP probe mutants, excitation light was 630 nm and emission light was 660 nm. The fluorescence intensity was obtained with and without ATP, and ΔF / F0 was calculated to obtain the probe response.

[0151] 3. Plot a concentration-dependent fluorescence response curve, with the ATP solution concentration on the x-axis and the fluorescence intensity or fluorescence response amplitude on the y-axis.

[0152] Method for calculating fluorescence response amplitude:

[0153] The fluorescence intensity was measured using an ELISA reader, and the fluorescence response amplitude of the probe before and after ATP or ADP binding was calculated using the following formula to evaluate the probe's responsiveness to ATP or ADP.

[0154] Fluorescence response amplitude = (F - F0) / F0 = ΔF / F0, where F is the fluorescence intensity measured when ATP or ADP has been added and the fluorescence intensity measured without the addition of ATP or ADP. Curve fitting was performed based on the fluorescence response amplitude using Origin software for nonlinear pharmacological concentration-dependent curve fitting, and the median effective concentration (EC50) was calculated based on the fitting results. 50 (For analytical methods, see Wu et al., 2022)

[0155] Fluorescence spectroscopy method for probe determination:

[0156] The assay was performed at room temperature using a multi-functional microplate reader. When screening for ATP probe mutants, an excitation wavelength of 630 nm and an emission wavelength of 660 nm were used. For the detection of ATP probes, the microplate reader settings are generally as follows: an excitation spectrum scanning range of 500–690 nm and a fixed emission wavelength of 710 nm; and an emission spectrum scanning range of 610–810 nm and a fixed excitation wavelength of 600 nm.

[0157] For ADP probe detection, the microplate reader is generally set as follows: the excitation spectrum scanning range is 400 – 510 nm, and the emission wavelength is fixed at 530 nm; the emission spectrum scanning range is 490 – 610 nm, and the excitation wavelength is fixed at 470 nm.

[0158] Confocal imaging methods:

[0159] Before imaging, the culture medium was replaced with cell imaging solution. Cells were then imaged using a confocal imaging microscope (NIKON A1&N-SIMS, Nikon, Japan) or a laser confocal high-content cell analysis system (CQ1, YOKOGAWA, Japan). Imaging with the laser confocal high-content cell analysis system typically involved acquiring images every 8-12 minutes. The raw image data was exported in TIF format, and the fluorescence intensity was quantified using ImageJ software. After subtracting the background signal, the fluorescence response amplitude before and after ATP or ADP binding was calculated using the following formula: Fluorescence response amplitude = (F - F0) / F0, where F is the fluorescence intensity measured when ATP or ADP is added and the fluorescence intensity measured without ATP or ADP.

[0160] In this embodiment of the invention, HeLa and HEK293T cells were cultured and grown in a 37°C, 5% CO2 incubator using a high-glucose DMEM medium containing 10% (v / v) fetal bovine serum and 1% penicillin-streptomycin. Before transfecting with exogenous DNA, when the cells reached approximately 70% confluency, a transfection mixture containing DNA and PEI (mass ratio 1:3) was added to the cell culture medium. Six to eight hours after transfection, the medium was replaced with fresh medium preheated to 37°C, and imaging or detection was performed 24 to 36 hours later.

[0161] Methods to inhibit intracellular ATP synthesis:

[0162] Inhibiting intracellular ATP synthesis by inhibiting glycolysis alone or simultaneously inhibiting glycolysis and oxidative phosphorylation, the specific exemplary experimental procedure is as follows:

[0163] 1. Cell preparation: Seed cells onto coverslips (for obtaining confocal static images) or 96-well plates (for measuring time-series curves), transfect with probes, and culture to an appropriate density;

[0164] 2. Buffer pretreatment: Discard the culture medium and replace it with normal Tyrode buffer containing glucose (unit: mM: 150 mM NaCl, 4 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, 10 mM glucose (pH 7.3-7.4)), and allow it to stand to stabilize;

[0165] 3. Baseline acquisition (0–30 minute Basal phase)

[0166] Cover glass: Nikon confocal microscope images of Expression and Basal fluorescence pseudocolor were captured.

[0167] 96-well plates: Continuous scanning was performed using a high-content device (high-content cell imaging analysis system), and baseline fluorescence ΔF / F0 was recorded;

[0168] 4. Grouped drug perfusion treatment: (30–60 minute phase)

[0169] (1) Experimental group 1: Perfusion with sugar-free Tyrode buffer containing 2-DG for 30 minutes, and simultaneously capture fluorescence images and record time-series fluorescence curves under 2DG treatment;

[0170] (2) Experimental group 2: Perfusion with sugar-free Tyrode buffer containing 2-DG and oligomycin for 30 minutes, and simultaneously photographing fluorescence images and recording time-series fluorescence curves under 2-DG & Oligo treatment;

[0171] 5. Glucose recovery treatment: (60–90 minutes Glc phase)

[0172] Each group was switched back to Tyrode buffer containing glucose (Glc) for 30 minutes; fluorescence pseudo-color images of the Glc phase were collected and fluorescence signal fallback time curves were recorded simultaneously.

[0173] 6. Imaging data analysis

[0174] Confocal static image: Export cell fluorescence intensity at each stage and generate intensity pseudo-color image;

[0175] Time-series curves: Calculate ΔF / F0 for the entire process, and plot time-fluorescence change curves to distinguish between the probe group and the control group.

[0176] The primary rat neurons-glial cells used in this embodiment of the invention were cultured in culture dishes coated with poly-D-lysine (to improve cell adhesion efficiency) in Neurobasal medium containing 2% B-27 supplement, 1% GlutaMAX, and 1% penicillin-streptomycin. To maintain stable growth of the co-culture system, on the fourth day of culture, the medium was changed at 50% concentration, and 0.2 μL of a 1×10⁻⁶ titer was added. 13 Adeno-associated virus (AAV) at a concentration of vg / mL was used to express the probe. Imaging experiments were performed 5–7 days after probe expression (Peng et al., 2020).

[0177] Data processing and statistical analysis methods:

[0178] ImageJ software was used to process the imaging data. Origin was used for data statistics and charting. Before analysis of variance, a normality test was performed. All data in this study passed the normality test; therefore, Student's-test or one-way ANOVA was used for statistical analysis. All data with error bars in this study are expressed as mean ± standard error (mean ± SEM). Statistical significance is indicated by * for p ≤ 0.05, ** for p ≤ 0.01, and ns for not significant (p > 0.05).

[0179] Example 1. Selection of linker peptides for HL-ATP probes and HL-ATP 0.9 probe

[0180] An ATP probe (HL-ATP probe) was designed, which is itself an ATP-binding protein comprising an ATP-binding domain and a dye-binding domain. The ATP-binding domain is derived from the PS3 ε subunit, and the dye-binding domain is derived from the cpHaloTag protein. The cpHaloTag is inserted at position 107 of the PS3 ε subunit and linked using a linker peptide (see [link to HL-ATP probe]). Figure 1 B).

[0181] This embodiment provides several HL-ATP probe variants, all employing the above-described structure, differing only in their linker peptides. The probe with the N-Linker sequence LEFLW (SEQ ID NO:27) and the C-Linker sequence YESAS (SEQ ID NO:28) is named HL-ATP0.3, and its sequence is shown in SEQ ID NO:29. The probe with the N-Linker sequence KRDHMLEFLW (SEQ ID NO:30) and the C-Linker sequence YESAS (SEQ ID NO:31) is named HL-ATP0.5, and its sequence is shown in SEQ ID NO:32. The probe with the N-Linker sequence KRDHTKEFLW (SEQ ID NO:33) and the C-Linker sequence YESAS (SEQ ID NO:34) is named HL-ATP0.7, and its sequence is shown in SEQ ID NO:35. The probe with N-Linker sequence KKESIKEFLW (SEQ ID NO:36) and C-Linker sequence YSAS (SEQ ID NO:37) was named HL-ATP0.9 probe, and its sequence is shown in SEQ ID NO: 38.

[0182] HL-ATP0.3, HL-ATP0.5, HL-ATP0.7, HL-ATP0.9, and HL-ATP0.9 probes were prepared using the above-described method. Each probe was labeled with the fluorescent dye JF635, and the fluorescence response amplitude of each ATP probe to 200 μM ATP was then measured. The results are as follows: Figure 3 As shown in A, all of them can produce fluorescence response amplitudes greater than 300%, namely 302%, 377%, 1159%, and 3550%.

[0183] The HL-ATP0.9 probe was labeled with various fluorescent dyes, and its ATP concentration-dependent response was tested. The results are as follows: Figure 3 B shows that the maximum response amplitudes when labeled with fluorescent dyes JF635, JF639, JF646, and SiR650 were 4900%, 1200%, 1410%, and 1780%, respectively, with half-maximum effective concentrations (EC50) of 100%. 50 The concentrations were 310 μM, 22 μM, 75 μM, and 51 μM, respectively. Figure 2 ).

[0184] The above results indicate that: (i) the HL-ATP0.9 probe maintains a high sensitivity fluorescence response to ATP under different HaloTag dye labeling conditions, with the maximum response amplitude spanning a dynamic range of 12 to 49 times; (ii) dye selection significantly affects the apparent ATP affinity of the probe, EC 100%. 50 The values ​​varied between 22 and 310 μM, spanning more than an order of magnitude; (iii) the unexpected synergistic effect of the dye-probe combination provides a technical basis for customizing the sensitivity and linear detection range of ATP detection under different physiological and pathological conditions. In some implementation schemes, for applications requiring high sensitivity to detect low concentrations of ATP (e.g., extracellular microenvironment ATP monitoring), JF639-labeled HL-ATP0.9 (EC) is preferred. 50 = 22 μM); for high-concentration ATP quantification scenarios requiring a wide linear range (e.g., intracellular ATP imaging), JF635-labeled HL-ATP 0.9 (EC) is preferred. 50 = 310 μM, with a maximum fluorescence response amplitude of 4900%.

[0185] Compared with the iATPSnFR2l probe, the HL-ATP0.9 probe of this invention, when labeled with JF635 dye, achieved a maximum response amplitude of 4900% to ATP, significantly higher than the iATPSnFR2l probe (1200%), with an apparent affinity EC50. 50 Approximately 310 μM, slightly lower than the iATPSnFR2l probe (EC). 50 (~530 μM). Given that intracellular ATP concentration can reach millimole levels under physiological conditions, appropriately reducing the apparent affinity of the probe is expected to yield a greater response amplitude. Therefore, the HL-ATP0.9 probe of this invention is significantly superior to the iATPSnFR2l probe in terms of ATP response.

[0186] Example 2. Selection of the ATP-binding domain of the HL-ATP probe and the HL-ATP1.0 and HL-ATPmut probes

[0187] 1. HL-ATP1.0 probe

[0188] Based on the HL-ATP0.9 probe, one site each was randomly selected on the α-helix and β-sheet structure of its PS3 ε subunit for mutation, and the resulting variant probes were detected and screened. Figure 4A). During the screening process, the fluorescence response amplitude of each mutant (labeled with the fluorescent dye JF635) to 1 mM ATP and 200 μM ATP was measured simultaneously. The ratio of the two fluorescence response amplitudes was defined as the affinity index, which ranges from 0 to 1, with a higher value indicating lower affinity. The results are as follows: Figure 3 As shown in B, when the 60th amino acid position of the HL-ATP0.9 probe is mutated from tyrosine to threonine (Y60T), it is named the HL-ATP1.0 probe (its sequence is shown in SEQ ID NO:1). It maintains a high response amplitude to 1 mM ATP while having a large affinity index. Figure 4 B).

[0189] Further measurements were performed on the ATP concentration-dependent curves of the HL-ATP1.0 probe binding to different dyes. The results showed that the highest response amplitude using the labeled fluorescent dye JF635 reached 7600%, EC50%. 50 Increased from 310 μM to 589 μM ( Figure 4 C).

[0190] When the HL-ATP1.0 probe was labeled with fluorescent dyes JF639, JF646, and SiR650, the highest response amplitudes of the probe to ATP were 2400%, 1890%, and 1810%, respectively, EC50. 50 The values ​​were 152 μM, 429 μM, and 340 μM, respectively. Figure 4 C).

[0191] 2. HL-ATPmut probe

[0192] To obtain mutants with no (or very low) fluorescence response to ATP (as a control probe to verify the authenticity of the probe's signal response), two point mutations, D89A and R92A, were introduced into the ATP-binding pocket of the HL-ATP1.0 probe. Figure 4 D), where the carbonyl and amide groups of D89 specifically recognize the adenine ring via Watson-Crick type hydrogen bonds, and the guanidino π-electron system of R92 stacks with the adenine ring. This residue also recognizes ribose and γ-phosphate groups via hydrogen bonds. Figure 4 E). Further testing of the ATP concentration-dependent curves of the probe binding to different dyes revealed that it lost its fluorescence response to ATP in all cases. Figure 4 F), named the mutant HL-ATPmut probe (its sequence is shown in SEQ ID NO:9), can be used as a control probe that does not respond to ATP.

[0193] 3. HL-ATP1.0 (R103A) probe

[0194] Mutations were introduced at two conserved ATP-binding regulatory arginine sites, R103 and R105, near the binding pocket of the PS3 ε subunit. Figure 4 G). The fluorescence response amplitude of each mutant (labeled with the fluorescent dye SiR650) to 400 μM ATP and 2 mM ATP was measured, and its affinity index was calculated. The mutant with arginine at position 103 mutated to alanine was named HL-ATP1.0 (R103A) probe (its sequence is shown in SEQ ID NO:3), which has both a high affinity index and a high fluorescence response to 2 mM ATP. Figure 4 H), and its concentration-dependent response curve shows that the EC50 of this mutant labeled with SiR650 dye is... 50 The concentration was 913 μM, approximately 2.4 times lower than that of HL-ATP1.0. Figure 4 (I), but its maximum response amplitude has decreased slightly.

[0195] Example 3. Selection of dye-binding domains for HL-ATP probes and HL-ATP1h and HL-ATP1l probes

[0196] 1. HL-ATP 1h probe

[0197] The dye-binding domain of the HL-ATP probe uses cpHaloTag. Eight sites near the "antennae" on both sides of the dye on cpHaloTag (amino acids 18-20 and 27-31 of cpHaloTag) were randomly mutated. The resulting variant probes were then detected and screened. Figure 5 During the screening process (A and B), the fluorescence response amplitude of each mutant (labeled with the fluorescent dye SiR650) to 200µM ATP was measured. The mutant HL-ATP1.0 (D18T Q19T) probe, which showed a 3000% response amplitude to 200µM ATP, was successfully obtained. Figure 5 B). Further measurements of the ATP concentration-dependent response curves when the mutant was labeled with different dyes showed that the maximum response amplitude when labeled with the fluorescent dye SiR650 reached 3500%, EC 100%. 50 450 μM ( Figure 5 C). Given that the ATP concentration is usually at the millimole level under physiological conditions, the probe has relatively high affinity, so it has been renamed the HL-ATP1h probe (its sequence is shown in SEQ ID NO:5), where h represents high affinity.

[0198] 2. HL-ATP1l probe

[0199] To create a probe that combines low affinity with high response amplitude, the R103A mutation of the HL-ATP1.0 (R103A) probe was combined with the D18T Q19T mutation of the HL-ATP1h probe. Figure 5 (D) Finally, a probe with both low affinity and high response amplitude was obtained. When labeled with the fluorescent dye SiR650, its affinity was 1072 μM and its maximum response amplitude reached 3200%. Figure 5 E), named the HL-ATP1l probe (its sequence is shown in SEQ ID NO:7), l indicates low affinity.

[0200] Example 4. Spectral characteristics of HL-ATP probes labeled with different fluorescent dyes

[0201] 1. Spectral characteristics of HL-ATP probes labeled with different dyes

[0202] To meet the application requirements of multicolor imaging, the fluorescent probe must be orthogonal to the green and red fluorescent probes in the spectrum, so as not to interfere with each other during detection. The excitation wavelength range of commonly used green fluorescent probes is usually 470 ± 25 nm, and the emission wavelength range is 525 ± 25 nm; the excitation wavelength range of commonly used red fluorescent probes is 560 ± 25 nm, and the emission wavelength range is 600 ± 25 nm.

[0203] The specific method for detecting the spectral properties of ATP probes is as follows:

[0204] (a) Provide a cell population expressing the test probe: Using the above-described method for preparing ATP probes, obtain purified probes and dilute the probe protein with test buffer to 0.25 mg / ml.

[0205] (b) Label the probe with a fluorescent dye and determine the excitation spectrum (Ex) of the fluorescent dye-labeled probe: Collect the fluorescence signal at the first emission wavelength, and scan multiple excitation wavelengths in 2 nm steps within the theoretical excitation peak range of the probe. Record the fluorescence intensity under the conditions of ATP presence and absence, and normalize it with its maximum value as 1. Plot the normalized fluorescence intensity value against the excitation wavelength to obtain the excitation spectrum.

[0206] (c) Label the probe with a fluorescent dye and determine the emission spectrum (Em) of the fluorescent dye-labeled probe: Excite the sensor with the peak excitation wavelength determined in step (b), scan multiple emission wavelengths in 2 nm steps within the theoretical emission peak range of the probe, record the fluorescence intensity under the conditions of ATP presence and absence, and normalize it with its maximum value as 1. Plot the normalized fluorescence intensity value against the emission wavelength to obtain the emission spectrum.

[0207] (d) Determine the optimal working wavelength pair: Select the excitation wavelength and emission wavelength with the largest response value from the excitation spectrum and emission spectrum as the optimal working wavelength pair of the probe.

[0208] The results are as follows Figure 6 The results showed that labeling HL-ATP1.0, HL-ATP1h, and HL-ATP1l probes with fluorescent dyes BD626, JF635, JF639, JF646, SiR650, and BD666, respectively, significantly enhanced fluorescence intensity in the presence of ATP compared to the absence of ATP, indicating that the HL-ATP probes have good far-red dye compatibility. The optimal excitation and emission wavelengths of the probes labeled with the above six dyes almost do not overlap with the excitation and emission spectra of commonly used green or red fluorescent probes (excitation wavelength is generally 650±25nm, emission wavelength is generally 670±25nm), proving that the probes of this invention can meet the requirements of multicolor imaging.

[0209] To characterize the dye adaptation properties of the HL-ATP probe, three key performance indicators were compared when labeling the aforementioned dyes.

[0210] Regarding affinity, the apparent affinity (EC) of HL-ATP1.0, HL-ATP1h, and HL-ATP1l probes labeled with the fluorescent dye BD666 was [not specified]. 50 The lowest values ​​were 649 μM, 1029 μM, and 1391 μM, respectively. Figure 7 (A, B, C). Different dyes endow probes with diverse affinity ranges, which can meet the detection needs of dynamic changes in ATP at different concentration ranges.

[0211] In terms of response amplitude, the HL-ATP1.0 probe showed the largest response amplitude when labeled with the fluorescent dye JF635. Figure 7 E); when labeled with fluorescent dyes, JF639, JF646, and SiR650 HLATP1h probes all showed high response amplitudes ( Figure 7F); the response amplitude was largest when the HL-ATP1l probe was labeled with the fluorescent dye JF646. The response amplitude was smallest when the HL-ATP1.0, HL-ATP1h, and HL-ATP1l probes were labeled with the fluorescent dye BD626. Figure 7 EG).

[0212] Regarding fluorescence intensity, compared to labeling with other fluorescent dyes, labeling the probe with fluorescent dye BD626 resulted in the strongest fluorescence intensity both before and after ATP binding, followed by labeling with fluorescent dye JF639. Figure 7 IP).

[0213] Considering the above characteristics, and taking into account that the fluorescent dye JF635 can achieve a response amplitude of up to 7600% when paired with the HL-ATP1.0 probe, it is the preferred fluorescent dye for the HL-ATP1.0 probe. On the other hand, the fluorescent dye SiR650 performs excellently in in vivo imaging, and its response amplitudes for labeling the HL-ATP1h and HL-ATP1l probes reach 3700% and 3400%, respectively. Therefore, it is preferable to pair the fluorescent dye SiR650 with these two probes, making it more suitable for application in living tissues.

[0214] Example 5. In vitro characterization of the HL-ATP probe

[0215] Each HL-ATP probe was synthesized using the ADP probe preparation method described above, and Ni was used. 2+ The HL-ATP probe was separated and purified using column affinity chromatography to characterize its detection performance in vitro. SDS-PAGE electrophoresis and Coomassie brilliant blue staining results showed that the in vitro purified HL-ATP probe had high purity, and the displayed band size was around 58 kDa, consistent with the theoretical molecular weight. Figure 8 A, B).

[0216] 1. Substrate selectivity of HL-ATP probes

[0217] To verify the substrate selectivity of the HL-ATP probe, this study tested its fluorescence response to various triphosphate analogs, including: thymidine triphosphate (TP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), uridine triphosphate (UTP) and its analogs uridine diphosphate (UDP) and uridine diphosphate glucose (UDP-G), and adenine analogs including: ADP, adenosine monophosphate (AMP), adenosine (ADO), adenine (Ade) and its analog inosine (Ino). The results showed that the responses of the HLATP1.0, HL-ATP1h, and HL-ATP1l probes to these analogues were all close to 0, significantly lower than their responses to ATP, indicating that the HL-ATP probe of the present invention is not affected by the above-mentioned substances during detection. Figure 9 ).

[0218] The HL-ATP probe exhibited a slight fluorescent response to ADP and AMP. Further analysis of their concentration-dependent curves revealed that even at higher concentrations of ADP or AMP, the HL-ATP probe showed almost no change in fluorescence signal, further demonstrating its high specificity for ATP. Figure 9 B, D, F).

[0219] 2. pH insensitivity of the HL-ATP probe

[0220] The effects of different pH conditions on the iATPSnFR2l probe and the HL-ATP probe were compared. The results are as follows: Figure 11 The results show that the relative fluorescence intensity of the iATPSnFR2l probe after binding to ATP decreases with decreasing pH (Figure 10A), and the negative logarithm of the acid dissociation constant, pKa, is approximately 7.0. In contrast, the HL-ATP1.0, HL-ATP1h, and HL-ATP1l probes all exhibit stable fluorescence intensity within a pH range of 6.0 to 8.0 (Figure 10B, C, D), demonstrating good resistance to pH interference. In summary, the HL-ATP probe of this invention overcomes the shortcomings of existing ATP fluorescent probes in terms of pH sensitivity, helps to eliminate false positive signals caused by pH fluctuations, and achieves accurate monitoring of dynamic changes in ATP.

[0221] 3. The HL-ATP probe is temperature insensitive.

[0222] The effects of different temperatures on the iATPSnFR2l probe and the HL-ATP probe were compared. The results showed that the fluorescence signal of the iATPSnFR2l probe decreased significantly during the temperature increase from 25℃ to 40℃. Figure 11 A); while the fluorescence signals of HL-ATP1.0, HL-ATP1h, and HL-ATP1l probes were relatively stable, with HL-ATP1h and HL-ATP1l probes showing good resistance to temperature interference in the 35-40℃ range (A). Figure 11 B, C, and D) are more suitable for detecting changes in ATP levels under the aforementioned temperature variations.

[0223] Example 6. In vivo characterization of the HL-ATP probe

[0224] 1. Localization of the HL-ATP probe

[0225] To further achieve specific localization of the probe in the cytoplasm, a nuclear export signal (NES) sequence was fused to the N-terminus of the HL-ATP1.0 probe sequence (NES sequence shown in SEQ ID NO:15). This sequence was constructed into a vector, introduced into host cells, positive clones were screened, and then expressed and purified to obtain the NES-fused HL-ATP probe (NES-HL-ATP1.0 probe, sequence shown in SEQ ID NO:14). This probe was transfected into the HeLa cell line, and confocal imaging results were obtained as shown in the image. Figure 12 As shown, the HL-ATP1.0 probe without NES is distributed throughout the entire cell; the HL-ATP1.0 probe with NES added exhibits fluorescence throughout the cytoplasm, but no expression is found in the nucleus, demonstrating the specific localization of the HL-ATP1.0 probe in the cytoplasm. Similarly, the same method can be used for other HL-ATP probes by fusing a nuclear output signal sequence to the N-terminus of the probe sequence to obtain HL-ATP probes fused with NES, which can then be used to achieve specific localization in the cytoplasm.

[0226] 2. The ability of the HL-ATP probe to detect ATP in living cells.

[0227] The HL-ATP probe was expressed in the HeLa cell line, and intracellular ATP synthesis was reduced by inhibiting glycolysis alone or by simultaneously inhibiting glycolysis and oxidative phosphorylation. The N-terminus of the sequences of HL-ATP1.0, HL-ATP1h, HL-ATP1l, or HL-ATPmut (as a control) were fused with the NES sequence and constructed into the pCMV plasmid to obtain a recombinant expression vector. This vector was then transfected into HEK293T cells and cultured at 37°C with 5% CO2 for 24-48 hours. For imaging assays, the probes were first labeled with the fluorescent dye JF635 (1 μM). Baseline measurements were taken in medium supplemented with 10 mM glucose for 30 minutes. At 30 minutes, the medium was switched to medium supplemented with 10 mM 2-deoxy-D-glucose (2-DG) (to inhibit glycolysis alone) or medium supplemented with 10 mM 2-DG and 2 μM oligomycin (to inhibit both glycolysis and oxidative phosphorylation). At 60 minutes, the medium was switched back to medium supplemented with 10 mM glucose. The medium contained 10% glucose. (v / v) fetal bovine serum and 1% penicillin-streptomycin in high-glucose DMEM medium were used for fluorescence detection throughout the process, and the fluorescence response amplitude was calculated.

[0228] The results are as follows Figure 13 The results showed that the HL-ATP1.0, HL-ATP1h, and HL-ATP1l probes carrying nuclear export sequences (NES) were all well expressed in the cytoplasm of HeLa cells. Figure 13 A). When treated alone with the glycolysis inhibitor 2-DG, the fluorescence signal intensity of all probes except the control was significantly reduced ( Figure 13 B), among which the decrease in the HL-ATP1.0 probe and the HLATP1l probe was greater than that of the HL-ATP1h probe ( Figure 13 C); however, after glucose supplementation, the fluorescence intensity significantly recovered ( Figure 13 B). As a control, the HL-ATPmut probe did not show significant signal changes under the same treatment ( Figure 13 (B, C). The above data indicate that the HL-ATP series probes can effectively respond to changes in cytoplasmic ATP concentration.

[0229] Furthermore, compared to inhibiting glycolysis alone, simultaneously inhibiting glycolysis and oxidative phosphorylation (i.e., adding 2-DG and oligomycin) caused a more significant decrease in fluorescence intensity, with the HL-ATP1.0 probe showing the largest decrease. After glucose refilling, the fluorescence intensity of all three probes significantly rebounded, while the HL-ATPmut probe did not show a significant signal change, further verifying the authenticity of the signal. Figure 13DF). In summary, the HL-ATP1.0, HL-ATP1h, and HL-ATP1l probes have the ability to specifically monitor the dynamic changes of ATP in living cells and can also locate ATP (for assessing the site of ATP production or changes).

[0230] 3. Dye compatibility of HL-ATP probes in live cell detection

[0231] The HL-ATP1.0 probe, containing a nuclear export sequence, was expressed in HeLa cells, labeled with the fluorescent dye BD666, and then subjected to confocal imaging to measure changes in fluorescence intensity. An HL-ATP1.0 probe labeled with the fluorescent dye JF635 was used as a positive control. Intracellular ATP synthesis was inhibited by either inhibiting glycolysis alone or by simultaneously inhibiting glycolysis and oxidative phosphorylation.

[0232] The results showed that the HL-ATP probe with the nuclear export sequence could be detected by fluorescence in the cytoplasm regardless of whether it was labeled with the fluorescent dyes JF635 or BD666, demonstrating good expression of the probe and successful binding of the dye. Figure 14 A). Treatment with the glycolysis inhibitor 2-DG alone significantly reduced the fluorescence signal intensity of the far-red channel in the cytoplasm using probes labeled with fluorescent dyes JF635 and BD666, with the decrease being greater when labeled with JF635 than when labeled with BD666; however, the fluorescence intensity recovered significantly after glucose supplementation. Figure 14 B).

[0233] Furthermore, compared to inhibiting glycolysis alone, simultaneous inhibition of glycolysis and oxidative phosphorylation (i.e., the addition of 2-DG and oligomycin) resulted in a more significant decrease in fluorescence intensity. Figure 14 C), among which the decrease was greater when labeled with fluorescent dye JF635 than when labeled with fluorescent dye BD666; after glucose refilling, a significant rebound in fluorescence intensity was observed in both cases. Figure 14 D).

[0234] The fluorescence intensity of the HL-ATP1.0 probe bound to the two dyes was also compared at baseline. The results showed that the fluorescence intensity labeled with the fluorescent dye JF635 was significantly higher than that of BD666. Figure 14 F). In summary, the HL-ATP1.0 probe exhibits good dye compatibility, being compatible not only with JF635 dye commonly used in live cells, but also with BD666 dye for detecting dynamic changes in ATP.

[0235] 4. Performance comparison of HL-ATP probe and iATPSnFR2l probe in cell detection

[0236] To systematically compare the response performance of the HL-ATP series probes with the reported iATPSnFR2l probe in intracellular ATP detection, the HL-ATP1l probe and the iATPSnFR2l probe were co-expressed in the cytoplasm of HeLa cells, and the red fluorescent protein mCherry was introduced as an internal control channel to verify the authenticity of the signal. Figure 15 A) Inhibit intracellular ATP synthesis by inhibiting glycolysis alone or by simultaneously inhibiting glycolysis and oxidative phosphorylation.

[0237] The results showed that treatment with the glycolysis inhibitor 2-DG alone significantly reduced the fluorescence signal intensity in both the green and far-red channels, while no significant signal change was observed in the red channel. Figure 15 B), among which the maximum decrease in the HL-ATP1l probe was significantly higher than that of the iATPSnFR2l probe (B). Figure 15 C). Furthermore, compared to inhibiting glycolysis alone, simultaneous inhibition of glycolysis and oxidative phosphorylation caused a more significant decrease in fluorescence intensity for both probes, and the decrease in fluorescence intensity for the HL-ATP1l probe was still significantly greater than that for the iATPSnFR2l probe; after glucose refilling, the fluorescence intensity of both probes recovered somewhat. Figure 15 D - E).

[0238] Furthermore, under the same experimental conditions, the responses of the HL-ATP1.0 probe, HL-ATP1h probe, and HL-ATP1mut probe to the above treatment were also tested. The results showed that the maximum response amplitudes of the HL-ATP1.0 probe and the HL-ATP1h probe were significantly higher than those of the iATPSnFR2l probe (…). Figure 15 GN).

[0239] In summary, the HL-ATP series probes are significantly superior to the iATPSnFR2l probe in detecting dynamic changes in ATP within living cells.

[0240] Example 7. The ability of the HL-ATP probe to detect ATP in primary cultured neurons and astrocytes.

[0241] Can HL-ATP be used to detect changes in ATP levels in neurons or astrocytes? To answer this question, in a neuron-astrocytocyte co-culture system, the hSyn promoter was added upstream of the HL-ATP1.0 probe expression sequence to specifically express it in neurons, or the GfaABC1D promoter was added upstream of the HL-ATP1.0 probe expression sequence, and a miRNA targeting cassette (e.g., 4×6T miR) sequence was added downstream of the HL-ATP1.0 probe expression sequence (to clear off-target expression and improve specific expression) to specifically express it in astrocytes. The sequence of the hSyn promoter is shown in SEQ ID NO: 39, the sequence of the GfaABC1D promoter is shown in SEQ ID NO: 40, and the sequence of the miRNA targeting cassette is shown in SEQ ID NO: 41. Intracellular ATP synthesis was inhibited by inhibiting glycolysis alone or simultaneously inhibiting glycolysis and oxidative phosphorylation.

[0242] Primary neuron-glial cell co-culture system: Rat primary neurons and glial cells were added to culture dishes coated with poly-D-lysine (to improve cell adhesion efficiency). The culture medium was Neurobasal medium (purchased from Thermo Fisher) containing 2% B-27 supplement, 1% GlutaMAX, and 1% penicillin-streptomycin. To maintain stable growth of the co-culture system, on the fourth day of culture, the medium was changed at 50% concentration, and 0.2 μL of a 1×10⁻⁶ titer was added. 13 Av / mL of AAV virus (adeno-associated virus, carrying an HL-ATP1.0 probe with an added hSyn promoter or GfaABC1D promoter and a 4×6T miR sequence) was used to express the probe; after 5-7 days of probe expression, it was labeled with the fluorescent dye JF635 and then confocal imaging was performed.

[0243] Imaging results showed that the HL-ATP1.0 probe was well expressed in both neurons and astrocytes. Figure 16 A, C). Furthermore, with treatment at 30 minutes using either a glycolysis inhibitor or a combination of both, a significant decrease in fluorescence signal was detected in both neurons and astrocytes. After the inhibition was lifted, some recovery of fluorescence signal was observed, but no recovery was observed when both glycolysis and oxidative phosphorylation (OXPHOX) in astrocytes were simultaneously inhibited. In the cytoplasm of neurons, the inhibition of glycolysis led to a significant decrease in ATP, and this decrease was greater than the decrease in ATP caused by the inhibition of oxidative phosphorylation. Figure 16(B) demonstrates that neuronal cell bodies are more dependent on glycolysis for ATP production. In astrocytes, inhibition of both glycolysis and oxidative phosphorylation leads to a significant decrease in ATP levels, suggesting that astrocytes exhibit a different energy metabolism mechanism than neurons under energy metabolic stress. Figure 16 D).

[0244] Example 8. The ability of the HL-ATP probe to detect ATP at the subcellular level

[0245] 1. The HL-ATP probe has the ability to detect ATP in mitochondria.

[0246] The HL-ATP series probes used in this study are soluble proteins that allow for sub-organelle localization through the fusion of specific targeting sequences. To detect mitochondrial ATP, four tandem Cox8 sequences (4×Cox8) were added to the N-terminus of the HL-ATP probe sequence. Cox8 is a subunit of cytochrome c oxidase (Complex IV). The protein it encodes exhibits typical mitochondrial targeting peptide (MTS) characteristics. Tandemly attaching the N-terminal localization sequences of the four Cox8 sequences to the target protein significantly enhances the mitochondrial localization signal. After the cell translates this fusion protein, the 4×Cox8 sequence is recognized by the mitochondrial protein introduction system, thus guiding the target protein to the mitochondria (its sequence is shown in SEQ ID NO:16), allowing it to anchor to the inner mitochondrial membrane of the cell. Figure 17 A), thus obtaining the Mito-HL-ATP1.0 probe (its sequence is shown in SEQ ID NO:17), the Mito-HL-ATP1h probe (its sequence is shown in SEQ ID NO:18), the Mito-HLATP1l probe (its sequence is shown in SEQ ID NO:19), and the control probe Mito_HL-ATPmut (its sequence is shown in SEQ ID NO:20). To verify their localization and function, the above probes were expressed in HeLa cells, and green fluorescent protein EGFP, which is localized to the inner mitochondrial membrane, was expressed as a mitochondrial localization marker. The cells were then labeled with the fluorescent dye JF635, followed by confocal imaging. Confocal microscopy imaging showed that the Mito-HL-ATP probe exhibited a punctate distribution consistent with the mitochondrial localization marker, and the two were co-localized ( Figure 17 B), indicating that the Cox8 sequence can effectively guide the probe to anchor in mitochondria.

[0247] Further functional experiments showed that treatment with the glycolysis inhibitor 2-DG resulted in decreased fluorescence signals in the Mito-HLATP1.0, Mito-HL-ATP1h, and Mito_HL-ATP1l probes labeled with the fluorescent dye JF635. Figure 18 A). Among them, the signal decrease of the Mito-HL-ATP1.0 and Mito-HL-ATP1l probes was the most significant ( Figure 18 B), which is consistent with their high ATP response range and low affinity as demonstrated in in vitro characterization. Figure 7 ).

[0248] In contrast, no significant change in fluorescence signal was observed in the Mito-HL-ATPmut probe used as a control, demonstrating the authenticity of the detection signal and the specificity of the probe response.

[0249] Based on the above results, a gene-encoded ATP fluorescent probe targeting mitochondria was successfully obtained, and it was confirmed that it can specifically monitor changes in ATP concentration in mitochondria, providing a powerful tool for studying mitochondrial energy metabolism and related physiological and pathological processes.

[0250] 2. The HL-ATP probe has the ability to detect ATP in the endoplasmic reticulum.

[0251] To detect ATP in the endoplasmic reticulum (ER), the HL-ATP probe was targeted to the ER compartment by adding an ER localization signal. Specifically, a calreticulin signal sequence (CSS) (SEQ ID NO. 21) was added to the N-terminus of the probe sequence, and an ER residency sequence (SEQ ID NO. 22) was added to the C-terminus of the probe sequence. This enabled the probe to localize to the ER, thus constructing the ER-HL-ATP1.0 probe (its sequence is shown in SEQ ID NO. 23) and its control ER-HL-ATPmut probe (its sequence is shown in SEQ ID NO. 24). Figure 19 A).

[0252] To determine whether the ER-HL-ATP probe successfully targeted the endoplasmic reticulum (ER), the HL-ATP1.0 probe was used. The constructed probe was expressed in HeLa cells, along with ER-mCherry (a red fluorescent protein with N-terminal CSS and C-terminal KDEL added, localizing to the ER) as an ER localization marker (shown in cyan). The probes were then labeled with the fluorescent dye JF635, followed by confocal microscopy imaging. Confocal microscopy results showed that the distribution of the ER-HL-ATP series probes exhibited a characteristic reticular structure similar to the ER (shown in red), and they co-localized with the ER localization marker (shown in gray). Figure 19 B).

[0253] Further functional experiments showed that treatment with the glycolysis inhibitor 2-DG significantly reduced the fluorescence signals of the ER-HL-ATP1.0, ER-HL-ATP1h, and ER-HL-ATP1l probes. The maximum decrease in fluorescence signal was significantly greater for ER-HL-ATP1.0 and ER-HL-ATP1h than for ER-HL-ATP1l, which is consistent with its higher apparent affinity and wider ATP response range observed in in vitro characterization. Figure 7 The fluorescence signal of the control probe ER-HLATPmut showed no significant change. Figure 19 B).

[0254] In summary, this study has enabled the application of HL-ATP probes to the endoplasmic reticulum, a key organelle, and established an imaging tool that can monitor changes in ATP concentration in the endoplasmic reticulum lumen in real time and with specificity. This provides new tool support for in-depth research on the relationship between endoplasmic reticulum energy metabolism and cell function and pathological processes.

[0255] Example 9. Coupling of HL-ATP probe with other probes

[0256] 1. Multicolor imaging capability of HL-ATP probes

[0257] To verify whether the HL-ATP probe of this invention can be used in combination with green and red fluorescent probes, a multicolor imaging experiment was performed on the same cell by co-expressing the HL-ATP1l probe labeled with the fluorescent dye JF635, the green glucose probe iGlucoSnFR (see Keller et al., 2021), and the red lactate probe R-iLACCO1.2 (see Nasu et al., 2023). The results showed that all three probes were well expressed in the same cell. Figure 20A). After treatment with a glycolysis inhibitor, the green fluorescence signal of glucose immediately decreased, while the red and far-red fluorescence signals of lactate and ATP showed a gradual decreasing trend; after the inhibition was lifted and glucose was replenished, the green fluorescence signal of glucose rapidly rebounded, lactate accumulated rapidly, while ATP slowly recovered. Figure 20 B).

[0258] Further simultaneous inhibition of glycolysis and oxidative phosphorylation showed that the green signal of glucose decreased rapidly, while the decrease in lactate and ATP signals was even more significant; after the inhibition was lifted and glucose was replenished, the green signal of glucose quickly recovered, while lactate and ATP slowly accumulated. Figure 20 (C and D).

[0259] The above results indicate that the HL-ATP probe can be used in conjunction with green and red fluorescent probes, and has the advantage of simultaneous detection of multiple probe molecules when exploring issues related to energy metabolism reprogramming.

[0260] 2. Coupling of HL-ATP probe with ADP probe

[0261] The inventors also developed an ADP probe for detecting ADP, which consists of two parts: ParM protein and green fluorescent protein (GFP), connected by linking peptides (N-linker and C-linker). The GFP is a circularly arranged enhanced green fluorescent protein (cpEGFP). The sequence of the ADP probe from the N-terminus to the C-terminus includes: amino acid sequences 1 to 196 of the ParM protein, the N-linker sequence, the cpEGFP sequence, the C-linker sequence, and amino acid sequences 197 to 320 of the ParM protein; the amino acid sequence of the ParM protein is shown in SEQ ID NO:42, and the amino acid sequence of the cpEGFP is shown in SEQ ID NO:43. The ParM protein undergoes a significant conformational change upon binding to ADP, thereby causing the GFP to emit light. The ADP probe used in this embodiment is an iADP0.7 probe, the sequence of which is shown in SEQ ID NO:25, and its preparation method is as described in the ADP probe preparation method above.

[0262] The HL-ATP1l probe sequence and the iADP0.7 probe sequence were constructed into the pCMV plasmid. The two plasmids were then transiently transfected into the same HEK293T cells at a 1:1 ratio. The HL-ATP1l probe was labeled with the fluorescent dye JF635, followed by confocal imaging. The imaging results showed ( Figure 21 A) Both probes can be well expressed in the same cell.

[0263] Inhibition of intracellular ATP synthesis was achieved by either inhibiting glycolysis alone or by simultaneously inhibiting glycolysis and oxidative phosphorylation. The results showed that ( Figure 21 (B) When glycolysis and oxidative phosphorylation are inhibited simultaneously, the fluorescence signal in the far-red channel decreases significantly, while the fluorescence signal in the green channel increases significantly. After the inhibition is lifted, the green fluorescence signal returns to the baseline level, and the far-red signal also returns to the baseline. This indicates that ADP levels increase significantly after the ATP production pathway is blocked. After the contact is interrupted, ADP levels gradually decrease as ATP recovers.

[0264] The above results indicate that the HL-ATP probe and ADP probe of the present invention can be used in combination in the same mammalian cell, enabling simultaneous detection of dynamic changes in ATP and ADP molecules.

[0265] Sequence listing (underlined sections indicate mutation sites, localization signal sequences, and linker peptide sequences)

[0266] SEQ ID NO.1: Amino acid sequence of the HL-ATP1.0 probe

[0267] MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQTIAVSGGNLEVRPDKVTIYAQAAERAEDIDVLRAKAAKERAER R LQSQ KKESIKEFLW ETFQAFRTTDVGRKLII DQ NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGGSGGTGGSG GTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEW YSAS DIDFKRAELALKRAMNRLSVAEMK

[0268] SEQ ID NO.2: Amino acid sequence of the ATP-binding domain of the HL-ATP1.0 probe

[0269] MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQTIAVSGGNLEVRPDKVTIYAQAAERAEDIDVLRAKAAKERAERRLQSQDIDFKRAELALKRAMNRLSVAEMK

[0270] SEQ ID NO.3: Amino acid sequence of HL-ATP1.0 (R103A) probe

[0271] MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQTIAVSGGNLEVRPDKVTIYAQAAERAEDIDVLRAKAAKERAER A LQSQ KKESIKEFLW ETFQAFR TT DVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGGSGGTGGSGGTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEW YSAS DIDFKRAELALKRAMNRLSVAEMK

[0272] SEQ ID NO.4: Amino acid sequence of the ATP binding domain of HL-ATP1.0 (R103A) probe

[0273] MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQTIAVSGGNLEVRPDKVTIYAQAAERAEDIDVLRAKAAKERAER A LQSQDIDFKRAELALKRAMNRLSVAEMK

[0274] SEQ ID NO.5: Amino acid sequence of HL-ATP1h probe

[0275] MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQTIAVSGGNLEVRPDKVTIYAQAAERAEDIDVLRAKAAKERAER R LQSQ KKESIKEFLW ETFQAFRTTDVGRKLII TT NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGGSGGTGGSGGTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEW YSAS DIDFKRAELALKRAMNRLSVAEMK

[0276] SEQ ID NO.6: Amino acid sequence of the ATP binding domain of HL-ATP1h probe

[0277] MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQTIAVSGGNLEVRPDKVTIYAQAAERAEDIDVLRAKAAKERAER R LQSQDIDFKRAELALKRAMNRLSVAEMK

[0278] SEQ ID NO.7: Amino acid sequence of HLATP1l probe

[0279] MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQTIAVSGGNLEVRPDKVTIYAQAAERAEDIDVLRAKAAKERAER A LQSQ KKESIKEFLWETFQAFRTTDVGRKLII TT NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGGSGGTGGSGGTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEW YSAS DIDFKRAELALKRAMNRLSVAEMK

[0280] SEQ ID NO. 8: Amino acid sequence of the ATP binding domain of the HLATP1l probe

[0281] MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQTIAVSGGNLEVRPDKVTIYAQAAERAEDIDVLRAKAAKERAER A LQSQDIDFKRAELALKRAMNRLSVAEMK

[0282] SEQ ID NO. 9: Amino acid sequence of the HL-ATPmut probe

[0283] MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQTIAVSGGNLEVRPDKVTIYAQAAERAEDI A VL A AKAAKERAERRLQSQ KKESIKEFLWETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGGSGGTGGSGGTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEW YSAS DIDFKRAELALKRAMNRLSVAEMK

[0284] SEQ ID NO.10: Amino acid sequence of the ATP binding domain of HL-ATPmut probe

[0285] MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQTIAVSGGNLEVRPDKVTIYAQAAERAEDI A VL A AKAAKERAERRLQSQDIDFKRAELALKRAMNRLSVAEMK

[0286] SEQ ID NO.11: Amino acid sequence of the dye binding domain cpHaloTag

[0287] ETFQAFRTTDVGRKLII DQ NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGGSGGTGGSGGTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEW

[0288] SEQ ID NO.12: Amino acid sequence of the dye-binding domain cpHaloTag variant (D18T Q19T mutation)

[0289] ETFQAFRTTDVGRKLII TT NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGGSGGTGGSG GTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEW

[0290] SEQ ID NO.13: Nucleotide sequence of the dye-binding domain cpHaloTag variant (D18T Q19T mutation)

[0291] GAGACCTTCCAGGCCTTCCGCACCACCGACGTCGGCCGCAAGCTGATCATCACGACTAACGTTTTTATCGAGGGTACGCTGCCGATGGGTGTCGTCCGCCCGCTGACTGAAGTCGAGATGGACCATTACCGCGAGCCGTTCCTGAATCCTGTTGACCGCGAGCCACTGTGGCGCTTCCCAAACGAGCTGCCAATCGCCGGTGAGCCAGCGAACATCGTCGCGCTGGTCGAAGAATACATGGACTGGCTGCACCAGTCCCCTGTCCCGAAGCTGCTGTTCTGGGGCACCCCAGGCGTTCTGATCCCACCGGCCGAAGCCGCTCGCCTGGCCAAAAGCCTGCCTAACTGCAAGGCTGTGGACATCGGCCCGGGTCTGAATCTGCTGCAAGAAGACAACCCGGACCTGATCGGCAGCGAGATCGCGCGCTGGCTGTCGACGCTCGAGATTTCCGGCGGAGGAACAGGTGGTTCTGGTGGAACAGGGGGTAGCGGAGGTACAGGAGGAAGTATGGCGGAGATCGGAACTGGATTCCCGTTTGATCCGCATTATGTGGAAGTTCTGGGAGAGCGCATGCATTATGTGGACGTTGGTCCTCGTGATGGGACACCAGTGCTGTTCCTTCACGGCAATCCGACATCGTCGTACGTGTGGCGTAATATCATCCCGCACGTTGCCCCCACGCACCGCTGCATTGCCCCTGACTTAATTGGTATGGGGAAAAGTGATAAGCCTGATCTGGGGTACTTCTTTGACGACCACGTACGCTTCATGGATGCTTTTATTGAAGCATTGGGTTTGGAGGAAGTAGTTTTGGTGATCCATGATTGGGGTAGTGCTCTGGGGTTCCATTGGGCCAAGCGTAACCCAGAACGCGTGAAAGGAATTGCCTTTATGGAGTTCATCCGTCCGATTCCAACATGGGACGAATGG

[0292] SEQ ID NO.14: Amino acid sequence of NES-HL-ATP1.0 probe

[0293] MGNLNELALKLAGLDI MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQTIAVSGGNLEVRPDKVTIYAQAAERAEDIDVLRAKAAKERAERRLQSQKKESIKEFLWETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGGSGGTGGSGGTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWYSASDIDFKRAELALKRAMNRLSVAEMK

[0294] SEQ ID NO.15: NES localization signal sequence

[0295] MGNLNELALKLAGLDI

[0296] SEQ ID NO.16: Mitochondrial localization signal (Mito) sequence

[0297] MSVLTPLLLRGLTGSARRLLPVPRAKIHSLGSVLTPLLLRGLTGSARRLLPVPRAKIHSLSVLTPLLLRGL TGSARLPVPRAKIHSLGSVLTPLLLRGLTGSARRLLPVPRAKIHSLTG

[0298] SEQ ID NO.17: Amino acid sequence of Mito-HL-ATP1.0 probe

[0299] MSVLTPLLLRGLTGSARRLLPVPRAKIHSLGSVLTPLLLRGLTGSARRLLPVPRAKIHSLSVLTPLLLRGL TGSARLPVPRAKIHSLGSVLTPLLLRGLTGSARRLLPVPRAKIHSLTG MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQTIAVSGGNLEVRPDKVTIYAQAAERAEDIDVLRAKAAKERAER RLQSQKKESIKEFLWETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGGSGGTGGSGGTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWYSASDIDFKRAELALKRAMNRLSVAEMK

[0300] SEQ ID NO. 18: Amino acid sequence of Mito-HL-ATP1h probe

[0301] MSVLTPLLLRGLTGSARRLLPVPRAKIHSLGSVLTPLLLRGLTGSARRLLPVPRAKIHSLSVLTPLLLRGL TGSARLPVPRAKIHSLGSVLTPLLLRGLTGSARRLLPVPRAKIHSLTG MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQTIAVSGGNLEVRPDKVTIYAQAAERAEDIDVLRAKAAKERAER R LQSQKKESIKEFLWETFQAFRTTDVGRKLII TT NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGGSGGTGGSGGTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWYSASDIDFKRAELALKRAMNRLSVAEMK

[0302] SEQ ID NO.19: Amino acid sequence of Mito-HL-ATP1l probe

[0303] MSVLTPLLLRGLTGSARRLLPVPRAKIHSLGSVLTPLLLRGLTGSARRLLPVPRAKIHSLSVLTPLLLRGL TGSARLPVPRAKIHSLGSVLTPLLLRGLTGSARRLLPVPRAKIHSLTG KNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQTIAVSGGNLEVRPDKVTIYAQAAERAEDIDVLRAKAAKERAER A LQSQKKESIKEFLWETFQAFRTTDVGRKLII TT NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGGSGGTGGSGGTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWYSASDIDFKRAELALKRAMNRLSVAEMK

[0304] SEQ ID NO.20: Amino acid sequence of Mito-HL-ATPmut probe

[0305] MSVLTPLLLRGLTGSARRLLPVPRAKIHSLGSVLTPLLLRGLTGSARRLLPVPRAKIHSLSVLTPLLLRGL TGSARLPVPRAKIHSLGSVLTPLLLRGLTGSARRLLPVPRAKIHSLTG MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQTIAVSGGNLEVRPDKVTIYAQAAERAEDI A VL AAKAAKERAERRLQSQKKESIKEFLWETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGG SGGTGGSGGTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWYSASDIDFKRAELALKRAMNRLSVAEMK

[0306] SEQ ID NO.21: Calreticulin signal sequence (CSS)

[0307] MLLPVPLLLGLLGAAAD

[0308] SEQ ID NO.22: Endoplasmic reticulum resident sequence

[0309] KDEL

[0310] SEQ ID NO.23: Amino acid sequence of the ER-HL-ATP1.0 probe

[0311] MLLPVPLLLGLLGAAADKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQTIAVSGGNLEVRPDKVTIYAQAAERAEDIDVLRAKAAKERAERRLQSQKKESIKEFLWETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGGSGGTGGSGGTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWYSASDIDFKRAELALKRAMNRLSVAEMKGGTGGSLQVDEQKLISEEDLN KDEL

[0312] SEQ ID NO.24: Amino acid sequence of ER-HL-ATPmut probe

[0313] MLLPVPLLLGLLGAAAD KNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQTIAVSGGNLEVRPDKVTIYAQAAERAEDI A VL AAKAAKERAERRLQSQKKESIKEFLWETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGGSGGTGGSGGTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWYSASDIDFKRAELALKRAMNRLSVAEMKGGTGGSLQVDEQKLISEEDLN KDEL

[0314] SEQ ID NO.25: Amino acid sequence of iADP0.7 probe

[0315] MLVFIDDGSTNIKLQWQESDGTIKQHISPNSFKREWAVSFGDKKVFNYTLNGEQYSFDPISPDAVVTTNIAWQYSDVNVVAVHHALLTSGLPVSEVDIVCTLPLTEYYDRNNQPNTENIERKKANFRKKITLNGGDTFTIKDVKVMPESIPAGYEVLQELDELDSLLIIDLGGTTLDISQVMGKLSGISKIYGDSS ALDLEEGG NVYIKADKQKNGIKANFTIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATEGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN TGAPILGVSLVTSAVKDALSLARTKGSSYLADDIIIHRKDNNYLKQRINDENKISIVTEAMNEALRKLEQRVLNTLNEFSGYTHVMVIGGGAELICDAVKKHTQIRDERFFKTNNSQYDLVNGMYLIGN

[0316] SEQ ID NO.26: Amino acid sequence of PS3 ε subunit protein

[0317] MKTIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLVAPLEISAARLKKGGKTQYIAVSGGFLEVRPDKVTILAQAAERAEDIDVLRAKAAKERAERRLQSQQDDIDFKRAELALKRAMNRLSVAEMK

[0318] SEQ ID NO.27: N-Linker of HL-ATP0.3 probe

[0319] LEFLW

[0320] SEQ ID NO.28: C-Linker of HL-ATP0.3 probe

[0321] YESAS

[0322] SEQ ID NO.29: Amino acid sequence of the HL-ATP0.3 probe

[0323] MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQ Y IAVSGGNLEVRPDKVTIYAQAAERAEDIDVLRAKAAKERAER R LQSQ LEFLW ETFQAFRTTDVGRKLII DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGGSGGTGGSGGTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEW YESAS DIDFKRAELALKRAMNRLSVAEMK

[0324] SEQ ID NO.30: N-Linker of HL-ATP0.5 probe

[0325] KRDHMLEFLW

[0326] SEQ ID NO.31: C-Linker of HL-ATP0.5 probe

[0327] YESAS

[0328] SEQ ID NO.32: Amino acid sequence of HL-ATP0.5 probe

[0329] MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQ Y IAVSGGNLEVRPDKVTIYAQAAERAEDIDVLRAKAAKERAER R LQSQ KRDHMLEFLW ETFQAFRTTDVGRKLII DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGGSGGTGGSGGTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEW YESAS DIDFKRAELALKRAMNRLSVAEMK

[0330] SEQ ID NO.33: N-Linker of HL-ATP0.7 probe

[0331] KRDHTKEFLW

[0332] SEQ ID NO.34: C-Linker of HL-ATP0.7 probe

[0333] YESAS

[0334] SEQ ID NO.35: Amino acid sequence of HL-ATP0.7 probe

[0335] MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQYIAVSGGNLEVRPDKVTIYAQAAERAEDIDVLRAKAAKERAER R LQSQ KRDHTKEFLW ETFQAFRTTDVGRKLII DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGGSGGTGGSGGTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEW YESAS DIDFKRAELALKRAMNRLSVAEMK

[0336] SEQ ID NO.36: N-Linker of HL-ATP0.9 probe

[0337] KKESIKEFLW

[0338] SEQ ID NO.37: C-Linker of HL-ATP0.9 probe

[0339] YSAS

[0340] SEQ ID NO.38: Amino acid sequence of HL-ATP0.9 probe

[0341] MKNIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLKAPLEISAARLKKGGKTQ Y IAVSGGNLEVRPDKVTIYAQAAERAEDIDVLRAKAAKERAER R LQSQ KKESIKEFLW ETFQAFRTTDVGRKLII DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGTGGSGGTGGSGGTGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEW YSAS DIDFKRAELALKRAMNRLSVAEMK

[0342] SEQ ID NO.39: Sequence of hSyn promoter

[0343] ATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGTCGAGAAACCGGTGCCACC

[0344] SEQ ID NO.40: Sequence of GfaABC1D promoter

[0345]

[0346] SEQ ID NO.41: Sequence of miRNA targeted expression cassette

[0347] GGCATTCACCGCGTGCCTTACTACGCGTATTCTTAAGCAATAAGCGAATATAACACGGTCGATCTAAAAAGGTTAGCTGGGTGTGTTAGAAGCCCTGCAAGACGACCTGCGCATTATTACTCACGGTACGACGCATTATTACTCACGGTACGAGGCATTCACCGCGTGCCTTACTACGCGTATTCTTAAGCAATAAGCGAATATAACACGGTCGATCTAAAAAGGTTAGCTGGGTGTGTTAGAAGCCCTGCAAGACGACCTGGGCATTCACCGCGTGCCTTACTACGCGTATTCTTAAGCAATAAGCGAATATAACACGGTCGATCTAAAAAGGTTAGCTGGGTGTGTTAGAAGCCCTGCAAGACGACCTGGGTACCGCATTATTACTCACGGTACGACGCATTATTACTCACGGTACGAGGCATTCACCGCGTGCCTTACTACGCGTATTCTTAAGCAATAAGCGAATATAACACGGTCGATCTAAAAAGGTTAGCTGGGTGTGTTAGAAGCCCTGCAAGACGACCTG

[0348] SEQ ID NO:42: Amino acid sequence of ParM protein

[0349] MLVFIDDGSTNIKLQWQESDGTIKQHISPNSFKREWAVSFGDKKVFNYTLNGEQYSFDPISPDAVVTTNIAWQYSDVNVVAVHHALLTSGLPVSEVDIVCTLPLTEYYDRNNQPNTENIERKKANFRKKITLNGGDTFTIKDVKVMPESIPAGYEVLQELDELDSLLIIDLGGTTLDISQVMGKLSGISKIYGDSSLGVSLVTSAVKDALSLARTKGSSYLADDIIIHRKDNNYLKQRINDENKISIVTEAMNEALRKLEQRVLNTLNEFSGYTHVMVIGGGAELICDAVKKHTQIRDERFFKTNNSQYDLVNGMYLIGN

[0350] SEQ ID NO:43: Amino acid sequence of cpEGFP

[0351] NVYIKADKQKNGIKANFTIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATEGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

[0352] SEQ ID NO.44: Sequence of Strep tag

[0353] WSHPQFEK

[0354] SEQ ID NO.45: Sequence of FLAG tag

[0355] DYKDDDDDK

[0356] SEQ ID NO.46: Sequence of HA tag

[0357] YPYDVPDYA

[0358] SEQ ID NO.47: Sequence of Myc tag

[0359] EQKLISEEDL

[0360] SEQ ID NO.48: Sequence of V5 tag

[0361] GKPIPNPLLGLDST

[0362] SEQ ID NO:49

[0363] GGGGS

[0364] SEQ ID NO:50

[0365] QSGS

[0366] References

[0367] ARAI S, KRISZT R, HARADA K, et al. 2018. RGB-Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells. Angew Chem Int Ed Engl [J], 57: 10873-10878.

[0368] CHEN W, ZHANG Y, YEO W S, et al. 2017. Rapid and efficient genome editing in staphylococcus aureus by using an engineered CRISPR / Cas9 system. J Am Chem Soc [J], 139: 3790-3795.

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Claims

1. An ATP-binding protein, the sequence of which from the N-terminus to the C-terminus includes: The amino acid sequences of the ATP-binding domain from position 1 to 107, the N-terminal linker sequence, the dye-binding domain sequence, the C-terminal linker sequence, and the amino acid sequences of the ATP-binding domain from position 108 to 131. The ATP-binding domain sequence is as follows: (1) The sequence shown in SEQ ID NO:2; (2) The sequence shown in SEQ ID NO:4; (3) The sequence shown in SEQ ID NO:6; (4) The sequence shown in SEQ ID NO:8; or (5) The sequence shown in SEQ ID NO:10; The dye-binding domain sequence is as follows: (A) The sequence shown in SEQ ID NO:11; or (B) The sequence shown in SEQ ID NO:12; The sequences of the N-terminal connector and the C-terminal connector are as follows: (a) The sequences shown in SEQ ID NO:27 and SEQ ID NO:28; (b) The sequences shown in SEQ ID NO:30 and SEQ ID NO:31; (c) The sequences shown in SEQ ID NO:33 and SEQ ID NO:34; or (d) The sequences shown in SEQ ID NO:36 and SEQ ID NO:

37.

2. The ATP-binding protein of claim 1, having the sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35 or SEQ ID NO:

38.

3. The ATP-binding protein of claim 1 or 2, further comprising a localization sequence, a purification tag, or an immunoblot tag.

4. The ATP-binding protein of claim 3, wherein the localization sequence is a nuclear export signal sequence, a mitochondrial localization sequence, an endoplasmic reticulum localization sequence, a neuronal localization sequence, an astrocyte localization sequence, a calreticulin signal sequence, and / or an endoplasmic reticulum resident sequence.

5. The ATP-binding protein of claim 4, wherein the nuclear output signal sequence is shown in SEQ ID NO:15, the mitochondrial localization sequence is shown in SEQ ID NO:16, the calreticulin signal sequence is shown in SEQ ID NO:21, the endoplasmic reticulum resident sequence is shown in SEQ ID NO:22, the neuronal localization sequence is shown in SEQ ID NO:39, and the astrocyte localization sequence is shown in SEQ ID NO:

40.

6. The ATP-binding protein of claim 3, wherein the purification tag includes a histidine tag, a Strep tag, or a FLAG tag.

7. The ATP-binding protein of claim 3, wherein the immunoblot tag includes an HA tag, a Myc tag, or a V5 tag.

8. A far-red fluorescent ATP probe comprising the ATP-binding protein of any one of claims 1-7 labeled with a far-red fluorescent dye.

9. The far-red fluorescent ATP probe of claim 8, wherein the far-red fluorescent dye is a dye with a chloroalkane group.

10. The far-red fluorescent ATP probe of claim 9, wherein the dye comprises JF635, JF639, JF646, SiR650, BD626 or BD666.

11. A nucleic acid molecule comprising: (1) The coding sequence of the ATP-binding protein according to any one of claims 1-7; (2) A sequence complementary to (1).

12. Nucleic acid constructs, including the nucleic acid molecule of claim 11.

13. The nucleic acid construct of claim 12, wherein the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.

14. A host cell comprising, expressing, or secreting: the ATP-binding protein of any one of claims 1-7, the nucleic acid molecule of claim 11, and / or the nucleic acid construct of claim 12 or 13.

15. A kit for detecting ATP, comprising: an ATP-binding protein according to any one of claims 1-7, a nucleic acid molecule according to claim 11, a nucleic acid construct according to claim 12 or 13, and / or a host cell according to claim 11, and a far-red fluorescent dye.

16. The kit of claim 12, wherein the far-red fluorescent dye is a dye with a chloroalkane group.

17. The kit of claim 16, wherein the dye comprises JF635, JF639, JF646, SiR650, BD626 or BD666.

18. A method for preparing the ATP-binding protein according to any one of claims 1-7, comprising: The host cell of claim 14 is cultured, and the ATP-binding protein is isolated from the culture.

19. Use of the far-red fluorescent ATP probe binding protein according to any one of claims 8-10 in the detection of ATP.

20. Use of the far-red fluorescent ATP probe-binding protein according to any one of claims 8-10 in the intracellular and / or extracellular localization of ATP.

21. A method for detecting ATP in a sample, the method comprising: The ATP-binding protein of any one of claims 1-7, the nucleic acid molecule of claim 11, or the nucleic acid construct of claim 12 or 13 is introduced into the sample and labeled with a far-red fluorescent dye. The amount of ATP is determined based on the generated fluorescence intensity; or The far-red fluorescent ATP probe of any one of claims 8-10 is introduced into the sample, and the amount of ATP is determined based on the generated fluorescence intensity.

22. A method for localizing ATP intracellularly and / or extracellularly, the method comprising: The ATP-binding protein of any one of claims 1-7, the nucleic acid molecule of claim 11, or the nucleic acid construct of claim 12 or 13 is introduced into the cells and / or extracellularly, and labeled with a far-red fluorescent dye, and the location of ATP is determined according to the location of fluorescence. or The far-red fluorescent ATP probe of any one of claims 8-10 is introduced into the sample, and the location of ATP is determined according to the location where fluorescence is generated.

23. A method for simultaneously detecting ATP and ADP in a sample, the method comprising: The ATP-binding protein of any one of claims 1-7, the nucleic acid molecule of claim 11, or the nucleic acid construct of claim 12 or 13 is introduced into the sample and labeled with a far-red fluorescent dye. An ADP-binding protein, whose sequence includes the sequence shown in SEQ ID NO. 25, is introduced into the sample, and the amounts of ATP and ADP are determined based on the generated fluorescence intensity; or... The far-red fluorescent ATP probe of any one of claims 8-10 is introduced into the sample, and the ADP-binding protein is introduced into the sample, the ADP-binding protein sequence comprising the sequence shown in SEQ ID NO.25, and the amounts of ATP and ADP are determined based on the generated fluorescence intensity.

24. The method of claim 23, wherein the ADP-binding protein further comprises a localization sequence, a purification tag, or an immunoblot tag.

25. The method of claim 24, wherein the purification tag includes a histidine tag, a Strep tag, or a FLAG tag; and the immunoblotting tag includes an HA tag, a Myc tag, or a V5 tag.

26. A method for simultaneously detecting ATP and glucose and / or lactate in a sample, the method comprising: The ATP-binding protein of any one of claims 1-7, the nucleic acid molecule of claim 11, or the nucleic acid construct of claim 12 or 13 is introduced into the sample and labeled with a far-red fluorescent dye. The glucose probe iGlucoSnFR and / or the lactate probe R-iLACCO1.2 are then introduced into the sample, and the amounts of ATP, glucose, and / or lactate are determined based on the generated fluorescence intensity; or The far-red fluorescent ATP probe of any one of claims 8-10 is introduced into the sample, and the glucose probe iGlucoSnFR and / or the lactate probe R-iLACCO1.2 is introduced into the sample. The amount of ATP and glucose and / or lactate is determined based on the generated fluorescence intensity.

27. The method of any one of claims 21-26, wherein the far-red fluorescent dye is a dye with a chloroalkane group.

28. The method of claim 27, wherein the dye comprises JF635, JF639, JF646, SiR650, BD626 or BD666.

Images