ATP (adenosine triphosphate) optical probe as well as preparation method and application thereof

Abstract

The invention relates to an adenosine triphosphate optical probe, in particular to an adenosine triphosphate (ATP) optical probe as well as a preparation method and application thereof. Specifically, the invention provides an adenosine triphosphate optical probe, which comprises an adenosine triphosphate sensitive polypeptide and an optically active polypeptide, and the optically active polypeptide is located in the sequence of the adenosine triphosphate sensitive polypeptide.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of optical probe, in particular to an ATP optical probe and a preparation method and application thereof. BACKGROUND

[0002] Adenosine triphosphate (ATP) is a nucleotide that acts as currency in energy transfer in cells, playing a function of storing and transferring chemical energy; ATP also plays an important role in the process of nucleic acid synthesis. The molecule is composed of adenosine and three phosphate groups, widely exists in all living things, and is involved in many biochemical processes, thus has a great influence on the physiological and pathological state of organisms, and once imbalance may cause diabetes, neurodegenerative diseases, neuromuscular diseases, inflammatory reactions and even cancer.

[0003] The most commonly used methods for detecting ATP at present include chromatography, electrophoresis, bioluminescence, quantum dot method, and gene-encoded ATP probes. Among them, the chromatography method is expensive in consumables and very strict in buffer selection, the electrophoresis method is high in consumable cost and cannot be applied to complex samples, the bioluminescence method is simple and intuitive, but the sensitivity is limited and the enzyme stability is poor, while the quantum dot method has high brightness and low detection limit, but the potential toxicity and the selection of surface functionalized aptamer also affect its popularization and use. The currently developed gene-encoded fluorescent probes have limited specificity and limited response range, and the affinity to the substrate cannot complete in vivo real-time monitoring, so it is urgent to develop a genetically encoded fluorescent probe that can monitor the dynamic changes of ATP in living cells in situ. SUMMARY

[0004] The purpose of the present application is to provide a probe and method for real-time positioning, high-throughput and quantitative detection of ATP in and out of cells.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0006] The first aspect of the present application provides an ATP binding protein variant, which:

[0007] (a) has the sequence shown in SEQ ID NO: 1 and has a mutation at 1, 2, 3, 4, 5 or more sites selected from I88, D89, V90, R92, R102, R122, the mutation including modification, substitution or deletion of amino acids,

[0008] (b) is a sequence having at least 70% sequence identity with the sequence of (a) and having (1) the mutation and retaining the ability to bind ATP.

[0009] In one or more embodiments, the ATP-binding protein variant mutation comprises a mutation at any 1, 2, 3, 4, 5 or more of the following positions: D89, I88, V90, R92, R102, R122.

[0010] In one or more embodiments, the mutation comprises a mutation at any of the following groups of positions: (1) R102 and D89, (2) R102 and I88, (3) R102 and R92, (4) R102 and R122, (5) R102 and V90.

[0011] In one or more embodiments, D89 is mutated to E, V, M, N, F, I, Q, S, H, Y, K, T or W. In one or more embodiments, I88 is mutated to V or L. In one or more embodiments, R92 is mutated to A, M, Q, H or K. In one or more embodiments, V90 is mutated to I, D, A, R, L, F, E, M, C, P, Q, N, G, H, Y, K, S or T. In one or more embodiments, R122 is mutated to N or K. In one or more embodiments, R102 is mutated to T.

[0012] In one or more embodiments, the mutation comprises a mutation selected from any one of the following groups: (1) D89E, (2) D89V, (3) D89M, (4) D89Q, (5) D89N, (6) D89Y, (7) D89S, (8) V90D, (9) V90A, (10) V90L, (11) V90E, (12) V90M, (13) V90C, (14) V90P, (15) V90H, (16) V90K, (17) V90S, (18) V90T, (19) R102T, (20) R122N, (21) R122K, (22) R102T and D89V, (23) R102T and D89F, (24) R102T and D89I, (25) R102T and D89Q, (26) R102T and D89H, (27) R102T and D89Y, (28) R102T and D89K, (29) R102T and D89T, (30) R102T and D89W, (31) R102T and E83R, (32) R102T and I88V, (33) R102T and I88L, (34) R102T and R92A, (35) R102T and R92H, (36) R102T and R92M, (37) R102T and R92Q (38) R102T and R92H, (39) R102T and R92K, (40) R102T and R122K, (41) R102T and V90I, (42) R102T and V90D (43) R102T and V90R, (44) R102T and V90L, (45) R102T and V90F, (46) R102T and V90E, (47) R102T and V90C, (48) R102T and V90P, (49) R102T and V90Q, (50) R102T and V90N, (51) R102T and V90G, (52) R102T and V90H, (53) R102T and V90Y, (54) R102T and V90K, (55) R102T and V90S, (56) R102T and V90T.

[0013] The first aspect of the present application also provides an ATP optical probe comprising an ATP-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located between residues 10-13, 19-21, 27-30, 33-38, 41-43, 46-48, 54-57, 61-63, 66-69, 71-74, 78-90 and 103-116 of the ATP-sensitive polypeptide, the ATP-sensitive polypeptide being an ATP-binding protein or a functional variant thereof, and the optically active polypeptide being a fluorescent protein or a functional variant thereof. The ATP-sensitive polypeptide is divided into a first part and a second part by the optically active polypeptide.

[0014] In one or more embodiments, the ATP-sensitive polypeptide comprises an ATP-binding protein or a functional variant thereof. In one or more embodiments, the ATP-sensitive polypeptide is derived from the epsilon subunit of the F0F1-ATP synthase of Bacillus thermophilus PS3.

[0015] In the optical probe of the present application, the ATP-sensitive polypeptide has:

[0016] (1) the sequence set forth in SEQ ID NO: 1, or a sequence having at least 70% sequence identity thereto and retaining ATP-binding activity,

[0017] (2) the sequence of the ATP-binding protein variant described in any one of the embodiments of the first aspect herein, or

[0018] (3) a sequence having at least 70% sequence identity to the sequence of (2) and having the mutations of (2) and retaining ATP-sensitivity.

[0019] In one or more embodiments, the optically active polypeptide is a fluorescent protein or a functional variant thereof. In one or more embodiments, the fluorescent protein is selected from yellow fluorescent protein (such as cpYFP set forth in SEQ ID NO: 2), orange fluorescent protein (such as cpmOrange set forth in SEQ ID NO: 3), red fluorescent protein (such as mKate set forth in SEQ ID NO: 4 or 8, mcherry set forth in SEQ ID NO: 5), green fluorescent protein (such as cpGFP set forth in SEQ ID NO: 6), blue fluorescent protein (such as cpBFP set forth in SEQ ID NO: 7), and apple red fluorescent protein (such as cpmApple set forth in SEQ ID NO: 9). Preferably, the optically active polypeptide is cpYFP. In one or more embodiments, the fluorescent protein has the sequence set forth in any one of SEQ ID NOs: 2-9.

[0020] In one or more embodiments, the functional variant of the fluorescent protein has: (1) the sequence set forth in SEQ ID NO: 2 and has a mutation at the Y1 site, the mutation comprising a modification, substitution or deletion of an amino acid, the numbering of the amino acid corresponding to the sequence of the fluorescent protein, or (2) a sequence having at least 70% sequence identity to the sequence of (1). In one or more embodiments, the mutation of the fluorescent protein is selected from any one or more of Y1R, Y1L, Y1V, Y1A, Y1G, Y1E, Y1W, Y1S, Y1K and Y1I. In one or more embodiments, the mutation of the fluorescent protein is Y1L.

[0021] In one or more embodiments, the optically active polypeptide is located between residues 10-13, 19-21, 27-30, 33-38, 41-43, 46-48, 54-57, 61-63, 66-69, 71-74, 78-90, and 103-116 of the ATP-sensitive polypeptide, numbering corresponding to the full length of the ATP-sensitive polypeptide.

[0022] In one or more embodiments, the optically active polypeptide is located at any one or more of the following positions of the ATP-sensitive polypeptide: 11 / 13, 12 / 13, 19 / 21, 27 / 30, 28 / 28, 28 / 30, 29 / 28, 29 / 29, 33 / 35, 33 / 36, 33 / 38, 34 / 34, 34 / 35, 35 / 34, 35 / 35, 35 / 36, 35 / 37, 36 / 34, 36 / 35, 36 / 36, 36 / 37, 36 / 38, 37 / 38, 41 / 42, 41 / 43, 41 / 44, 42 / 42, 42 / 43, 43 / 42, 43 / 43, 46 / 46, 46 / 47, 47 / 46, 47 / 47, 47 / 48, 54 / 55, 55 / 55, 55 / 57, 56 / 55, 56 / 57, 61 / 63, 62 / 62, 63 / 62, 66 / 67, 66 / 69, 67 / 67, 67 / 69, 68 / 67, 68 / 69, 71 / 72, 71 / 73, 72 / 73, 72 / 74, 73 / 73, 73 / 74, 78 / 80, 78 / 81, 80 / 80, 80 / 81, 84 / 85, 84 / 91, 85 / 88, 85 / 91, 86 / 85, 86 / 86, 86 / 88, 86 / 90, 87 / 88, 87 / 89, 87 / 90, 87 / 91, 88 / 86, 88 / 87, 88 / 88, 88 / 89, 89 / 88, 89 / 90, 89 / 91, 90 / 89, 90 / 90, 103 / 104, 103 / 108, 103 / 109, 103 / 110, 103 / 112, 103 / 113, 103 / 115, 103 / 116, 104 / 104, 104 / 105, 104 / 106, 104 / 107, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 104 / 112, 104 / 114, 104 / 116, 105 / 104, 105 / 105, 105 / 106, 105 / 107, 105 / 108, 105 / 109, 105 / 110, 105 / 112, 105 / 114, 105 / 116, 106 / 104, 106 / 107, 106 / 111, 106 / 113, 106 / 114, 106 / 115, 106 / 116, 107 / 111, 107 / 113, 107 / 116, 108 / 109, 108 / 110, 108 / 111, 108 / 112, 108 / 113, 108 / 115, 108 / 113, 109 / 109, 109 / 110, 109 / 112, 109 / 113, 109 / 116, 110 / 104, 110 / 112, 110 / 113, 110 / 115, 110 / 116, 111 / 107, 110 / 109, 111 / 110,111 / 111, 111 / 112, 111 / 113, 111 / 116, 112 / 104, 112 / 105, 112 / 110, 112 / 114, 112 / 115, 112 / 116, 113 / 108, 113 / 109, 113 / 110, 113 / 113, 113 / 115, 113 / 116, 114 / 104, 114 / 105, 114 / 106, 114 / 108, 114 / 109, 114 / 110, 114 / 111, 114 / 115, 114 / 116, 115 / 108, 115 / 111, 115 / 113, 115 / 115, and 115 / 116. More preferably, the optically active polypeptide is located at any one or more of the following positions of the ATP- sensitive polypeptide: 12 / 13, 55 / 56, 56 / 57, 67 / 69, 103 / 110, 105 / 104, and 109 / 112.

[0023] In one or more embodiments, the fluorescent protein has the sequence set forth in SEQ ID NO: 2 or is a variant thereof having any one or more of the following mutations at position 1 of the amino acid: 1R, 1L, 1V, 1A, 1G, 1E, 1W, 1S, 1K, and 1I, and the optically active polypeptide is located at any one or more of the following positions of the ATP-sensitive polypeptide: 12 / 13, 55 / 56, 56 / 57, 67 / 69, 103 / 110, 105 / 104, 109 / 112, 33 / 36, 108 / 109, and 113 / 115.

[0024] In one or more embodiments, the fluorescent protein has the sequence set forth in SEQ ID NO: 7 or is a variant thereof having any one or more of the following mutations at position 1 of the amino acid: 1R, 1L, 1V, 1A, 1G, 1E, 1W, 1S, 1K, and 1I, and the optically active polypeptide is located at any one or more of the following positions of the ATP-sensitive polypeptide: 12 / 13, 55 / 56, 56 / 57, 67 / 69, 103 / 110, 105 / 104, 109 / 112, 35 / 35, 107 / 113, and 108 / 109.

[0025] In one or more embodiments, the fluorescent protein has the sequence set forth in SEQ ID NO: 9 or is a variant thereof having any one or more of the following mutations at position 1 of the amino acid: 1R, 1L, 1V, 1A, 1G, 1E, 1W, 1S, 1K, and 1I, and the optically active polypeptide is located at any one or more of the following positions of the ATP-sensitive polypeptide: 12 / 13, 55 / 56, 56 / 57, 67 / 69, 103 / 110, 105 / 104, 109 / 112, 35 / 35, 47 / 46, and 110 / 109.

[0026] In one embodiment, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linker according to the present application can be any amino acid sequence of any length. In one embodiment, the optically active polypeptide is flanked by a linker of no more than 5 amino acids, such as a linker of 0, 1, 2, 3, 4 amino acids. In one embodiment, the linker flanking the optically active polypeptide comprises the amino acid Y. In one embodiment, the linker Y is located at the N-terminus and / or C-terminus of the optically active polypeptide. In one embodiment, the optical probe is as follows: first part of the ATP-sensing polypeptide B1, first linker Y1, optically active polypeptide A, second linker Y2, second part of the ATP-sensing polypeptide B2. In one embodiment, the optical probe according to the present application does not comprise a linker.

[0027] In one embodiment, the optical probe according to the present application further comprises a localization sequence for localizing the probe to a specific organelle, such as a cell. Preferred organelles are subcellular organelles, more preferred are cytoplasm, nucleus and mitochondria.

[0028] In one or more embodiments, the amino acid sequence of the optical probe is as set forth in any one of SEQ ID NOs: 10-11.

[0029] In one or more embodiments, in the optical probe, the ATP-sensing polypeptide is as set forth in SEQ ID NO: 1, the optically active polypeptide is as set forth in any one of SEQ ID NOs: 2-9 (preferably SEQ ID NOs: 2, 6, 7, 9) or is a variant thereof having at the amino acid corresponding to amino acid position 1 of SEQ ID NO: 2 a mutation selected from any one or more of the following: Y1R; Y1L; Y1V; Y1A; Y1G; Y1E; Y1W; Y1S; Y1K and Y1I, and the optically active polypeptide is located at any one or more of the following positions of the ATP-sensing polypeptide: 12 / 13, 55 / 56, 56 / 57, 67 / 69, 103 / 110, 105 / 104 and 109 / 112. Preferably, the optically active polypeptide is located at any one or more of the following positions of the ATP-sensing polypeptide: 12 / 13 or 103 / 110.

[0030] In one or more embodiments, the optical probe, the ATP-sensitive polypeptide is as set forth in SEQ ID NO: 1 and has one or more mutations of: D89, I88, V90, R92, R102 and R122, the optically active polypeptide is as set forth in SEQ ID NO: 2-9 (preferably SEQ ID NO: 2, 6, 7, 9) or is a variant thereof having at the amino acid corresponding to amino acid position 1 of SEQ ID NO: 2 one or more mutations selected from the group consisting of: Y1R; Y1L; Y1V; Y1A; Y1G; Y1E; Y1W; Y1S; Y1K and Y1I, and the optically active polypeptide is located at the 103 / 110 site of the ATP-sensitive polypeptide. Preferably, the mutations of the ATP-sensitive polypeptide comprise mutations selected from the group consisting of: (1) D89E, (2) D89V, (3) D89M, (4) D89Q, (5) D89N, (6) D89Y, (7) D89S, (8) V90D, (9) V90A, (10) V90L, (11) V90E, (12) V90M, (13) V90C, (14) V90P, (15) V90H, (16) V90K, (17) V90S, (18) V90T, (19) R102T, (20) R122N, (21) R122K, (22) R102T and D89V, (23) R102T and D89F, (24) R102T and D89I, (25) R102T and D89Q, (26) R102T and D89H, (27) R102T and D89Y, (28) R102T and D89K, (29) R102T and D89T, (30) R102T and D89W, (31) R102T and E83R, (32) R102T and I88V, (33) R102T and I88L, (34) R102T and R92A, (35) R102T and R92H, (36) R102T and R92M, (37) R102T and R92Q, (38) R102T and R92H, (39) R102T and R92K, (40) R102T and R122K, (41) R102T and V90I, (42) R102T and V90D, (43) R102T and V90R, (44) R102T and V90L, (45) R102T and V90F, (46) R102T and V90E, (47) R102T and V90C, (48) R102T and V90P, (49) R102T and V90Q, (50) R102T and V90N, (51) R102T and V90G, (52) R102T and V90H, (53) R102T and V90Y, (54) R102T and V90K, (55) R102T and V90S, (56) R102T and V90T.

[0031] In one or more embodiments, the optical probe, wherein the optically active polypeptide is at position 103 / 110 of an ATP-sensing polypeptide having a sequence as set forth in SEQ ID NO: 1 or a sequence having at least 70% sequence identity thereto and retaining ATP binding activity, the optically active polypeptide is as set forth in SEQ ID NO: 2-9 (preferably SEQ ID NO: 2, 6, 7, 9), and the optical probe has a mutation selected from the group consisting of: (1) D89E of the ATP-sensing polypeptide, (2) D89V of the ATP-sensing polypeptide, (3) D89M of the ATP-sensing polypeptide, (4) D89Q of the ATP-sensing polypeptide, (5) D89N of the ATP-sensing polypeptide, (6) D89Y of the ATP-sensing polypeptide, (7) D89S of the ATP-sensing polypeptide, (8) V90D of the ATP-sensing polypeptide, (9) V90A of the ATP-sensing polypeptide, (10) V90L of the ATP-sensing polypeptide, (11) V90E of the ATP-sensing polypeptide, (12) V90M of the ATP-sensing polypeptide, (13) V90C of the ATP-sensing polypeptide, (14) V90P of the ATP-sensing polypeptide, (15) V90H of the ATP-sensing polypeptide, (16) V90K of the ATP-sensing polypeptide, (17) V90S of the ATP-sensing polypeptide, (18) V90T of the ATP-sensing polypeptide, (19) R102T of the ATP-sensing polypeptide, (20) R122N of the ATP-sensing polypeptide, (21) R122K of the ATP-sensing polypeptide, (22) R102T, D89V and optically active polypeptide 1L of the ATP-sensing polypeptide, (23) R102T, D89F and optically active polypeptide 1L of the ATP-sensing polypeptide, (24) R102T, D89I and optically active polypeptide 1L of the ATP-sensing polypeptide, (25) R102T, D89Q and optically active polypeptide 1L of the ATP-sensing polypeptide, (26) R102T, D89H and optically active polypeptide 1L of the ATP-sensing polypeptide, (27) R102T, D89Y and optically active polypeptide 1L of the ATP-sensing polypeptide, (28) R102T, D89K and optically active polypeptide 1L of the ATP-sensing polypeptide, (29) R102T, D89T and optically active polypeptide 1L of the ATP-sensing polypeptide, (30) R102T, D89W and optically active polypeptide 1L of the ATP-sensing polypeptide, (31) R102T, E83R and optically active polypeptide 1L of the ATP-sensing polypeptide, (32) R102T, I88V and optically active polypeptide 1L of the ATP-sensing polypeptide, (33) R102T, I88L and optically active polypeptide 1L of the ATP-sensing polypeptide, (34) R102T, R92A and optically active polypeptide 1L of the ATP-sensing polypeptide, (35) R102T, R92H and optically active polypeptide 1L of the ATP-sensing polypeptide, (36) R102T, R92M and optically active polypeptide 1L of the ATP-sensing polypeptide.(37) R102T, R92Q and optically active polypeptide 1L of an ATP-sensitive polypeptide, (38) R102T, R92H and optically active polypeptide 1L of an ATP-sensitive polypeptide, (39) R102T, R92K and optically active polypeptide 1L of an ATP-sensitive polypeptide, (40) R102T, R122K and optically active polypeptide 1L of an ATP-sensitive polypeptide, (41) R102T, V90I and optically active polypeptide 1L of an ATP-sensitive polypeptide, (42) R102T, V90D and optically active polypeptide 1L of an ATP-sensitive polypeptide, (43) R102T, V90R and optically active polypeptide 1L of an ATP-sensitive polypeptide, (44) R102T, V90L and optically active polypeptide 1L of an ATP-sensitive polypeptide, (45) R102T, V90F and optically active polypeptide 1L of an ATP-sensitive polypeptide, (46) R102T, V90E and optically active polypeptide 1L of an ATP-sensitive polypeptide, (47) R102T, V90C and optically active polypeptide 1L of an ATP-sensitive polypeptide, (48) R102T, V90P and optically active polypeptide 1L of an ATP-sensitive polypeptide, (49) R102T, V90Q and optically active polypeptide 1L of an ATP-sensitive polypeptide, (50) R102T, V90N and optically active polypeptide 1L of an ATP-sensitive polypeptide, (51) R102T, V90G and optically active polypeptide 1L of an ATP-sensitive polypeptide, (52) R102T, V90H and optically active polypeptide 1L of an ATP-sensitive polypeptide, (53) R102T, V90Y and optically active polypeptide 1L of an ATP-sensitive polypeptide, (54) R102T, V90K and optically active polypeptide 1L of an ATP-sensitive polypeptide, (55) R102T, V90S and optically active polypeptide 1L of an ATP-sensitive polypeptide, (56) R102T, V90T and optically active polypeptide 1L of an ATP-sensitive polypeptide.

[0032] In one or more embodiments, the optical probe, wherein the ATP-sensing polypeptide is as set forth in SEQ ID NO: 1, the optically active polypeptide is as set forth in SEQ ID NO: 2-9 (preferably SEQ ID NO: 2, 6, 7, 9), the optically active polypeptide is located at the 103 / 110 site of the ATP-sensing polypeptide, and the optical probe has the following mutations: (1) R102T, D89Q of the ATP-sensing polypeptide and optically active polypeptide 1L, (2) R102T, D89H of the ATP-sensing polypeptide and optically active polypeptide 1L, (3) R102T, D89K of the ATP-sensing polypeptide and optically active polypeptide 1L, (4) R102T, D89T of the ATP-sensing polypeptide and optically active polypeptide 1L, (5) R102T, R122K of the ATP-sensing polypeptide and optically active polypeptide 1L, (6) R102T, V90E of the ATP-sensing polypeptide and optically active polypeptide 1L, (7) R102T, V90Q of the ATP-sensing polypeptide and optically active polypeptide 1L, (8) R102T, V90G of the ATP-sensing polypeptide and optically active polypeptide 1L, (9) R102T, V90Y of the ATP-sensing polypeptide and optically active polypeptide 1L, (10) R102T, V90S of the ATP-sensing polypeptide and optically active polypeptide 1L.

[0033] In one or more embodiments, the optical probe is suitable for use in a mammalian cell system, wherein the ATP-sensing polypeptide is as set forth in SEQ ID NO: 1, the optically active polypeptide is as set forth in SEQ ID NO: 2-9 (preferably SEQ ID NO: 2, 6, 7, 9), the optically active polypeptide is located at the 103 / 110 site of the ATP-sensing polypeptide, and the optical probe has the following mutations: (1) R122N of the ATP-sensing polypeptide, (2) R102T, I88V of the ATP-sensing polypeptide and optically active polypeptide 1L, (3) R102T, I88L of the ATP-sensing polypeptide and optically active polypeptide 1L, (4) R102T, R92A of the ATP-sensing polypeptide and optically active polypeptide 1L, (5) R102T, R92Q of the ATP-sensing polypeptide and optically active polypeptide 1L, (6) R102T, R92M of the ATP-sensing polypeptide and optically active polypeptide 1L, (7) R102T, R92H of the ATP-sensing polypeptide and optically active polypeptide 1L.

[0034] In one or more embodiments, the optical probe comprises any one of the amino acid sequences of SEQ ID NOs: 10-11, or a variant thereof. In one or more embodiments, the optical probe provided herein comprises a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 10-11. Preferably, the optical probe provided herein comprises a sequence substantially similar or identical to any one of the amino acid sequences of SEQ ID NOs: 10-11.

[0035] The present application also provides fusion polypeptides comprising the optical probes described in any of the embodiments herein and other polypeptides, including localization sequences, tags to facilitate purification, or tags for use in immunological reactions. In some embodiments, the optical probes described herein further comprise other polypeptides fused thereto. The other polypeptides described herein do not affect the properties of the optical probes. In some embodiments, the other polypeptides are located at the N-terminus and / or C-terminus of the optical probes. In some embodiments, the other polypeptides include localization sequences (e.g., polypeptides that localize the optical probes to different organelles or subcellular compartments), tags to facilitate purification, or tags for use in immunological reactions (e.g., immunoblotting). There can be linkers between the optical probes and the other polypeptides in the fusion polypeptides described herein.

[0036] The present application also provides nucleic acid molecules comprising: (a) a coding sequence of the ATP-binding protein variant, the optical probe, or the fusion polypeptide described in any of the embodiments herein, or (b) a complement of (a), or (c) a fragment of (a) or (b). The fragment is a primer.

[0037] The present application also relates to variants of the nucleic acid molecules described above, including nucleic acid sequences encoding protein variants, optical probes, or fusion polypeptides of the present application, fragments, analogs, derivatives, soluble fragments, and variants thereof, or complements thereof.

[0038] The present application also provides nucleic acid constructs comprising the nucleic acid molecules described herein. The nucleic acid sequences encode the protein variants, the optical probes, or the fusion polypeptides described in any of the embodiments of the present application.

[0039] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.

[0040] In one or more embodiments, the nucleic acid molecule is operably linked to an expression control sequence.

[0041] In some embodiments, the expression vector is selected from the group consisting of a prokaryotic expression vector, a eukaryotic expression vector, and a viral vector.

[0042] In another aspect, the present application provides a host cell that: (1) comprises, expresses or secretes an optical probe or fusion polypeptide of any of the embodiments described herein; (2) comprises a nucleic acid molecule of any of the embodiments described herein; or (3) comprises a nucleic acid construct of any of the embodiments described herein. Preferably, the host cell is E. coli.

[0043] In another aspect, the present application provides an ATP detection kit comprising an optical probe or fusion polypeptide or a nucleic acid molecule or a nucleic acid construct or a host cell described herein.

[0044] In one or more embodiments, the kit further comprises one or more reagents selected from the group consisting of: a buffer, a medium, an ATP standard.

[0045] In another aspect, the present application provides a method of making an optical probe or fusion polypeptide described herein, comprising: culturing a host cell described herein, and isolating the optical probe or fusion polypeptide from the culture.

[0046] In one or more embodiments, the method comprises the steps of: 1) incorporating a nucleic acid molecule encoding an optical probe or fusion polypeptide described herein into an expression vector; 2) transferring the expression vector into a host cell; 2) culturing the host cell under conditions suitable for expression of the expression vector, 3) isolating the optical probe or fusion polypeptide.

[0047] In another aspect, the present application provides a method of detecting ATP in a sample, comprising: contacting an optical probe or fusion polypeptide or a host cell described herein with the sample, and detecting a change in the optical activity of the polypeptide. The detection can be in vivo, in vitro, subcellular or in situ. The sample can be, for example, blood.

[0048] In another aspect, the present application provides a method of quantifying ATP in a sample, comprising: contacting an optical probe or fusion polypeptide or a host cell described herein with the sample, detecting an optical change in the optical activity of the polypeptide, and quantifying the ATP in the sample based on the optical change in the optical activity of the polypeptide.

[0049] In another aspect, the present application provides a method of screening for a compound (e.g., a drug), comprising: contacting an optical probe or fusion polypeptide or a host cell described herein with a candidate compound in an ATP-containing system, detecting an optical change in the optical activity of the polypeptide, and screening for the compound based on the optical change in the optical activity of the polypeptide. The method can be used to screen for compounds in high throughput.

[0050] In one or more embodiments, a host cell described herein is contacted with a candidate compound in an ATP-containing system, and an optical change in the optical activity of the polypeptide indicates whether the candidate compound is capable of modulating cellular uptake of ATP.

[0051] In another aspect, the present application provides a method for intracellular and / or extracellular localization of ATP, comprising: contacting an ATP-containing system with the optical probe or the host cell, and detecting the optical change of the optically active polypeptide.

[0052] In one or more embodiments, the system is a solution system, a cellular system, a subcellular system.

[0053] In another aspect, the present application provides the use of the ATP optical probe or the fusion polypeptide or the host cell as described herein in the detection of ATP in a sample, in the screening of compounds, or in the intracellular and / or extracellular localization of ATP. In one or more embodiments, the localization is real-time localization.

[0054] In another aspect, the present application provides the use of the ATP optical probe or the fusion polypeptide or the polynucleotide or the nucleic acid construct as described herein in the preparation of a kit for the detection of ATP in a sample, for the screening of compounds, or for the intracellular and / or extracellular localization of ATP. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 SDS-PAGE of an exemplary ATP optical probe.

[0056] Figure 2 Fluorescence spectral properties of an exemplary ATP optical probe.

[0057] Figure 3 Titration curve of an exemplary ATP optical probe against different concentrations of ATP.

[0058] Figure 4 Titration curve of an exemplary ATP optical probe against different concentrations of ATP.

[0059] Figure 5 Titration curve of an exemplary ATP optical probe against different concentrations of ATP.

[0060] Figure 6 Bar graph of specificity of an exemplary ATP optical probe against another substrate and sugar metabolism intermediates and analogs.

[0061] Figure 7 Photograph of subcellular organelle localization of an exemplary ATP optical probe in mammalian cells.

[0062] Figure 8 Schematic diagram of dynamic monitoring of ATP concentration in cytosol of an exemplary ATP optical probe in mammalian cells.

[0063] Figure 9 Dot plot of high-throughput compound screening of an exemplary ATP optical probe at live cell level.

[0064] Figure 10 Bar graphs of exemplary ATP optical probes quantifying ATP in mouse and human blood. DETAILED DESCRIPTION

[0065] The term "about" as used herein when given a numerical value or range means that the numerical value or range is within 20%, within 10%, and within 5% of the given numerical value or range.

[0066] The terms "comprising", "including" and grammatical variants thereof used herein, such as "comprise", "includes" and "comprising", "including", are inclusive after- meaninged and do not exclude additional, un-recited elements or method steps. The term "consisting essentially of" when used herein in relation to a composition, means that the composition can contain additional ingredients, provided that the additional ingredients do not materially alter the basic and novel characteristics of the claimed composition. The term "consisting of when used herein in relation to a composition, means that the composition can contain only the specified ingredients.

[0067] The term "ATP-sensitive polypeptide" as used herein refers to a polypeptide that responds to ATP, including any response of a chemical, biological, electrical, or physiological parameter of the polypeptide associated with the interaction of the sensitive polypeptide with ATP. The response includes small changes, such as, for example, changes in the orientation of an amino acid or peptide fragment of the polypeptide and changes in, for example, the primary, secondary, or tertiary structure of the polypeptide, including, for example, changes in protonation, electrochemical potential, and / or conformation. "Conformation" is the three-dimensional arrangement of the primary, secondary, and tertiary structure of a molecule that includes side groups in the molecule; a change in conformation occurs when the three-dimensional structure of the molecule changes. Examples of changes in conformation include a change from an alpha-helix to a beta-sheet or a change from a beta-sheet to an alpha-helix. It is understood that the detectable change need not be a change in conformation so long as the fluorescence of the fluorescent protein moiety is changed. The ATP-sensitive polypeptides described herein can also include functional variants thereof. Functional variants of the ATP-sensitive polypeptides include, but are not limited to, variants that can interact with ATP such that the same or similar changes as the parent ATP-sensitive polypeptide occur.

[0068] As used herein, the term "optical probe" refers to an ATP-sensitive polypeptide fused to an optically active polypeptide, such as a fluorescent protein, operably inserted into an ATP-sensitive polypeptide, such as an ATP-binding protein. The ATP-binding protein can be responsive to changes in ATP concentration, and the spatial conformation of the ATP-binding protein can change during dynamic changes in ATP concentration. The inventors have discovered that when an optically active polypeptide is fused to an ATP-sensitive polypeptide, such as an ATP-binding protein, the conformational change in the ATP-sensitive polypeptide that specifically occurs upon binding of ATP at physiological concentrations can cause a conformational change in the optically active polypeptide, such as a fluorescent protein, which results in a change in the optical properties of the optically active polypeptide. By measuring the fluorescence of the fluorescent protein at different ATP concentrations and plotting a standard curve, the presence and / or level of ATP can be detected and analyzed. The ATP-sensitive polypeptides described herein include, but are not limited to, the ε subunit of the F0F1-ATP synthase from Thermophilic Bacillus PS3 or a variant having more than 90% homology thereto. An exemplary ε subunit of the F0F1-ATP synthase is set forth in SEQ ID NO: 1, which is the full-length sequence of an exemplary SEQ ID NO: 1. When describing the optical probes, ATP-sensitive proteins, or ATP-binding proteins of the present invention, such as when describing insertion sites or mutation sites, reference to the amino acid residue number is made with reference to SEQ ID NO: 1.

[0069] A protein-based "optically active polypeptide" is a polypeptide that has the ability to emit fluorescence. Fluorescence is an optical property of the optically active polypeptide that can be used as a means to detect responsiveness of the optical probes of the present invention. As used herein, the term "fluorescent property" refers to the molar extinction coefficient at an appropriate excitation wavelength, the fluorescence quantum efficiency, the shape of the excitation or emission spectrum, the excitation and emission wavelength maxima, the amplitude of excitation at two different wavelengths, the ratio of emission amplitudes at two different wavelengths, the excited state lifetime, or the fluorescence anisotropy. A measurable difference in any of these properties between the active and inactive states is sufficient for the utility of the fluorescent protein substrate of the present invention in an activity assay. The measurable difference can be determined by determining the amount of any quantitative fluorescent property, e.g., the fluorescence amount at a particular wavelength or the integral of the fluorescence over the emission spectrum. Preferably, the protein substrate is selected to have fluorescent properties that are easily distinguishable between the unactivated and activated conformational states. The optically active polypeptides described herein can also include functional variants thereof. Functional variants of the optically active polypeptides include, but are not limited to, variants that can undergo the same or similar changes in fluorescent properties as the parent optically active polypeptide.

[0070] Herein, "Response Fold" is the normalized fluorescence ratio. The more the response fold of the probe deviates from 1 (whether it is larger or smaller), the greater the change fold or the response ability of the probe to the substrate relative to the control. For example, the embodiments of the present application calculate the response fold by detecting the change in the fluorescence intensity ratio at 420 nm excitation and 528 nm emission (Normalized Ratio 420 / 485 ) and 485 nm excitation and 528 nm emission, as follows:

[0071] The fluorescence signal value is corrected by subtracting the detection signal value of the cell not expressing the probe protein. The probe detection signal in the parallel experimental group is divided by the control detection signal to eliminate pH-sensitive interference and obtain the corrected data.

[0072] F = F sample - F BLK

[0073]

[0074]

[0075]

[0076]

[0077] F represents the fluorescence intensity (Fluorescence intensity), F sample represents the total fluorescence intensity of the sample expressing the fluorescent probe, F BLK represents the background fluorescence intensity of the sample not expressing the fluorescent probe, F cpYFP represents the fluorescence intensity of the sample as a pH control. F 485 represents the fluorescence intensity of the fluorescent protein sample at 485 nm excitation and 528 nm emission, F 420 represents the fluorescence intensity of the fluorescent protein sample at 420 nm excitation and 528 nm emission. Ratio sensor represents the fluorescence intensity ratio of the probe, Ratio cpYFP represents the fluorescence intensity ratio of the pH control fluorescent protein corresponding to the probe. Normalized Ratio 420 / 485 is the change fold or the response fold of the probe. Normalized Ratio 420 / 485 The more the response fold of the probe deviates from 1 (whether it is larger or smaller) indicates that the change fold or the response fold of the probe is greater.

[0078] "Linker" or "junction" refers to the amino acid or nucleotide sequence that connects two parts in the polypeptide, protein or nucleic acid of the present application. Exemplarily, the number of amino acids at the amino terminal of the junction between the ATP-sensitive polypeptide and the optically active polypeptide in the present application is selected from 0-3, and the number of amino acids at the carboxyl terminal is selected from 0-2; when the recombinant optical probe is connected to the functional protein as a basic unit, it can be fused to the amino terminal or carboxyl terminal of the recombinant optical probe. The linker sequence can be a short peptide chain composed of one or more flexible amino acids, such as Y.

[0079] The term "fluorescent protein" as used herein refers to a protein that emits fluorescence under excitation light irradiation. Fluorescent proteins are basic detection means in the field of biological science, such as green fluorescent protein GFP commonly used in the field of biotechnology and circularly permuted blue fluorescent protein (cpBFP), circularly permuted green fluorescent protein (cpGFP), circularly permuted yellow fluorescent protein (cpYFP) and the like derived from mutation of the protein; and red fluorescent protein RFP commonly used in the technical field, and circularly permuted proteins derived from the protein, such as cpmApple, cpmOrange, cpmKate and the like. Exemplary sequences of fluorescent proteins are shown in any one of SEQ ID NOs: 2-9.

[0080] The ATP optical probe described in the present application comprises an ATP-sensitive polypeptide B, such as an ATP-binding protein or a variant thereof, and an optically active polypeptide A, such as a fluorescent protein or a variant thereof. The optically active polypeptide A is inserted into the ATP-sensitive polypeptide B, which divides B into a first part B1 and a second part B2, forming a probe structure of the form B1-A-B2; the interaction between the ATP-sensitive polypeptide B and ATP causes the optical signal of the optically active polypeptide A to become stronger.

[0081] In the optical probe of the present application, the optically active polypeptide can be located at any position of the ATP-sensitive polypeptide. In one or more embodiments, the optically active polypeptide is located in the N-C direction at the following regions of the ATP-sensitive polypeptide in the N-C direction: amino acid residues 10-13, 19-21, 27-30, 33-38, 41-43, 46-48, 54-57, 61-63, 66-69, 71-74, 78-90, and 103-116 regions. Illustratively, the optically active polypeptide is located at 11 / 13, 12 / 13, 19 / 21, 27 / 30, 28 / 28, 28 / 30, 29 / 28, 29 / 29, 33 / 35, 33 / 36, 33 / 38, 34 / 34, 34 / 35, 35 / 34, 35 / 35, 35 / 36, 35 / 37, 36 / 34, 36 / 35, 36 / 36, 36 / 37, 36 / 38, 37 / 38, 41 / 42, 41 / 43, 41 / 44, 42 / 42, 42 / 43, 43 / 42, 43 / 43, 46 / 46, 46 / 47, 47 / 46, 47 / 47, 47 / 48, 54 / 55, 55 / 55, 55 / 57, 56 / 55, 56 / 57, 61 / 63, 62 / 62, 63 / 62, 66 / 67, 66 / 69, 67 / 67, 67 / 69, 68 / 67, 68 / 69, 71 / 72, 71 / 73, 72 / 73, 72 / 74, 73 / 73, 73 / 74, 78 / 80, 78 / 81, 80 / 80, 80 / 81, 84 / 85, 84 / 91, 85 / 88, 85 / 91, 86 / 85, 86 / 86, 86 / 88, 86 / 90, 87 / 88, 87 / 89, 87 / 90, 87 / 91, 88 / 86, 88 / 87, 88 / 88, 88 / 89, 89 / 88, 89 / 90, 89 / 91, 90 / 89, 90 / 90, 103 / 104, 103 / 108, 103 / 109, 103 / 110, 103 / 112, 103 / 113, 103 / 115, 103 / 116, 104 / 104, 104 / 105, 104 / 106, 104 / 107, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 104 / 112, 104 / 114, 104 / 116, 105 / 104, 105 / 105, 105 / 106, 105 / 107, 105 / 108, 105 / 109, 105 / 110, 105 / 112, 105 / 114, 105 / 116, 106 / 104, 106 / 107, 106 / 111, 106 / 113, 106 / 114, 106 / 115, 106 / 116, 107 / 111, 107 / 113, 107 / 116, 108 / 109, 108 / 110,108 / 111, 108 / 112, 108 / 113, 108 / 115, 108 / 113, 109 / 109, 109 / 110, 109 / 112, 109 / 113, 109 / 116, 110 / 104, 110 / 112, 110 / 113, 110 / 115, 110 / 116, 111 / 107, 110 / 109, 111 / 110, 111 / 111, 111 / 112, 111 / 113, 111 / 116, 112 / 104, 112 / 105, 112 / 110, 112 / 114, 112 / 115, 112 / 116, 113 / 108, 113 / 109, 113 / 110, 113 / 113, 113 / 115, 113 / 116, 114 / 104, 114 / 105, 114 / 106, 114 / 108, 114 / 109, 114 / 110, 114 / 111, 114 / 115, 114 / 116, 115 / 108, 115 / 111, 115 / 113, 115 / 115 and 115 / 116. In exemplary embodiments, the optically active polypeptide is located at 12 / 13, 55 / 56, 56 / 57, 67 / 69, 103 / 110, 105 / 104, 109 / 112 of the amino acid sequence of the ATP-binding protein.

[0082] In the context of a site denoted in the form "X / Y", the ends of the optically active polypeptide are flanked by portions of the ATP-sensitve polypeptide, wherein the N-terminus of the optically active polypeptide is the N-terminal starting amino acid (e.g., any amino acid from position 1 to position 12) to the amino acid at position X of the ATP-sensitve polypeptide sequence, and the C-terminus of the optically active polypeptide is the amino acid at position Y to the C-terminal ending amino acid (e.g., any amino acid from position Y to position 133) of the ATP-sensitve polypeptide sequence. If the two numbers in the site denoted in the form "X / Y" are consecutive integers, then the optically active polypeptide is located between the amino acids denoted by the numbers. For example, insertion site 12 / 13 indicates that the optically active polypeptide is located between amino acids 12 and 13 of the ATP-sensitve polypeptide. If the two numbers in the site denoted in the form "X / Y" are not consecutive integers and X is less than Y, then the optically active polypeptide replaces the amino acids between the amino acids denoted by the numbers. For example, insertion site 55 / 57 indicates that the optically active polypeptide replaces amino acid 56 of the ATP-sensitve polypeptide, and insertion site 103 / 110 indicates that the optically active polypeptide replaces amino acids 104-109 of the ATP-sensitve polypeptide. If X is greater than or equal to Y in the site denoted in the form "X / Y", then the portion of the ATP-sensitve polypeptide at the N-terminus of the optically active polypeptide ends at the amino acid at position X of the ATP-sensitve polypeptide sequence, and the portion of the ATP-sensitve polypeptide at the C-terminus of the optically active polypeptide begins at the amino acid at position Y of the ATP-sensitve polypeptide sequence. For example, insertion site 29 / 28 indicates that the N-terminus of the optically active polypeptide is fused to the N-terminal starting amino acid (e.g., any amino acid from position 1 to position 15) to the amino acid at position 29 of the ATP-sensitve polypeptide sequence, and the C-terminus of the optically active polypeptide is fused to the amino acid at position 28 to the C-terminal ending amino acid (e.g., the amino acid at position 133) of the ATP-sensitve polypeptide sequence, and an exemplary structure is: (amino acids 15-29 of the ATP-sensitve polypeptide sequence)-(optically active polypeptide)-(amino acids 28-133 of the ATP-sensitve polypeptide sequence).

[0083] In one or more embodiments, the optical probe comprises, in order from N- to C-terminus, residues 1-X of SEQ ID NO: 1, an optically active polypeptide set forth in any one of SEQ ID NOs: 2-9 or a variant thereof, and residues Y-133 of SEQ ID NO: 1, wherein X and Y are selected from any one of the following groups: (1) X is 11 and Y is 13, (2) X is 12 and Y is 13, (3) X is 19 and Y is 21, (4) X is 27 and Y is 30, (5) X is 28 and Y is 28, (6) X is 28 and Y is 30, (7) X is 29 and Y is 28, (8) X is 29 and Y is 29, (9) X is 33 and Y is 35, (10) X is 33 and Y is 36, (11) X is 33 and Y is 38, (12) X is 34 and Y is 34, (13) X is 34 and Y is 35, (14) X is 35 and Y is 34, (15) X is 35 and Y is 35, (16) X is 35 and Y is 36, (17) X is 35 and Y is 37, (18) X is 36 and Y is 34, (19) X is 36 and Y is 35, (20) X is 36 and Y is 36, (21) X is 36 and Y is 37, (22) X is 36 and Y is 38, (23) X is 37 and Y is 38, (24) X is 41 and Y is 42, (25) X is 41 and Y is 43, (26) X is 41 and Y is 44, (27) X is 42 and Y is 42, (28) X is 42 and Y is 43, (29) X is 43 and Y is 42, (30) X is 43 and Y is 43, (31) X is 46 and Y is 46, (32) X is 46 and Y is 47, (33) X is 47 and Y is 46, (34) X is 47 and Y is 47, (35) X is 47 and Y is 48, (36) X is 54 and Y is 55, (37) X is 55 and Y is 55, (38) X is 55 and Y is 57, (39) X is 56 and Y is 55, (40) X is 56 and Y is 57, (41) X is 61 and Y is 63, (42) X is 62 and Y is 62, (43) X is 63 and Y is 62, (44) X is 66 and Y is 67, (45) X is 66 and Y is 69, (46) X is 67 and Y is 67, (47) X is 67 and Y is 69, (48) X is 68 and Y is 67, (49) X is 68 and Y is 69, (50) X is 71 and Y is 72, (51) X is 71 and Y is 73, (52) X is 72 and Y is 73, (53) X is 72 and Y is 74, (54) X is 73 and Y is 73, (55) X is 73 and Y is 74, (56) X is 78 and Y is 80, (57) X is 78 and Y is 81, (58) X is 80 and Y is 80, (59) X is 80 and Y is 81, (60) X is 84 and Y is 85, (61) X is 84 and Y is 91, (62) X is 85 and Y is 88, (63) X is 85 and Y is 91, (64) X is 86 and Y is 91, (65) X is 86 and Y is 92, (66) X is 87 and Y is 91, (67) X is 87 and Y is 92, (68) X is 88 and Y is 91, (69) X is 88 and Y is 92, (70) X is 89 and Y is 91, (71) X is 89 and Y is 92, (72) X is 90 and Y is 91, (73) X is 90 and Y is 92, (74) X is 91 and Y is 91, (75) X is 91 and Y is 92, (76) X is 92 and Y is 91, (77) X is 92 and Y is 92, (78) X is 93 and Y is 93, (79) X is 94 and Y is 94, (80) X is 95 and Y is 95, (81) X is 96 and Y is 96, (82) X is 97 and Y is 97, (83) X is 98 and Y is 98, (84) X is 99 and Y is 99, (85) X is 100 and Y is 100, (86) X is 101 and Y is 101, (87) X is 102 and Y is 102, (88) X is 103 and Y is 103, (89) X is 104 and Y is 104, (90) X is 105 and Y is 105, (91) X is 106 and Y is 106, (92) X is 107 and Y is 107, (93) X is 108 and Y is 108, (94) X is 109 and Y is 109, (95) X is 110 and Y is 110, (96) X is 111 and Y is 111, (97) X is 112 and Y is 112, (98) X is 113 and Y is 113, (99) X is 114 and Y is 114, (100) X is 115 and Y is 115, (101) X is 116 and Y is 116, (102) X is 117 and Y is 117, (103) X is 118 and Y is 118, (104) X is 119 and Y is 119, (105) X is 120 and Y is 120, (106) X is 121 and Y is 121, (107) X is 122 and Y is 122, (108) X is 123 and Y is 123, (109) X is 124 and Y is 124, (110) X is 125 and Y is 125, (111) X is 126 and Y is 126, (112) X is 127 and Y is 127, (113) X is 128 and Y is 128, (114) X is 129 and Y is 129, (115) X is 130 and Y is 130, (116) X is 131 and Y is 131, (117) X is 132 and Y is 132, (118) X is 133 and Y is 133.Y is 85, (65) X is 86, Y is 86, (66) X is 86, Y is 88, (67) X is 86, Y is 90, (68) X is 87, Y is 88, (69) X is 87, Y is 89, (70) X is 87, Y is 90, (71) X is 87, Y is 91, (72) X is 88, Y is 86, (73) X is 88, Y is 87, (74) X is 88, Y is 88, (75) X is 88, Y is 89, (76) X is 89, Y is 88, (77) X is 89, Y is 90, (78) X is 89, Y is 91, (79) X is 90, Y is 89, (80) X is 90, Y is 90, (81) X is 103, Y is 104, (82) X is 103, Y is 108, (83) X is 103, Y is 109, (84) X is 103, Y is 110, (85) X is 103, Y is 112, (86) X is 103, Y is 113, (87) X is 103, Y is 115, (88) X is 103, Y is 116, (89) X is 104, Y is 104, (90) X is 104, Y is 105, (91) X is 104, Y is 106, (92) X is 104, Y is 107, (93) X is 104, Y is 108, (94) X is 104, Y is 109, (95) X is 104, Y is 110, (96) X is 104, Y is 111, (97) X is 104, Y is 112, (98) X is 104, Y is 114, (99) X is 104, Y is 116, (100) X is 105, Y is 104, (101) X is 105, Y is 105, (102) X is 105, Y is 106, (103) X is 105, Y is 107, (104) X is 105, Y is 108, (105) X is 105, Y is 109, (106) X is 105, Y is 110, (107) X is 105, Y is 112, (108) X is 105, Y is 114, (109) X is 105, Y is 116, (110) X is 106, Y is 104, (111) X is 106, Y is 107, (112) X is 106, Y is 111, (113) X is 106, Y is 113, (114) X is 106, Y is 114, (115) X is 106, Y is 115, (116) X is 106, Y is 116, (117) X is 107, Y is 111, (118) X is 107, Y is 113, (119) X is 107, Y is 116, (120) X is 108, Y is 109, (121) X is 108, Y is 110, (122) X is 108, Y is 111, (123) X is 108, Y is 112, (124) X is 108, Y is 113, (125) X is 108, Y is 115, (126) X is 108, Y is 113,(127) X is 109, Y is 109, (128) X is 109, Y is 110, (129) X is 109, Y is 112, (130) X is 109, Y is 113, (131) X is 109, Y is 116, (132) X is 110, Y is 104, (133) X is 110, Y is 112, (134) X is 110, Y is 113, (135) X is 110, Y is 115, (136) X is 110, Y is 116, (137) X is 111, Y is 107, (138) X is 110, Y is 109, (139) X is 111, Y is 110, (140) X is 111, Y is 111, (141) X is 111, Y is 112, (142) X is 111, Y is 113, (143) X is 111, Y is 116, (144) X is 112, Y is 104, (145) X is 112, Y is 105, (146) X is 112, Y is 110, (147) X is 112, Y is 114, (148) X is 112, Y is 115, (149) X is 112, Y is 116, (150) X is 113, Y is 108, (151) X is 113, Y is 109, (152) X is 113, Y is 110, (153) X is 113, Y is 113, (154) X is 113, Y is 115, (155) X is 113, Y is 116, (156) X is 114, Y is 104, (157) X is 114, Y is 105, (158) X is 114, Y is 106, (159) X is 114, Y is 108, (160) X is 114, Y is 109, (161) X is 114, Y is 110, (162) X is 114, Y is 111, (163) X is 114, Y is 115, (164) X is 114, Y is 116, (165) X is 115, Y is 108, (166) X is 115, Y is 111, (167) X is 115, Y is 113, (168) X is 115, Y is 115, (169) X is 115, Y is 116.

[0084] Preferably, the optically active polypeptide is located at 12 / 13, 55 / 56, 56 / 57, 67 / 69, 103 / 110, 105 / 104, 109 / 112 of the amino acid sequence of the ATP binding protein, and an exemplary optically active polypeptide is located at 103 / 110 of the amino acid sequence of the ATP binding protein, and has the sequence of SEQ ID NO: 10.

[0085] As used herein, the term "variant" or "mutant" with respect to a polypeptide or protein includes variants having the same function as the polypeptide or protein but having a different sequence. Variants of a polypeptide or protein can include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. These variants include, but are not limited to, sequences in which one or more (typically 1-30, preferably 1-20, more preferably 1-10, most preferably 1-5) amino acids have been deleted, inserted, and / or substituted in the sequence of the polypeptide or protein, as well as sequences in which one or more (typically 20 or fewer, preferably 10 or fewer, more preferably 5 or fewer) amino acids have been added at the carboxy terminus and / or amino terminus. These variants can also include polypeptides or proteins having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence of the polypeptide or protein. Without wishing to be bound by theory, amino acid residues are changed without changing the overall conformation and function of the polypeptide or protein, i.e., function-conservative mutations. For example, in the art, substitutions with amino acids having similar or analogous properties are typically made without changing the function of the polypeptide or protein. In the art, amino acids with similar properties are often referred to as families of amino acids having similar side chains, which are well defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), uncharged, nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Also, for example, adding one or more amino acids at the amino terminus and / or carboxy terminus also typically does not change the function of the polypeptide or protein. Conservative substitutions of non-genetically encoded amino acids are known in the art. Other conservative substitutions of non-encoded amino acids can be determined based on a comparison of their physical properties with those of genetically encoded amino acids. It is well known to those skilled in the art that, in genetic cloning procedures, it is often necessary to engineer appropriate restriction sites, which necessarily introduce one or more extraneous residues at the termini of the expressed polypeptide or protein, without affecting the activity of the polypeptide or protein of interest.For example, to construct a fusion protein, to facilitate expression of a recombinant protein, to obtain a recombinant protein that is secreted into the extracellular space of a host cell, or to facilitate purification of a recombinant protein, it is often desirable to add some amino acids to the N-terminus, C-terminus, or other suitable region within the recombinant protein, such as, but not limited to, a suitable linker peptide, a signal peptide, a leader peptide, a terminal extension, glutathione S-transferase (GST), maltose E binding protein, protein A, a tag such as 6His or Flag, or a proteolytic enzyme site for Factor Xa or thrombin or enterokinase. Variants of a polypeptide or protein can include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. These variants can also include polypeptides or proteins having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the sequence of the polypeptide or protein. An exemplary ε subunit of F0F1-ATP synthase is the full-length amino acid sequence set forth in SEQ ID NO: 1, which retains the binding function of the ε subunit of F0F1-ATP synthase to ATP and does not affect the optical property change of the optically active polypeptide inserted therein in response to the binding of ATP.

[0086] The optical probe of the present application can comprise an ATP-sensitive polypeptide with mutations. ATP-binding protein variants with mutations at positions selected from the following of SEQ ID NO: 1 or a truncated variant thereof exhibit different binding activities to ATP: D89, I88, V90, R92, R102, R122. The amino acid mutations include modification, substitution, or deletion of an amino acid. In preferred embodiments, the mutations of the ATP-binding protein variants include mutations at positions selected from any one of the following groups: (1) R102 and D89, (2) R102 and I88, (3) R102 and R92, (4) R102 and R122, (5) R102 and V90.

[0087] In one or more embodiments, D89 is mutated to E, V, M, N, F, I, Q, S, H, Y, K, T, or W. In one or more embodiments, I88 is mutated to V or L. In one or more embodiments, R92 is mutated to A, M, Q, H, or K. In one or more embodiments, V90 is mutated to I, D, A, R, L, F, E, M, C, P, Q, N, G, H, Y, K, S, or T. In one or more embodiments, R122 is mutated to N or K.

[0088] In one or more embodiments, the mutation comprises a mutation selected from any one of the following groups: (1) D89E, (2) D89V, (3) D89M, (4) D89Q, (5) D89N, (6) D89Y, (7) D89S, (8) V90D, (9) V90A, (10) V90L, (11) V90E, (12) V90M, (13) V90C, (14) V90P, (15) V90H, (16) V90K, (17) V90S, (18) V90T, (19) R102T, (20) R122N, (21) R122K, (22) R102T and D89V, (23) R102T and D89F, (24) R102T and D89I, (25) R102T and D89Q, (26) R102T and D89H, (27) R102T and D89Y, (28) R102T and D89K, (29) R102T and D89T, (30) R102T and D89W, (31) R102T and E83R, (32) R102T and I88V, (33) R102T and I88L, (34) R102T and R92A, (35) R102T and R92H, (36) R102T and R92M, (37) R102T and R92Q (38) R102T and R92H, (39) R102T and R92K, (40) R102T and R122K, (41) R102T and V90I, (42) R102T and V90D (43) R102T and V90R, (44) R102T and V90L, (45) R102T and V90F, (46) R102T and V90E, (47) R102T and V90C, (48) R102T and V90P, (49) R102T and V90Q, (50) R102T and V90N, (51) R102T and V90G, (52) R102T and V90H, (53) R102T and V90Y, (54) R102T and V90K, (55) R102T and V90S, (56) R102T and V90T. The present invention provides ATP binding protein variants having these mutations and optical probes comprising such ATP binding protein variants as ATP sensitive polypeptides.

[0089] The optical probes of the present application can comprise an optically active polypeptide having a mutation. In some embodiments, the mutated optically active polypeptide has a mutation at the Y1 site, which mutation comprises a modification, substitution or deletion of an amino acid, in one or more embodiments, the mutation is Y1L. In one or more embodiments, the optical probe comprises an amino acid sequence of any one of SEQ ID NOs: 10-11 or a variant thereof. In one or more embodiments, the optical probe provided by the present application comprises a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 10-11. In preferred embodiments, the optical probe provided by the present application comprises a sequence substantially similar or identical to any one of the amino acid sequences of SEQ ID NOs: 10-11.

[0090] In some embodiments, the optically active polypeptide is at position 103 / 110 of the ATP binding protein, the ATP sensitive polypeptide has a sequence as set forth in SEQ ID NO: 1 or a sequence with at least 70% sequence identity thereto and retains ATP binding activity, the optically active polypeptide has a sequence as set forth in any one of SEQ ID NOS: 2-9, and the ATP sensitive polypeptide has a mutation as set forth in any one of: (1) D89E, (2) D89V, (3) D89M, (4) D89Q, (5) D89N, (6) D89Y, (7) D89S, (8) V90D, (9) V90A, (10) V90L, (11) V90E, (12) V90M, (13) V90C, (14) V90P, (15) V90H, (16) V90K, (17) V90S, (18) V90T, (19) R102T, (20) R122N, (21) R122K, (22) R102T and D89V, (23) R102T and D89F, (24) R102T and D89I, (25) R102T and D89Q, (26) R102T and D89H, (27) R102T and D89Y, (28) R102T and D89K, (29) R102T and D89T, (30) R102T and D89W, (31) R102T and E83R, (32) R102T and I88V, (33) R102T and I88L, (34) R102T and R92A, (35) R102T and R92H, (36) R102T and R92M, (37) R102T and R92Q, (38) R102T and R92H, (39) R102T and R92K, (40) R102T and R122K, (41) R102T and V90I, (42) R102T and V90D, (43) R102T and V90R, (44) R102T and V90L, (45) R102T and V90F, (46) R102T and V90E, (47) R102T and V90C, (48) R102T and V90P, (49) R102T and V90Q, (50) R102T and V90N, (51) R102T and V90G, (52) R102T and V90H, (53) R102T and V90Y, (54) R102T and V90K, (55) R102T and V90S, (56) R102T and V90T; and optionally the optically active polypeptide mutation is (57) Y1L.

[0091] The terms "identity" or "percent identity," in the context of two or more polypeptide or nucleic acid molecule sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm known in the art, such as by manual alignment and visual inspection. Preferred algorithms are BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol. 215:403, respectively.

[0092] It is well known to those skilled in the art that in gene cloning procedures, it is often necessary to engineer appropriate restriction sites, which will necessarily introduce one or more extraneous residues at the termini of the expressed polypeptide or protein, without affecting the activity of the polypeptide or protein of interest. Also, for example, in order to construct fusion proteins, to facilitate expression of recombinant proteins, to obtain a recombinant protein that is secreted automatically outside the host cell, or to facilitate purification of a recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable region of the recombinant protein, such as, but not limited to, a suitable linker peptide, a signal peptide, a leader peptide, a terminal extension, glutathione S-transferase (GST), maltose E binding protein, protein A, a tag such as 6His or Flag, or a proteolytic enzyme site for factor Xa or thrombin or enterokinase.

[0093] The terms "functional fragment," "derivative," and "analog" as used herein refer to a protein that substantially maintains the same biological function or activity as the original polypeptide or protein (e.g., ATP-binding protein or fluorescent protein). Functional variants, derivatives, or analogs of the polypeptides or proteins (e.g., ATP-binding protein or fluorescent protein) of the present application can be (i) proteins having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues can or can not be encoded by the genetic code, or (ii) proteins having a substituent group at one or more amino acid residues, or (iii) proteins formed by fusing the mature protein to another compound (such as a compound that prolongs the half-life of the protein, e.g., polyethylene glycol), or (iv) proteins formed by fusing additional amino acid sequences to the protein sequence (such as a secretion sequence or a sequence or protein for purifying the protein or a proprotein sequence, or a fusion protein with an antigen IgG fragment). These functional variants, derivatives, and analogs are within the scope of one of ordinary skill in the art in light of the teachings herein.

[0094] The analogs also include those having residues other than naturally occurring L-amino acids, for example, D-amino acids, and those having non-naturally occurring or synthetic amino acids, for example, beta, gamma, etc. It is understood that the ATP-sensitive polypeptides of the present application are not limited to the representative proteins, variants, derivatives, and analogs listed above. Modified (typically not changing the primary structure) forms include chemically derivatized forms of the protein in vivo or in vitro, such as acetylated or carboxylated. Modifications also include glycosylated, such as those produced by glycosylation modifications during or further to the synthesis and processing of the protein. Such modifications can be accomplished by exposing the protein to enzymes that glycosylate or deglycosylate, such as mammalian glycosylation enzymes. Modified forms also include sequences having phosphorylated amino acid residues, such as phosphotyrosine, phosphoserine, phosphothreonine. Proteins modified to increase their resistance to proteolysis or to optimize solubility are also included.

[0095] The fusion polypeptides of the present application comprise the optical probes described herein and other polypeptides, including localization sequences, tags for purification or for immunoreactions. In some embodiments, the optical probes described herein further comprise other polypeptides fused thereto. The other polypeptides described herein do not affect the properties of the optical probes. The other polypeptides can be located at the N- and / or C-terminus of the optical probes. In some embodiments, the other polypeptides include polypeptides that localize the optical probes to different organelles or subcellular compartments, tags for purification or for immunoblotting. There can be linkers between the optical probes and the other polypeptides in the fusion polypeptides described herein.

[0096] Subcellular organelles described herein include cytoplasm, mitochondria, nucleus, endoplasmic reticulum, and cell membrane. In some embodiments, the tag for purification or the tag for immunoblotting includes 6 histidines (6*His), glutathione S-transferase (GST), Flag.

[0097] The present application also provides a method for preparing the ATP optical probe described above, comprising the following steps: 1) incorporating a nucleic acid sequence encoding the ATP optical probe described herein into an expression vector; 2) transferring the expression vector into a host cell; 2) culturing the host cell under conditions suitable for expression of the expression vector, 3) isolating the ATP optical probe.

[0098] The present application includes nucleic acid molecules encoding the ATP-sensitive polypeptides or optical probes or fusion polypeptides described herein. The term "nucleic acid" or "nucleotide" or "polynucleotide" or "nucleic acid sequence" as used herein can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand. When referring to nucleic acid, the term "variant" as used herein can be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is an alternative form of a nucleic acid that can result from natural or induced DNA changes. It can be a substitution of one or more nucleotides, a deletion or an insertion, but does not substantially alter the functional properties of the encoded protein. The nucleic acid of the present application can comprise a nucleotide sequence having at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the sequence of the nucleic acid. The present application also relates to nucleic acid fragments that hybridize to the above sequences. As used herein, a "nucleic acid fragment" has a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. The nucleic acid fragment can be used in nucleic acid amplification techniques (e.g., PCR).

[0099] The full-length sequence of the optical probe or fusion protein of the present application or fragments thereof can generally be obtained by PCR amplification, artificial synthesis, or recombination. The steps and reagents used in conventional PCR, synthesis, and recombination are known in the art. In addition, mutations can be introduced into the protein sequence of the present application by methods such as mutagenic PCR or chemical synthesis.

[0100] The present application also relates to nucleic acid constructs comprising the nucleic acid molecules described herein, and one or more control sequences operably linked to the sequences. The polynucleotides of the present application can be manipulated in a variety of ways to produce the polypeptides or proteins of interest. Manipulation of the nucleic acid constructs prior to their insertion into a vector can be desirable for a variety of reasons, e.g., to direct the expression of the gene product in a host cell, to assemble several genes into one vector, etc. Techniques for modifying nucleic acid sequences using recombinant DNA methods are known in the art.

[0101] In certain embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a homologous recombination vector. The polynucleotides of the present application can be cloned into a variety of types of vectors, e.g., plasmids, phagemids, phage derivatives, animal viruses, and cosmids.

[0102] Typical expression vectors will incorporate one or more expression control sequences that are operably linked to the nucleic acid sequences of the present application or their complements to control their expression. As used herein, the term "expression control sequence" refers to nucleic acid sequences that direct transcription and / or translation of a gene product, and can include origins of replication, promoters, selectable markers, or translation control elements, including enhancers, operators, terminators, ribosome binding sites, and the like. The choice of expression control sequences depends on the host cell used. In a recombinant expression vector, "operably linked" means that the nucleic acid sequence of interest is linked to the expression control sequences in a manner that allows for expression of the nucleic acid sequence. Methods are known in the art for ligating nucleic acid sequences to expression control sequences, e.g., by ligation of adaptors. The DNA sequences of the present application can be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are the lac or trp promoter of E. coli, the PL promoter of bacteriophage lambda, eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTR from retrovirus, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In one embodiment, the expression vector can employ commercially available pCDF vector without any special requirements. Illustratively, the nucleotide sequence encoding the optical probe and the expression vector are double digested with BamHI and EcoRI, respectively, and the digested products are ligated to obtain a recombinant expression vector. The present application does not have any special limitation on the specific steps and parameters for the digestion and ligation, and the steps and parameters of the art can be employed.

[0103] After obtaining the recombinant expression vector, the vector is transformed into a host cell to produce the protein or peptide including the optical probe or fusion protein. Such transformation process can be performed by using conventional techniques well known to those skilled in the art, such as transformation or transfection. The host cell of the present application refers to a cell capable of receiving and accommodating a recombinant DNA molecule, and is a place for amplification of a recombinant gene. The ideal recipient cell should satisfy two conditions of easy access and proliferation. The "host cell" of the present application can include prokaryotic cells and eukaryotic cells, and specifically includes bacterial cells, yeast cells, insect cells and mammalian cells. The host cell is preferably various cells which are beneficial to expression of a gene product or fermentation production, and such cells are well known and commonly used in the art. Specifically, the cells can be bacterial cells of Escherichia coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells of Drosophila S2 or Sf9, animal cells of CHO, COS, HEK293, HeLa cells, or Bowes melanoma cells, etc. The exemplary host cell used in the examples of the present application is Escherichia coli BL21-DE3 strain. Those skilled in the art are aware of how to select appropriate vectors, promoters, enhancers and host cells.

[0104] The method for transferring into a host cell of the present application is a conventional method in the art, including calcium phosphate or calcium chloride coprecipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemical-mediated transfer or electroporation. When the host is a prokaryote such as Escherichia coli, the method is preferably CaCl2or MgCl2treatment, and the steps used are well known in the art. When the host cell is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0105] After the expression vector is transferred into the host cell, the host cell into which the expression vector is transferred is subjected to amplification expression culture, and the ATP optical probe or fusion polypeptide of the present application is isolated from the culture. The amplification expression culture of the host cell can be performed using conventional methods. Depending on the type of host cell used, the medium used in the culture can be various conventional media. The culture is performed under conditions suitable for growth of the host cell.

[0106] In the present application, the optical probe is expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated or purified by various separation methods using its physical, chemical and other properties. The present application does not have special limitations on the method for isolating the ATP fluorescent protein, and the conventional separation method for fusion protein in the art can be used. These methods are well known to those skilled in the art, including but not limited to: conventional renaturation treatment, salting-out method, centrifugation, osmotic lysis, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques, and combinations of these methods. In one embodiment, the His-tag affinity chromatography method is used for the separation of the optical probe.

[0107] The present application also provides the use of the ATP optical probe or fusion polypeptide or host cell in detecting ATP in a sample, screening compounds, or intracellular and / or extracellular localization of ATP. In one aspect, the ATP optical probe is preferably linked to a signal peptide at different parts of the cell, and is introduced into the cell. By detecting the strength of the fluorescent signal in the cell, real-time localization of ATP is performed; by combining the standard addition curve of ATP with the change of the fluorescent signal, the corresponding ATP is quantitatively detected. The change of the fluorescent signal is shown by, for example, the ratio of the normalized fluorescent signal, and in the embodiment involving cpYFP, the ratio is the ratio of the 420 nm fluorescent signal to the 485 nm fluorescent signal of the sample to the corresponding ratio of the control. The ATP standard addition curve of the present application is drawn according to the fluorescent signal of the ATP optical probe under different concentrations of ATP. The ATP optical probe of the present application is directly introduced into the cell, and in the process of real-time localization and quantitative detection of ATP, there is no time-consuming sample processing process, and it is more accurate. In the process of high-throughput compound screening, different compounds are added to the cell culture medium, and the change of ATP content is determined, so as to screen out the compounds that affect the change of ATP content. The application of the ATP optical probe in the present application in real-time localization, quantitative detection and high-throughput compound screening is for non-diagnostic and therapeutic purposes, and is not related to the diagnosis and treatment of diseases.

[0108] The present application also provides a detection kit comprising the optical probe, fusion polypeptide, nucleic acid molecule, nucleic acid construct and / or host cell described herein. The kit also comprises other reagents required for detecting ATP. The other reagents are well known in the art, such as buffer, cell culture medium, ATP standard. Exemplary buffer, such as 100 mM HEPES and 100 mM NaCl, pH 7.4.

[0109] The ATP optical probe provided by the application is easy to mature, has large fluorescence dynamic change, good specificity, can be expressed in cells through gene operation, can be used for real-time positioning, high-throughput and quantitative detection of ATP in and outside cells, and saves time-consuming sample processing steps. Experimental results show that the highest response of the ATP optical probe provided in the application to ATP is more than 10 times that of a control, and the ATP optical probe can be used for positioning, qualitative and quantitative detection of cells in cytoplasm, nuclei, mitochondria and other subcellular structures, and can be used for high-throughput compound screening and quantitative detection of ATP in blood.

[0110] In this document, concentration, amount, percentage and other numerical values can be expressed in the form of ranges. It should also be understood that the use of such range forms is only for convenience and brevity, and should be flexibly interpreted as including the numerical values explicitly mentioned in the upper and lower limits of the range, and also including all individual numerical values or sub-ranges included in the range.

[0111] Examples

[0112] The ATP optical probe provided by the application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the application.

[0113] I. Experimental materials and reagents

[0114] In the examples, conventional genetic engineering molecular biology cloning methods, cell culture and imaging methods and the like are mainly used, which are well known to those skilled in the art, for example: Jane Ross-Kenmeyer et al., Molecular Biology Laboratory Reference Manual, J. Sambrook, D. W. Russell, Huang Peitang et al., Molecular Cloning Laboratory Guide (3rd Edition, August 2002, Science Press, Beijing); Ferenczi et al., Animal Cell Culture: Basic Technical Guide (5th Edition), Zhang Jingbo, Xu Cunqian et al., J. S. Bonifacino, M. Daso et al., Compact Cell Biology Experiment Guide, Zhang Jingbo et al.

[0115] The pCDF-cpYFP, pCDF-PS3 plasmids used in the examples were constructed by the Protein Lab of East China University of Technology, and the pCDF plasmid vector was purchased from Invitrogen Company. All primers used for PCR were synthesized, purified and identified correctly by mass spectrometry by Shanghai Jeery Bioengineering Technology Co., Ltd. and Huada Gene. The expression plasmids constructed in the examples were all sequenced, and the sequence determination was completed by Huada Gene Company and Jiali Sequencing Company. The Taq DNA polymerase used in each example was purchased from Dongsheng Bio, the pfu DNA polymerase was purchased from Tiangeng Biochemical Technology (Beijing) Co., Ltd., and the primeSTAR DNA polymerase was purchased from TaKaRa Company. The three kinds of polymerases purchased at the same time are attached with the corresponding polymerase buffer and dNTPs. The restriction endonucleases such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, SpeI, T4 ligase, T4 phosphorylase (T4 PNK) were purchased from Fermentas Company, and the corresponding buffer was attached when purchased. The transfection reagent Lip2000 Kit was purchased from Invitrogen Company. ATP and other compounds were purchased from Sigma Company. Unless otherwise stated, inorganic salts and other chemical reagents were purchased from Sigma-Aldrich Company. HEPES salt, ampicillin (Amp) and puromycin were purchased from Ameresco Company. 96-well detection blackboard and 384-well fluorescence detection blackboard were purchased from Grenier Company.

[0116] The DNA purification kit used in the examples was purchased from BBI Company (Canada), and the general plasmid small extraction kit was purchased from Tiangeng Biochemical Technology (Beijing) Co., Ltd. The cloning strain Mach1 was purchased from Invitrogen Company. The nickel column affinity chromatography column and desalting column filler were from GE healthcare company.

[0117] The main instruments used in the examples include: Biotek Synergy 2 multifunctional enzyme marker (Bio-Tek Company, USA), X-15R high-speed refrigerated centrifuge (Beckman Company, USA), Microfuge22R table top high-speed refrigerated centrifuge (Beckman Company, USA), PCR amplifier (Biometra Company, Germany), ultrasonic crusher (Ningbo Xinzhi Company), nucleic acid electrophoresis instrument (Shengneng Bochai Company), fluorescence spectrophotometer (Agilent Company, USA), CO2 constant temperature cell incubator (SANYO), inverted fluorescence microscope (Nikon Company, Japan).

[0118] II. Molecular biology methods and cell experimental methods

[0119] II.1 Polymerase chain reaction (PCR):

[0120] 1. PCR for fragment amplification

[0121] This method is mainly used for gene fragment amplification and positive clone identification by colony PCR. The reaction system of PCR amplification is as follows: template sequence 0.5-1 μL, forward primer (25 μM) 0.5 μL, reverse primer (25 μM) 0.5 μL, 10×pfu buffer 5 μL, pfu DNA polymerase 0.5 μL, dNTP (10 mM) 1 μL, sterilized ultrapure water (ddH2O) 41.5-42 μL, total volume 50 μL. The PCR amplification program is as follows: denaturation at 95°C for 2-10 minutes, 30 cycles of 94-96°C for 30-45 seconds, 50-65°C for 30-45 seconds, 72°C for a certain time (600 bp / min), and extension at 72°C for 10 minutes.

[0122] 2. PCR for long fragment (>2500 bp) amplification

[0123] The long fragment amplification used in the present application is mainly reverse PCR amplification vector, which is used in the following examples to obtain a site-directed mutation technique. Reverse PCR primers are designed at the mutation site, wherein the 5' end of one primer contains a mutated nucleotide sequence. The amplified product contains the corresponding mutation site. The reaction system of long fragment amplification PCR is as follows: template sequence (10 pg-1 ng) 1 μL, forward primer (25 μM) 0.5 μL, reverse primer (25 μM) 0.5 μL, 5×PrimerSTAR buffer 10 μL, PrimerSTAR DNA polymerase 0.5 μL, dNTP (2.5 mM) 4 μL, sterilized ultrapure water (ddH2O) 33.5 μL, total volume 50 μL. The PCR amplification program is as follows: denaturation at 95°C for 5 minutes, 30 cycles of 98°C for 10 seconds, 50-68°C for 5-15 seconds, and 72°C for a certain time (1000 bp / min), and extension at 72°C for 10 minutes; or denaturation at 95°C for 5 minutes, 30 cycles of 98°C for 10 seconds and 68°C for a certain time (1000 bp / min), and extension at 72°C for 10 minutes.

[0124] II.2 Endonuclease enzyme digestion reaction

[0125] The system for double enzyme digestion of plasmid vector is as follows: plasmid vector 20 μL (about 1.5 μg), 10×buffer 5 μL, restriction endonuclease 1 11-2 μL, restriction endonuclease 2 11-2 μL, and sterilized ultrapure water to a total volume of 50 μL. The reaction condition is 37°C for 1-7 hours.

[0126] II.3 DNA fragment 5' end phosphorylation reaction

[0127] The plasmid or genome extracted from microorganism contains phosphate group at the end, but the PCR product does not, so the phosphate group addition reaction is needed for the 5' end base of the PCR product, and only the DNA molecule with phosphate group at the end can have the ligation reaction. The phosphorylation reaction system is as follows: PCR product segment DNA sequence 5-8 μL, 10 x T4 ligase buffer 1 μL, T4 polynucleotide kinase (T4 PNK) 1 μL, sterilized ultrapure water 0-3 μL, total volume 10 μL. The reaction condition is 37°C for 30 minutes-2 hours, and then inactivated at 72°C for 20 minutes.

[0128] II.4 Purpose fragment and vector ligation reaction

[0129] The ligation methods between different fragments and vectors are different, and three ligation methods are used in the present application

[0130] 1. Blunt end ligation of blunt end short fragment and linearized vector

[0131] The principle of this method is that the blunt end product obtained by PCR is phosphorylated at the 5' end of the DNA fragment under the action of T4 PNK, and then connected with the linearized vector under the action of PEG4000 and T4 DNA ligase to obtain the recombinant plasmid. The homologous recombination ligation system is as follows: T4 PNK treated DNA fragment 4 μL, linearized vector fragment 4 μL, PEG4000 1 μL, 10 x T4 ligase buffer 1 μL, T4 DNA ligase 1 μL, total 10 μL. The reaction condition is 22°C for 30 minutes.

[0132] 2. Ligation of DNA fragment containing sticky end and vector fragment containing sticky end

[0133] The DNA fragment cut by restriction endonuclease usually produces protruding sticky end, so it can be connected with the vector fragment containing sequence complementary sticky end to form the recombinant plasmid. The ligation reaction system is as follows: PCR product fragment DNA after enzyme cutting 1-7 μL, plasmid after enzyme cutting 0.5-7 μL, 10 x T4 ligase buffer 1 μL, T4 DNA ligase 1 μL, sterilized ultrapure water to a total volume of 10 μL. The reaction condition is 16°C for 4-8 hours.

[0134] 3. Ligation reaction of 5' end phosphorylated DNA fragment product after introducing site-directed mutation by reverse PCR

[0135] The 5' end phosphorylated DNA fragment is connected with the 3' end and 5' end of the linearized vector by self-circular ligation reaction to obtain the recombinant plasmid. The self-circular ligation reaction system is as follows: phosphorylation reaction system 10 μL, T4 ligase (5 U / μL) 0.5 μL, total volume 10.5 μL. The reaction condition is 16°C for 4-16 hours.

[0136] II.5 Preparation and transformation of competent cells

[0137] Preparation of competent cells:

[0138] 1. Pick a single colony (e.g. Mach 1) and inoculate into 5 mL LB medium, 37°C, overnight.

[0139] 2. Take 0.5-1 mL of the overnight culture and inoculate into 50 mL LB medium, 37°C, 220 rpm for 3-5 hours until OD600 reaches 0.5.

[0140] 3. Pre-chill the cells in ice bath for 2 hours.

[0141] 4. Centrifuge at 4°C, 4000 rpm for 10 minutes.

[0142] 5. Discard the supernatant, resuspend the cells with 5 mL pre-chilled resuspension buffer, and add more resuspension buffer to a final volume of 50 mL after the cells are evenly resuspended.

[0143] 6. Incubate in ice bath for 45 minutes.

[0144] 7. Centrifuge at 4°C, 4000 rpm for 10 minutes, and resuspend the bacteria with 5 mL ice-pre-chilled storage buffer.

[0145] 8. Place 100 μL of the bacterial solution in each EP tube, and store at -80°C or in liquid nitrogen.

[0146] Resuspension buffer: CaCl2(100 mM), MgCl2(70 mM), NaAc(40 mM)

[0147] Storage buffer: 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10x CaCl2(1 M), 1 mL 10x MgCl2(700 mM), 1 mL 10x NaAc(400 mM), 4.6 mL ddH2O

[0148] Transformation of competent cells:

[0149] 1. Thaw 100 μL of competent cells on ice.

[0150] 2. Add the appropriate volume of ligation product, mix gently, and incubate in ice bath for 30 minutes. Usually, the volume of ligation product added is less than 1 / 10 of the volume of competent cells.

[0151] 3. Heat shock the bacterial solution in a 42°C water bath for 90 seconds, and quickly transfer to ice bath for 5 minutes.

[0152] 4. Add 500 μL LB, incubate at 37°C in a shaker at 200 rpm for 1 hour.

[0153] 5. Centrifuge the bacterial solution at 4000 rpm for 3 minutes, take 200 μL supernatant, blow the bacterial body evenly, and evenly spread on the surface of agar plate containing appropriate antibiotic, and plate in 37°C constant temperature incubator overnight.

[0154] II.6 Expression, purification and fluorescence detection of protein

[0155] 1. Transform the expression vector (for example, ATP optical probe expression vector based on pCDF) into BL21 (DE3) cells, invert and culture overnight, pick clones from the plate into 250 ml conical flask, and place in 37°C shaker, 220 rpm, to culture until OD = 0.4-0.8, add 1 / 1000 (v / v) IPTG (1M), and induce expression at 18°C for 24-36 hours.

[0156] 2. After the induction of expression is completed, centrifuge the bacteria at 4000 rpm for 30 minutes, add 50 mM phosphate buffer to resuspend the bacterial body precipitate, and ultrasonically break the bacteria to clarify the bacterial body. Centrifuge at 9600 rpm at 4°C for 20 minutes.

[0157] 3. Centrifuge the supernatant to purify the protein through a self-assembled nickel column affinity chromatography column, and then obtain the protein dissolved in 100 mM HEPES buffer (pH 7.4) through a self-assembled desalting column after nickel column affinity chromatography.

[0158] 4. After the purified protein is identified by SDS-PAGE, dilute the probe to a final concentration of 0.2-5 μM of protein solution using the assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4). Prepare ATP to a final concentration of 50 mM stock solution using the assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4).

[0159] 5. Take 100 μl of 1 μM protein solution, incubate at 37°C for 10 minutes, add ATP titration, and measure the fluorescence intensity of the protein at 420 nm light excitation and 528 nm emission and at 485 nm light excitation and 528 nm emission. The fluorescence excitation and emission of the sample are measured by using a multifunctional fluorescence enzyme marker.

[0160] 6. Take 100 μl of 1 μM protein solution, incubate at 37°C for 10 minutes, add ATP, and measure the absorption spectrum and fluorescence spectrum of the protein. The absorption spectrum and fluorescence spectrum of the sample are measured by using a spectrophotometer and a fluorescence spectrophotometer.

[0161] II.7 Transfection of mammalian cells and fluorescence detection

[0162] 1. The pCDNA3.1+ based ATP optical probe plasmid was transfected into HEK293 by transfection reagent Lipofectamine 2000 (Invitrogen) and cultured in a cell incubator at 37°C, 5% CO2. After 24-36 hours of sufficient expression of the exogenous gene, fluorescence detection was performed.

[0163] 2. After the induction of expression was completed, the adherent HEK293 cells were washed with PBS three times and placed in HBSS solution for fluorescence microscopy and enzyme marker detection, respectively.

[0164] Example 1: ATP binding protein plasmid

[0165] The ε subunit gene of F0F1-ATP synthase in Bacillus caldovelox was amplified by PCR, and the PCR product was recovered after gel electrophoresis and digested with HindIII and XhoI. The pCDF vector was also double-digested with the corresponding enzymes. After ligation with T4 DNA ligase, the product was used to transform DH5α, and the transformed DH5α was plated on LB plates (streptomycin 100 ug / mL) and incubated at 37°C overnight. After plasmid extraction of the growing DH5α transformant, PCR identification was performed. After sequencing of the positive plasmid, subsequent plasmid construction was performed.

[0166] Example 2: Expression and detection of cpYFP optical probes with different insertion sites

[0167] In this embodiment, the following sites were selected for insertion of cpYFP based on pCDF-PS3: 10 / 11, 10 / 12, 10 / 13, 11 / 11, 11 / 12, 11 / 13, 12 / 11, 12 / 12, 12 / 13, 19 / 20, 19 / 21, 20 / 20, 20 / 21, 27 / 28, 27 / 29, 27 / 30, 28 / 28, 28 / 29, 28 / 30, 29 / 30, 33 / 34, 33 / 35, 33 / 36, 33 / 37, 33 / 38, 34 / 34, 34 / 35, 34 / 36, 34 / 37, 34 / 38, 35 / 34, 35 / 35, 35 / 36, 35 / 37, 35 / 38, 36 / 34, 36 / 35, 36 / 36, 36 / 37, 36 / 38, 37 / 34, 37 / 35, 37 / 36, 37 / 37, 37 / 38, 41 / 42, 41 / 43, 41 / 44, 42 / 42, 42 / 43, 42 / 44, 43 / 43, 43 / 44, 45 / 46, 45 / 47, 45 / 48, 46 / 46, 46 / 47, 46 / 48, 47 / 46, 47 / 47, 47 / 48, 54 / 55, 54 / 56, 54 / 57, 55 / 55, 55 / 56, 55 / 57, 56 / 55, 56 / 56, 56 / 57, 61 / 62, 61 / 63, 61 / 64, 62 / 62, 62 / 63, 62 / 64, 63 / 62, 63 / 63, 63 / 64, 66 / 67, 66 / 68, 66 / 69, 67 / 67, 67 / 68, 67 / 69, 68 / 67, 68 / 68, 68 / 69, 71 / 72, 71 / 73, 71 / 74, 72 / 72, 72 / 73, 72 / 74, 73 / 72, 73 / 73, 73 / 74, 78 / 79, 78 / 80, 78 / 81, 79 / 79, 79 / 80, 79 / 81, 80 / 79, 80 / 80, 80 / 81, 84 / 85, 84 / 86, 84 / 87, 84 / 88, 84 / 89, 84 / 90, 84 / 91, 85 / 85, 85 / 86, 85 / 87, 85 / 88, 85 / 89, 85 / 90, 85 / 91, 86 / 85, 86 / 86, 86 / 87, 86 / 88, 86 / 89, 86 / 90, 86 / 91, 87 / 85, 87 / 86, 87 / 87, 87 / 88, 87 / 89, 87 / 90, 87 / 91, 88 / 85, 88 / 86, 88 / 87, 88 / 88, 88 / 89, 88 / 90, 88 / 91, 89 / 85, 89 / 86, 89 / 87, 89 / 88, 89 / 89, 89 / 90, 89 / 91, 90 / 85, 90 / 86, 90 / 87, 90 / 88, 90 / 89, 90 / 90, 90 / 891, 103 / 104,103 / 105、103 / 106、103 / 107、103 / 108、103 / 109、103 / 110、103 / 111、103 / 112、103 / 113、103 / 114、103 / 115、103 / 116、104 / 104、104 / 105、104 / 106、104 / 107、104 / 108、104 / 109、104 / 110、104 / 111、104 / 112、104 / 113、104 / 114、104 / 115、104 / 116、105 / 104、105 / 105、105 / 106、105 / 107、105 / 108、105 / 109、105 / 110、105 / 111、105 / 112、105 / 113、105 / 114、105 / 115、105 / 116、106 / 104、106 / 105、106 / 106、106 / 107、106 / 108、106 / 109、106 / 110、106 / 111、106 / 112、106 / 113、106 / 114、106 / 115、106 / 116、107 / 104、107 / 105、107 / 106、107 / 107、107 / 108、107 / 109、107 / 110、107 / 111、107 / 112、107 / 113、107 / 114、107 / 115、107 / 116、108 / 104、108 / 105、108 / 106、108 / 107、108 / 108、108 / 109、108 / 110、108 / 111、108 / 112、108 / 113、108 / 114、108 / 115、108 / 116、109 / 104、109 / 105、109 / 106、109 / 107、109 / 108、109 / 109、109 / 110、109 / 111、109 / 112、109 / 113、109 / 114、109 / 115、109 / 116、110 / 104、110 / 105、110 / 106、110 / 107、110 / 108、110 / 109、110 / 110、110 / 111、110 / 112、110 / 113、110 / 114、110 / 115、110 / 116、111 / 104、111 / 105、111 / 106、111 / 107、111 / 108、111 / 109、111 / 110、111 / 111、111 / 112、111 / 113、111 / 114、111 / 115、111 / 116、112 / 104、112 / 105、112 / 106、112 / 107、112 / 108、112 / 109、112 / 110、112 / 111、112 / 112、112 / 113, 112 / 114, 112 / 115, 112 / 116, 113 / 104, 113 / 105, 113 / 106, 113 / 107, 113 / 108, 113 / 109, 113 / 110, 113 / 111, 113 / 112, 113 / 113, 113 / 114, 113 / 115, 113 / 116, 114 / 104, 114 / 105, 114 / 106, 114 / 107, 114 / 108, 114 / 109, 114 / 110, 114 / 111, 114 / 112, 114 / 113, 114 / 114, 114 / 115, 114 / 116, 115 / 104, 115 / 105, 115 / 106, 115 / 107, 115 / 108, 115 / 109, 115 / 110, 115 / 111, 115 / 112, 115 / 113, 115 / 114, 115 / 115, and / or 115 / 116. By way of example only, the amino acid sequence of 103 / 110-PS3-cpYFP is shown in SEQ ID NO: 10.

[0168] The DNA fragment of cpYFP was generated by PCR, and cpYFP end homologous sequences were introduced by 5' end of primers. The PCR amplification generated pCDF-ATP binding protein linearization vector, which had the same sequences (15bp-20bp) at 5' and 3' ends as the corresponding ends of cpYFP. The linearized pCDF-PS3 and cpYFP fragments were subjected to homologous recombination under the action of Hieff Clone Enzyme. The product was transformed into DH5a, and the transformed DH5a was coated on LB plates (streptomycin 100ug / mL) and incubated at 37°C overnight. The positive clones identified by PCR were sequenced after plasmid extraction. Sequencing was completed by Ji Li Sequencing Company.

[0169] After sequencing was correct, the recombinant plasmid was transformed into BL21(DE3) to induce expression, and the protein was purified. The size of the protein was about 70Kda by SDS-PAGE electrophoresis. The size was consistent with the size of PS3-cpYFP fusion protein expressed by pCDF-PS3-cpYFP, which contained His-tag purification label. The results are shown in Figure 1

[0170] ​ATP-responsive screening was performed using the broken supernatant of E. coli expressing PS3-cpYFP fusion protein, and the detection signal of the fusion fluorescent protein containing 100 mM ATP was divided by the detection signal of the fusion fluorescent protein without ATP. As shown in Table 1, the detection results showed that the broken supernatant expressing PS3-cpYFP fusion protein had an optical probe with an ATP response of more than 1.2 times or less than 0.83 times in 11 / 13, 12 / 13, 19 / 21, 27 / 30, 28 / 28, 28 / 30, 29 / 28, 29 / 29, 33 / 35, 33 / 38, 34 / 34, 34 / 35, 35 / 34, 35 / 35, 35 / 36, 35 / 37, 36 / 34, 36 / 35, 36 / 36, 36 / 37, 36 / 38, 37 / 38, 33 / 35, 33 / 38, 41 / 42, 41 / 43, 41 / 44, 42 / 42, 42 / 43, 43 / 42, 43 / 43, 46 / 46, 46 / 47, 47 / 47, 47 / 48, 54 / 55, 55 / 55, 55 / 57, 56 / 55, 56 / 57, 61 / 63, 62 / 62, 63 / 62, 66 / 67, 66 / 69, 67 / 67, 67 / 69, 68 / 67, 68 / 69, 71 / 72, 71 / 73, 72 / 73, 72 / 74, 73 / 73, 73 / 74, 78 / 80, 78 / 81, 80 / 80, 80 / 81, 84 / 85, 84 / 91, 85 / 88, 85 / 91, 86 / 85, 86 / 86, 86 / 88, 86 / 90, 87 / 88, 87 / 89, 87 / 90, 87 / 91, 88 / 86, 88 / 87, 88 / 88, 88 / 89, 89 / 88, 89 / 90, 89 / 91, 90 / 89, 90 / 90, 103 / 104, 103 / 108, 103 / 109, 103 / 110, 103 / 112, 103 / 113, 103 / 115, 103 / 116, 104 / 104, 104 / 105, 104 / 106, 104 / 107, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 104 / 112, 104 / 114, 104 / 116, 105 / 104, 105 / 105, 105 / 106, 105 / 107, 105 / 108, 105 / 109, 105 / 110, 105 / 112, 105 / 114, 105 / 116, 106 / 104, 106 / 107, 106 / 111, 106 / 113, 106 / 114, 106 / 115, 106 / 116, 107 / 111, 107 / 113, 107 / 116, 108 / 109, 108 / 110, 108 / 111, 108 / 112, 108 / 113, 108 / 115, 108 / 113, 109 / 109, 109 / 110, 109 / 111, 109 / 112, 109 / 113, 109 / 115, 109 / 116, 110 / 109, 110 / 110, 110 / 111, 110 / 112, 110 / 113, 110 / 115, 110 / 116, 111 / 110, 111 / 111, 111 / 112, 111 / 113, 111 / 115, 111 / 116, 112 / 110, 112 / 111, 112 / 112, 112 / 113, 112 / 115, 112 / 116, 113 / 110, 113 / 111, 113 / 112, 113 / 113, 113 / 115, 113 / 116, 114 / 110, 114 / 111, 114 / 112, 114 / 113, 114 / 115, 114 / 116, 115 / 110, 115 / 111, 115 / 112, 115 / 113, 115 / 115, 115 / 116, 116 / 110, 116 / 111, 116 / 112, 116 / 113, 116 / 115, 116 / 116, 117 / 117, 117 / 118, 117 / 119, 117 / 120, 117 / 121, 117 / 122, 117 / 123, 117 / 124, 117 / 125, 117 / 126, 117 / 127, 117 / 128, 117 / 129, 117 / 130, 117 / 131, 117 / 132, 117 / 133, 117 / 134, 117 / 135, 117 / 136, 117 / 137, 117 / 138, 117 / 139, 117 / 140, 117 / 141, 117 / 142, 117 / 143, 117 / 144, 117 / 145, 117 / 146, 117 / 147, 117 / 148, 117 / 149, 117 / 150, 117 / 151, 117 / 152, 117 / 153, 117 / 154, 117 / 155, 117 / 156, 117 / 157, 117 / 158, 117 / 159, 117 / 160, 117 / 161, 117 / 162, 117 / 163, 117 / 164, 117 / 165, 117 / 166, 117 / 167, 117 / 168, 117 / 169, 117 / 170, 117 / 171, 117 / 172, 117 / 173, 117 / 174, 117 / 175, 117 / 176, 117 / 177, 117 / 178, 117 / 179, 117 / 180, 117 / 181, 117 / 182, 117 / 183, 117 / 184, 117 / 185, 117 / 186, 117 / 187, 117 / 188, 117 / 189, 117 / 190, 117 / 191, 117 / 192, 117 / 193, 117 / 194, 117 / 195, 117 / 196, 117 / 197, 117 / 198, 117 / 199, 117 / 200, 117 / 201, 117 / 202, 117 / 203, 117 / 204, 117 / 205, 117 / 206, 117 / 207, 117 / 208, 117 / 209, 117 / 210, 117 / 211, 117 / 212, 117 / 213, 117 / 214, 117 / 215, 117 / 216, 117 / 217, 117 / 218, 117 / 219, 117 / 220, 117 / 221, 117 / 222, 117 / 223, 117 / 224, 117 / 225, 117 / 226, 117 / 227, 117 / 228, 117 / 229, 117 / 230, 117 / 231, 117 / 232, 117 / 233, 117 / 234, 117 / 235, 117 / 236, 117 / 237, 117 / 238, 117 / 239, 117 / 240, 117 / 241, 117 / 242, 117 / 243, 117 / 244, 117 / 245, 117 / 246, 117 / 247, 117 / 248, 117 / 249, 117 / 250, 117 / 251, 117 / 252, 117 / 253, 117 / 254, 117 / 255, 117 / 256, 117 / 257, 117 / 258, 117 / 259, 117 / 260, 117 / 261, 117 / 262, 117 / 263, 117 / 264, 117 / 265, 117 / 266, 117 / 267, 117 / 268, 117 / 269, 117 / 270, 117 / 271, 117 / 272, 117 / 273, 117 / 274, 117 / 275, 117 / 276, 117 / 277, 117 / 278, 117 / 279, 117 / 280, 117 / 281, 117 / 282, 117 / 283, 117 / 284, 117 / 285, 117 / 286, 117 / 287, 117 / 288, 117 / 289, 117 / 290, 117 / 291, 117 / 292, 117 / 293, 117 / 294, 117 / 295, 117 / 296, 117 / 297, 117 / 298, 117 / 299, 117 / 300, 117 / 301, 117 / 302, 117 / 303, 117 / 304, 117 / 305, 117 / 306, 117 / 307, 117 / 308, 117 / 309, 117 / 310, 117 / 311, 117 / 312, 117 / 313, 117 / 314, 117 / 315, 117 / 316, 117 / 317, 117 / 318, 117 / 319, 117 / 320, 117 / 321, 117 / 322, 117 / 323, 117 / 324, 117 / 325, 117 / 326, 117 / 327, 117 / 328, 117 / 109 / 110, 109 / 112, 109 / 113, 109 / 116, 110 / 104, 110 / 112, 110 / 113, 110 / 115, 110 / 116, 111 / 107, 111 / 110, 111 / 111, 111 / 112, 111 / 113, 111 / 116, 112 / 104, 112 / 105, 112 / 110, 112 / 114, 112 / 115, 112 / 116, 113 / 108, 113 / 109, 113 / 110, 113 / 113, 113 / 116, 114 / 104, 114 / 105, 114 / 106, 114 / 108, 114 / 109, 114 / 110, 114 / 111, 114 / 115, 114 / 116, 115 / 108, 115 / 111, 115 / 113, 115 / 115, and 115 / 116.

[0171] Table 1

[0172]

[0173] Example 3: Expression and detection of cpGFP optical probes with different insertion sites

[0174] The ATP green fluorescent protein fluorescent probe was constructed by replacing cpYFP with cpGFP according to the method in Example 2. As shown in Table 2, the detection results showed that the optical probes with insertion at 33 / 36, 108 / 109, and 113 / 115 sites or the corresponding amino acid sites of the family proteins of the optical probes had more than 1.3-fold or less than 0.77-fold ATP response to the broken supernatant expressing PS3-cpGFP fusion protein.

[0175] Table 2

[0176]

[0177] Example 4: Expression and detection of cpBFP optical probes with different insertion sites

[0178] The ATP blue fluorescent protein fluorescent probe was constructed by replacing cpYFP with cpBFP according to the method in Example 2. As shown in Table 3, the detection results showed that the optical probes with insertion at 35 / 35, 107 / 113, and 108 / 109 sites or the corresponding amino acid sites of the family proteins of the optical probes had more than 1.3-fold or less than 0.77-fold ATP response to the broken supernatant expressing PS3-cpBFP fusion protein.

[0179] Table 3

[0180]

[0181] Example 5: Expression and detection of cpmApple optical probes with different insertion sites

[0182] ATP red fluorescent protein fluorescent probes were constructed by replacing cpYFP with cpmApple according to the method in Example 2. As shown in Table 4, the detection results showed that the optical probes with 35 / 35, 47 / 46 and 110 / 109 sites or the corresponding amino acid sites of the family protein of the optical probes were inserted into the optical probes with more than 1.3 times or less than 0.77 times of ATP response to the broken supernatant of PS3-cpmApple fusion protein.

[0183] Table 4

[0184]

[0185] Example 6: Performance of optical probes

[0186] For the optical probes obtained in Example 2 that responded to ATP more than 1.2-fold or less than 0.83-fold, i.e. at: 11 / 13, 12 / 13, 19 / 21, 27 / 30, 28 / 28, 28 / 30, 29 / 28, 29 / 29, 33 / 35, 33 / 38, 34 / 34, 34 / 35, 35 / 34, 35 / 35, 35 / 36, 35 / 37, 36 / 34, 36 / 35, 36 / 36, 36 / 37, 36 / 38, 37 / 38, 33 / 35, 33 / 38, 41 / 42, 41 / 43, 41 / 44, 42 / 42, 42 / 43, 43 / 42, 43 / 43, 46 / 46, 46 / 47, 47 / 47, 47 / 48, 54 / 55, 55 / 55, 55 / 57, 56 / 55, 56 / 57, 61 / 63, 62 / 62, 63 / 62, 66 / 67, 66 / 69, 67 / 67, 67 / 69, 68 / 67, 68 / 69, 71 / 72, 71 / 73, 72 / 73, 72 / 74, 73 / 73, 73 / 74, 78 / 80, 78 / 81, 80 / 80, 80 / 81, 84 / 85, 84 / 91, 85 / 88, 85 / 91, 86 / 85, 86 / 86, 86 / 88, 86 / 90, 87 / 88, 87 / 89, 87 / 90, 87 / 91, 88 / 86, 88 / 87, 88 / 88, 88 / 89, 89 / 88, 89 / 90, 89 / 91, 90 / 89, 90 / 90, 103 / 104, 103 / 108, 103 / 109, 103 / 110, 103 / 112, 103 / 113, 103 / 115, 103 / 116, 104 / 104, 104 / 105, 104 / 106, 104 / 107, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 104 / 112, 104 / 114, 104 / 116, 105 / 104, 105 / 105, 105 / 106, 105 / 107, 105 / 108, 105 / 109, 105 / 110, 105 / 112, 105 / 114, 105 / 116, 106 / 104, 106 / 107, 106 / 111, 106 / 113, 106 / 114, 106 / 115, 106 / 116, 107 / 111, 107 / 113, 107 / 116, 108 / 109, 108 / 110, 108 / 111, 108 / 112, 108 / 113, 108 / 115, 108 / 113, 109 / 109, 109 / 110, 109 / 112, 109 / 113, 109 / 116, 110 / 104, 110 / 112, 110 / 113, 110 / 115, 110 / 116, 111 / 107, 111 / 110, 111 / 111, 111 / 112,The 166 optical probes inserted at the 111 / 113, 111 / 116, 112 / 104, 112 / 105, 112 / 110, 112 / 114, 112 / 115, 112 / 116, 113 / 108, 113 / 109, 113 / 110, 113 / 113, 113 / 116, 114 / 104, 114 / 105, 114 / 106, 114 / 108, 114 / 109, 114 / 110, 114 / 111, 114 / 115, 114 / 116, 115 / 108, 115 / 111, 115 / 113, 115 / 115, and / or 115 / 116 sites are used to detect ATP at a concentration gradient (0-100 mM) and the change in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to that at 485 nm excitation and 528 nm emission. The Kd (binding constant) of the 7 ATP optical probes with insertion sites at 12 / 13, 55 / 56, 56 / 57, 67 / 69, 103 / 110, 105 / 104, and 109 / 112 is 12.42, 15.48, 9.912, 2.15, 60.91, 12.94, and 49.84 μM, respectively.

[0187] Example 7: Expression and detection of mutant cpYFP optical probes

[0188] Mutant optical probes are constructed based on PS3-103 / 110-cpYFP. The plasmid pCDF-PS3-103 / 110-cpYFP is linearized by PCR using primers containing the base sequences of the desired mutation sites, and the PCR product is subjected to homologous recombination to obtain mutant plasmids of the 9 sites D89, E83, I88, R92, R126, R122, V90, R102 of the ATP-sensitive polypeptide and Y1 of the optically active polypeptide, and sequencing is completed by Ji Li Sequencing Company. An exemplary 103 / 110-PS3-R102T / R92Q-cpYFP-Y1L sequence is shown in SEQ ID NO: 11.

[0189] The successfully constructed mutant plasmids are transformed into BL21(DE3) for expression, and the supernatant of the broken E. coli expressing the probe protein is used for response screening of ATP and other non-specific substrates, and the detection signal of the fusion fluorescent protein containing ATP or other non-specific substrates is divided by the detection signal of the fusion fluorescent protein without ATP. The detection results show that the optical probes with more than 3-fold response to ATP and better specificity are shown in Table 5.

[0190] When both the fluorescent protein and the ATP-sensitive polypeptide have mutations, the mutants show greater changes in ATP. Mutant plasmids with a change of more than 6-fold in ATP include PS3-103 / 110-cpYFP-R102T / D89Q / Y1L, PS3-103 / 110-cpYFP-R102T / D89H / Y1L, PS3-103 / 110-cpYFP-R102T / D89K / Y1L, PS3-103 / 110-cpYFP-R102T / D89T / Y1L, PS3-103 / 110-cpYFP-R102T / R122K / Y1L, PS3-103 / 110-cpYFP-R102T / V90E / Y1L, PS3-103 / 110-cpYFP-R102T / V90Q / Y1L, PS3-103 / 110-cpYFP-R102T / V90G / Y1L, PS3-103 / 110-cpYFP-R102T / V90Y / Y1L, and PS3-103 / 110-cpYFP-R102T / V90S / Y1L. In addition to these, mutant plasmids suitable for use in mammalian cell systems include PS3-103 / 110-cpYFP-R122N, PS3-103 / 110-cpYFP-R102T / I88V / Y1L, PS3-103 / 110-cpYFP-R102T / I88L / Y1L, PS3-103 / 110-cpYFP-R102T / R92A / Y1L, PS3-103 / 110-cpYFP-R102T / R92Q / Y1L, PS3-103 / 110-cpYFP-R102T / R92M / Y1L, and PS3-103 / 110-cpYFP-R102T / R92H / Y1L.

[0191] Table 5

[0192] Insertion site PS3 mutation cpYFP mutation R 420 / 485 Kd / μM Insertion site PS3 mutation cpYFP mutation R 420 / 485 Kd / μM 103 / 110 D89E - 3.44 229.2 103 / 110 R102T / D89K Y1L 6.61 27.87 103 / 110 D89V - 3.03 79.89 103 / 110 R102T / D89T Y1L 6.55 237.9 103 / 110 D89M - 3.15 301.8 103 / 110 R102T / D89W Y1L 4.98 224.7 103 / 110 D89Q - 3.45 249.7 103 / 110 R102T / I88V Y1L 4.87 1619 103 / 110 D89N - 3.54 79.4 103 / 110 R102T / I88L Y1L 4.44 2114 103 / 110 D89Y - 3.28 249.1 103 / 110 R102T / R92A Y1L 5.47 2542 103 / 110 D89S - 3.21 121 103 / 110 R102T / R92M Y1L 5.65 3109 103 / 110 R122N - 3.04 1540 103 / 110 R102T / R92Q Y1L 5.81 1152 103 / 110 R122K - 3.75 167.8 103 / 110 R102T / R92H Y1L 4.86 1359 103 / 110 V90D - 3.39 684.2 103 / 110 R102T / R92K Y1L 5.00 111.9 103 / 110 V90A - 3.04 282.4 103 / 110 R102T / R122K Y1L 7.28 41.56 103 / 110 V90L - 3.08 97.11 103 / 110 R102T / V901 Y1L 5.99 90.05 103 / 110 V90E - 3.33 90.5 103 / 110 R102T / V90D Y1L 5.24 401.6 103 / 110 V90M - 3.6 171.5 103 / 110 R102T / V90R Y1L 5.64 33.42 103 / 110 V90C - 3.23 329.2 103 / 110 R102T / V90L Y1L 4.61 20.04 103 / 110 V90P - 3.35 124.7 103 / 110 R102T / V90F Y1L 5.64 674.3 103 / 110 V90H - 3.14 112.1 103 / 110 R102T / V90E Y1L 6.5 48.59 103 / 110 V90K - 3.24 47.3 103 / 110 R102T / V90C Y1L 4.82 35.22 103 / 110 V90S - 3.16 94.23 103 / 110 R102T / V90P Y1L 4.87 30.83 103 / 110 V90T - 3.46 76.78 103 / 110 R102T / V90Q Y1L 6.46 107 103 / 110 R102T Y1L 4.71 474 103 / 110 R102T / V90N Y1L 5.11 214.8 103 / 110 R102T / D89V Y1L 4.92 69.58 103 / 110 R102T / V90G Y1L 6.09 67.02 103 / 110 R102T / D89F Y1L 5.21 180.3 103 / 110 R102T / V90H Y1L 5.7 45.36 103 / 110 R102T / D891 Y1L 5.79 130.8 103 / 110 R102T / V90Y Y1L 6.59 32.96 103 / 110 R102T / D89Q Y1L 6.13 135.5 103 / 110 R102T / V90K Y1L 5.86 4.46 103 / 110 R102T / D89H Y1L 6.21 37.24 103 / 110 R102T / V90S Y1L 6.27 142.5 103 / 110 R102T / D89Y Y1L 5.92 85.95 103 / 110 R102T / V90T Y1L 4.64 16.75

[0193] Example 8: Performance of optical probe mutants

[0194] The ATP optical probes in Table 5 described in Example 7 were each subjected to ATP detection at a concentration gradient (0-100 mM). After 10 minutes of probe treatment, the change in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to fluorescence intensity at 485 nm excitation and 528 nm emission was detected. The results of the probe titration are shown in Figure 3 、 Figure 4 and Figure 5 , which show that different mutants have different affinities for ATP.

[0195] The ATP probe in Table 5 was subjected to specific detection, respectively, with UTP, CTP, GTP, AMP, ADP, NADP + , NAD + , NADH, NADPH for reactive detection, and the results showed good specificity, as shown in Figure 6 .

[0196] Example 9: Subcellular organelle localization of optical probes and performance of optical probes in subcellular organelles

[0197] In this example, different localization signal peptides were used to fuse with the optical probe 103 / 110-PS3-R102T / R92Q-cpYFP-Y1L to localize the optical probe to different organelles.

[0198] After transfecting 293 cells with optical probe plasmids fused with different localization signal peptides for 36 hours, PBS was used for flushing, and the cells were placed in HBSS solution for fluorescence detection under the FITC channel using an inverted fluorescence microscope. The results are shown in Figure 7 . The ATP optical probe can be localized to subcellular organelles including cytoplasm, nucleus, mitochondria, etc. by fusing with different specific localization signal peptides. Different subcellular structures show fluorescence, and the distribution and intensity of fluorescence are different.

[0199] After transfecting HEK293 cells with cytoplasm-expressed optical probe plasmids for 36 hours, PBS was used for flushing, and the cells were placed in HBSS solution for 30 minutes to detect the change in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to fluorescence intensity at 485 nm excitation and 528 nm emission. The results are shown in Figure 8 . 5 mM glucose was added, and the detection was continued for another 30 minutes. The 420 / 485 of the sample with added glucose gradually increased, reaching up to 1.8 times the initial value, while the 420 / 485 of the control group without added Oxalate remained unchanged.

[0200] Example 10: High-throughput compound screening based on optical probes in living cells

[0201] In this example, HeLa cells expressing 103 / 110-PS3-R102T / R92Q-cpYFP-Y1L in the cytoplasm were used for high-throughput compound screening.

[0202] The transfected 293 cells were washed with PBS, treated with HBSS solution (without glucose) for 1 hour, and then treated with 10 μM of the compound for 1 hour. Glucose was added to each sample. The change in the ratio of the fluorescence intensity at 420 nm excitation and 528 nm emission to that at 485 nm excitation and 528 nm emission was recorded using a microplate reader. The sample without any compound was used as a control for normalization. The results are shown in Table 1. Figure 9 Among the 2000 compounds used, most of the compounds had little effect on the energy metabolism of the cells. 21 compounds could significantly improve the ability of the cells to produce ATP, and another 33 compounds could significantly reduce the energy metabolism of the cells.

[0203] Example 11: Quantitative detection of ATP in blood using optical probes

[0204] In this example, purified 67 / 69-PS3-cpYFP with a Kd of 2.15 μM was used to analyze ATP in the supernatant of mouse and human blood.

[0205] After 67 / 69-PS3-cpYFP was mixed with the diluted blood supernatant and treated for 10 minutes, the fluorescence intensity at 420 nm excitation and 528 nm emission and the ratio of the fluorescence intensity at 485 nm excitation and 528 nm emission were detected using a microplate reader. The results are shown in Table 2. Figure 10 As shown in Table 2, the ATP content in mouse blood was about 30 nM, and the ATP content in human blood was about 1.6 nM.

[0206] As can be seen from the above examples, the ATP optical probe provided by the present application has a relatively small protein molecular weight and is easy to mature, has a large dynamic change in fluorescence, has good specificity, and can be expressed in cells by genetic manipulation, and can be used for real-time localization and quantitative detection of ATP in and outside cells; and can be used for high-throughput compound screening.

[0207] Other embodiments

[0208] This specification describes many embodiments. However, it should be understood that various modifications made by those skilled in the art based on the specification, without departing from the concept and scope of the present application, should also be included in the scope of the appended claims.

[0209] Some sequences in this document

[0210] 1> ε subunit of F0F1-ATP synthase of Bacillus thermophilus PS3 (1-133 full length)

[0211] MKTIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLVAPLEISAARLKKGGKTQYIAVSGGFLEVRPDKVTILAQAAERAEDIDVLRAKAAKERAERRLQSQQDDIDFKRAELALKRAMNRLSVAEMK

[0212] 2>cpYFP

[0213] YNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

[0214] 3>cpmOrange

[0215] VSERMYPEDGVLKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHPTGGRDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFTYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEA

[0216] 4>cpmKate

[0217] MGGRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHKLNGGTGGSMVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLYPADGGLEGRSDMALKLVGGGHLICNLKTTYRSKK

[0218] 5 > mCherry

[0219] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0220] 6 > cpGFP

[0221] NVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

[0222] 7 > cpBFP

[0223] NVYIKADKQKNGIKANFKIRHNIEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSESMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPGGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

[0224] 8> mKate

[0225] MSELITENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLYPADGGLEGRADMALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHKLN

[0226] 9> cpmApple

[0227] VSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHSTGGMDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEA

[0228] 10> 103 / 110-PS3-cpYFP

[0229] MKTIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLVAPLEISAARLKKGGKTQYIAVSGGFLEVRPDKVTILAQAAERAEDIDVLRAKAAKERAERRYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNDIDFKRAELALKRAMNRLSVAEMK

[0230] 11 > 103 / 110-PS3-R102T / R92Q-cpYFP-Y1L

[0231] MKTIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLVAPLEISAARLKKGGKTQYIAVSGGFLEVRPDKVTILAQAAERAEDIDVLQAKAAKERAETRLNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNDIDFKRAELALKRAMNRLSVAEMK

Claims

1. An ATP-binding protein variant which: (a) has the sequence set forth in SEQ ID NO: 1 and has a mutation at 1, 2, 3, 4, 5 or more positions selected from I88, D89, V90, R92, R102, R122, the mutation comprising a modification, substitution or deletion of an amino acid, (b) is at least 70% sequence identical to the sequence of (a) and has (1) the mutation and retains the ability to bind ATP, and Preferably, the ATP-binding protein variant mutation comprises a mutation at any 1, 2, 3, 4, 5 or more positions selected from I88, D89, V90, R92, R102, R122; More preferably, the mutation comprises a mutation at a position selected from any one of the following groups: (1) R102 and D89, (2) R102 and I88, (3) R102 and R92, (4) R102 and R122, (5) R102 and V90; More preferably, the D89 mutation is E, V, M, N, F, I, Q, S, H, Y, K, T or W; the I88 mutation is V or L; the R92 mutation is A, M, Q, H or K; the V90 mutation is I, D, A, R, L, F, E, M, C, P, Q, N, G, H, Y, K, S or T; the R122 mutation is N or K; the R102 mutation is T; More preferably, the mutation comprises a mutation selected from any one of the following groups: (1) D89E, (2) D89V, (3) D89M, (4) D89Q, (5) D89N, (6) D89Y, (7) D89S, (8) V90D, (9) V90A, (10) V90L, (11) V90E, (12) V90M, (13) V90C, (14) V90P, (15) V90H, (16) V90K, (17) V90S, (18) V90T, (19) R102T, (20) R122N, (21) R122K, (22) R102T and D89V, (23) R102T and D89F, (24) R102T and D89I, (25) R102T and D89Q, (26) R102T and D89H, (27) R102T and D89Y, (28) R102T and D89K, (29) R102T and D89T, (30) R102T and D89W, (31) R102T and E83R, (32) R102T and I88V, (33) R102T and I88L, (34) R102T and R92A, (35) R102T and R92H, (36) R102T and R92M, (37) R102T and R92Q (38) R102T and R92H, (39) R102T and R92K, (40) R102T and R122K, (41) R102T and V90I, (42) R102T and V90D (43) R102T and V90R, (44) R102T and V90L, (45) R102T and V90F, (46) R102T and V90E, (47) R102T and V90C, (48) R102T and V90P, (49) R102T and V90Q, (50) R102T and V90N, (51) R102T and V90G, (52) R102T and V90H, (53) R102T and V90Y, (54) R102T and V90K, (55) R102T and V90S, (56) R102T and V90T.

2. An optical probe comprising an ATP-sensitive polypeptide and an optically active polypeptide, wherein, the optically active polypeptide is located between residues 10-13, 19-21, 27-30, 33-38, 41-43, 46-48, 54-57, 61-63, 66-69, 71-74, 78-90, and 103-116 of an ATP-sensing polypeptide, which is an ATP-binding protein or a functional variant thereof, the optically active polypeptide being a fluorescent protein or a functional variant thereof, the ATP-sensing polypeptide has: (i) the sequence set forth in SEQ ID NO: 1, or a sequence having at least 70% sequence identity thereto and retaining ATP-binding activity, (ii) the sequence of the ATP-binding protein variant of claim 1, or (iii) the sequence of the ATP-binding protein variant of claim 1, or (iii) a sequence having at least 70% sequence identity to the sequence of (ii) and having the mutation of (ii) and retaining sensitivity to ATP, Preferably, the optically active polypeptide has: (a) a sequence as set forth in any one of SEQ ID NOs: 2-9, (b) a sequence as set forth in SEQ ID NO: 2 and having a mutation at the Y1 position, the mutation comprising a modification, substitution or deletion of an amino acid; preferably, the mutation is selected from any one or more of Y1R, Y1L, Y1V, Y1A, Y1G, Y1E, Y1W, Y1S, Y1K and Y1I, or (c) a variant sequence having at least 70% sequence identity to (a) or (b) and retaining fluorescent protein function.

3. The optical probe of claim 2, wherein, The optically active polypeptide is located at one or more of the following positions in the ATP-sensitive polypeptide: 11 / 13, 12 / 13, 19 / 21, 27 / 30, 28 / 28, 28 / 30, 29 / 28, 29 / 29, 33 / 35, 33 / 36, 33 / 38, 34 / 34, 34 / 35, 35 / 34, 35 / 35, 35 / 36, 35 / 37, 36 / 34, 36 / 35, 36 / 36, 36 / 37, 36 / 38, 37 / 38, 41 / 42, 41 / 43, 41 / 44, 42 / 42, 42 / 43, 43 / 42, 43 / 43, 46 / 46, 46 / 47, 47 / 46, 47 / 47, 47 / 48, 54 / 55, 55 / 55, 55 / 57, 56 / 55, 56 / 57, 61 / 63, 62 / 62, 63 / 62, 66 / 67, 66 / 69, 67 / 67, 67 / 69, 68 / 67, 68 / 69, 71 / 72, 71 / 73, 72 / 73, 72 / 74, 73 / 73, 73 / 74, 78 / 80, 78 / 81, 80 / 80, 80 / 81, 84 / 85, 84 / 91, 85 / 88, 85 / 91, 86 / 85, 86 / 86, 86 / 88, 86 / 90, 87 / 88, 87 / 89, 87 / 90, 87 / 91, 88 / 86, 88 / 87, 88 / 88, 88 / 89, 89 / 88, 89 / 90, 89 / 91, 90 / 89, 90 / 90, 103 / 104, 103 / 108, 103 / 109, 103 / 110, 103 / 112, 103 / 113, 103 / 115, 103 / 116, 104 / 104, 104 / 105, 104 / 106, 104 / 107, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 104 / 112, 104 / 114, 104 / 116, 105 / 104, 105 / 105, 105 / 106, 105 / 107, 105 / 108, 105 / 109, 105 / 110, 105 / 112, 105 / 114, 105 / 116, 106 / 104, 106 / 107, 106 / 111, 106 / 113, 106 / 114, 106 / 115, 106 / 116, 107 / 111, 107 / 113, 107 / 116, 108 / 109, 108 / 110, 108 / 111, 108 / 112, 108 / 113, 108 / 115, 108 / 113, 109 / 109, 109 / 110, 109 / 112, 109 / 113, 109 / 116, 110 / 104, 110 / 112, 110 / 113, 110 / 115, 110 / 116, 111 / 107, 110 / 109, 111 / 110, 111 / 111,111 / 112，111 / 113，111 / 116，112 / 104，112 / 105，112 / 110，112 / 114，112 / 115，112 / 116，113 / 108，113 / 109，113 / 110，113 / 113，113 / 115，113 / 116，114 / 104，114 / 105，114 / 106，114 / 108，114 / 109，114 / 110，114 / 111，114 / 115，114 / 116，115 / 108，115 / 111，115 / 113，115 / 115，115 / 116。， 4. The optical probe of claim 2 or 3, wherein the optical fiber is a single mode fiber. The optically active polypeptide is located at the 103 / 110 site of the ATP-binding protein, the ATP-sensitive polypeptide has a sequence shown in SEQ ID NO: 1 or a sequence having at least 70% sequence identity thereto and retaining ATP-binding activity, the optically active polypeptide has a sequence shown in any one of SEQ ID NOs: 2-9, and the optical probe comprises mutations selected from any one of the following groups: (1) D89E of the ATP-sensitive polypeptide, (2) D89V of the ATP-sensitive polypeptide, (3) D89M of the ATP-sensitive polypeptide, (4) D89Q of the ATP-sensitive polypeptide, (5) D89N of the ATP-sensitive polypeptide, (6) D89Y of the ATP-sensitive polypeptide, (7) D89S of the ATP-sensitive polypeptide, (8) V90D of the ATP-sensitive polypeptide, (9) V90A of the ATP-sensitive polypeptide, (10) V90L of the ATP-sensitive polypeptide, (11) V90E of the ATP-sensitive polypeptide, (12) V90M of the ATP-sensitive polypeptide, (13) V90C of the ATP-sensitive polypeptide, (14) V90P of the ATP-sensitive polypeptide, (15) V90H of the ATP-sensitive polypeptide, (16) V90K of the ATP-sensitive polypeptide, (17) V90S of the ATP-sensitive polypeptide, (18) V90T of the ATP-sensitive polypeptide, (19) R102T of the ATP-sensitive polypeptide, (20) R122N of the ATP-sensitive polypeptide, (21) R122K of the ATP-sensitive polypeptide, (22) R102T, D89V and optically active polypeptide 1L of the ATP-sensitive polypeptide, (23) R102T, D89F and optically active polypeptide 1L of the ATP-sensitive polypeptide, (24) R102T, D89I and optically active polypeptide 1L of the ATP-sensitive polypeptide, (25) R102T, D89Q and optically active polypeptide 1L of the ATP-sensitive polypeptide, (26) R102T, D89H and optically active polypeptide 1L of the ATP-sensitive polypeptide, (27) R102T, D89Y and optically active polypeptide 1L of the ATP-sensitive polypeptide, (28) R102T, D89K and optically active polypeptide 1L of the ATP-sensitive polypeptide, (29) R102T, D89T and optically active polypeptide 1L of the ATP-sensitive polypeptide, (30) R102T, D89W and optically active polypeptide 1L of the ATP-sensitive polypeptide, (31) R102T, E83R and optically active polypeptide 1L of the ATP-sensitive polypeptide, (32) R102T, I88V and optically active polypeptide 1L of the ATP-sensitive polypeptide, (33) R102T, I88L and optically active polypeptide 1L of the ATP-sensitive polypeptide, (34) R102T, R92A and optically active polypeptide 1L of the ATP-sensitive polypeptide, (35) R102T, R92H and optically active polypeptide 1L of the ATP-sensitive polypeptide, (36) R102T, R92M and optically active polypeptide 1L of the ATP-sensitive polypeptide,(37) R102T, R92Q and optically active polypeptide 1L of an ATP-sensitive polypeptide, (38) R102T, R92H and optically active polypeptide 1L of an ATP-sensitive polypeptide, (39) R102T, R92K and optically active polypeptide 1L of an ATP-sensitive polypeptide, (40) R102T, R122K and optically active polypeptide 1L of an ATP-sensitive polypeptide, (41) R102T, V90I and optically active polypeptide 1L of an ATP-sensitive polypeptide, (42) R102T, V90D and optically active polypeptide 1L of an ATP-sensitive polypeptide, (43) R102T, V90R and optically active polypeptide 1L of an ATP-sensitive polypeptide, (44) R102T, V90L and optically active polypeptide 1L of an ATP-sensitive polypeptide, (45) R102T, V90F and optically active polypeptide 1L of an ATP-sensitive polypeptide, (46) R102T, V90E and optically active polypeptide 1L of an ATP-sensitive polypeptide, (47) R102T, V90C and optically active polypeptide 1L of an ATP-sensitive polypeptide, (48) R102T, V90P and optically active polypeptide 1L of an ATP-sensitive polypeptide, (49) R102T, V90Q and optically active polypeptide 1L of an ATP-sensitive polypeptide, (50) R102T, V90N and optically active polypeptide 1L of an ATP-sensitive polypeptide, (51) R102T, V90G and optically active polypeptide 1L of an ATP-sensitive polypeptide, (52) R102T, V90H and optically active polypeptide 1L of an ATP-sensitive polypeptide, (53) R102T, V90Y and optically active polypeptide 1L of an ATP-sensitive polypeptide, (54) R102T, V90K and optically active polypeptide 1L of an ATP-sensitive polypeptide, (55) R102T, V90S and optically active polypeptide 1L of an ATP-sensitive polypeptide, (56) R102T, V90T and optically active polypeptide 1L of an ATP-sensitive polypeptide.

5. A fusion polypeptide comprising the optical probe of any one of claims 2-4 and a further polypeptide, the further polypeptide comprising a localization sequence, a tag to facilitate purification or a tag for an immunological reaction.

6. A nucleic acid molecule comprising: (a) a coding sequence for the ATP-binding protein variant of claim 1, a coding sequence for the optical probe of any one of claims 2-4 or a coding sequence for the fusion polypeptide of claim 5, or (b) a complement of (a).

7. A nucleic acid construct comprising the nucleic acid molecule of claim 6, Preferably, the nucleic acid construct is a cloning vector, an expression vector or a recombinant vector.

8. A host cell, the host cell: (1) comprising, expressing or secreting the optical probe of any one of claims 2-4 or the fusion polypeptide of claim 5; (2) comprising the nucleic acid molecule of claim 6; and / or (3) comprising the nucleic acid construct of claim 7.

9. A detection kit comprising: (1) the optical probe of any one of claims 2-4 or the fusion polypeptide of claim 5, (2) the nucleic acid molecule of claim 6, (3) the nucleic acid construct of claim 7, (4) the host cell of claim 8, the detection kit optionally further comprising other reagents required for detection of ATP using the optical probe, Preferably, the detection kit further comprises one or more reagents selected from the group consisting of: a buffer, a medium, an ATP standard.

10. A method of making the optical probe of any one of claims 2-4 or the fusion polypeptide of claim 5, comprising: culturing the host cell of claim 8, and isolating the optical probe or fusion polypeptide from the culture.

11. Use of the optical probe of any one of claims 2-4, the fusion polypeptide of claim 5, the nucleic acid molecule of claim 6, the nucleic acid construct of claim 7 and / or the host cell of claim 8 in detecting ATP in a sample, in screening compounds or in intracellular and / or extracellular localization of ATP.

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