Novel acetyl coenzyme A optical probe as well as preparation method and application thereof

Abstract

The invention relates to an acetyl coenzyme A probe as well as a preparation method and application thereof. Specifically, the acetyl coenzyme A optical probe comprises acetyl coenzyme A sensitive polypeptide and optical active polypeptide, and the optical active polypeptide is located in the sequence of the acetyl coenzyme A sensitive polypeptide.

Description

Technical Field

[0001] This invention relates to the field of optical probe technology, and in particular to a novel acetyl-CoA optical probe, its preparation method, and its application. Background Technology

[0002] Acetyl-CoA plays a crucial role within cells, serving as a key intermediate in metabolic processes and playing a central role in a variety of biological functions. It not only participates in cellular energy metabolism, such as the tricarboxylic acid cycle and oxidative phosphorylation, providing energy for cells, but is also an important precursor in the synthesis of fatty acids, cholesterol, and other bioactive molecules. Furthermore, acetyl-CoA acts as a regulator in epigenetics, modulating gene expression through acetylation of histones. Notably, changes in acetyl-CoA levels are closely linked to various metabolic diseases, including cancer, obesity, and neurodegenerative diseases.

[0003] Currently, the main methods used for detecting acetyl-CoA include GC-MS, LC-MS, HPLC, NMR, and enzyme-linked reactions. Conventional chemical analysis methods directly or indirectly measure total acetyl-CoA in cells, but these procedures are complex and cannot meet the requirements for real-time dynamic in vivo monitoring of acetyl-CoA levels in living cells or even organelles. In recent years, researchers have developed fluorescent probes for acetyl-CoA based on different principles. For example, a detection method for acetyl-CoA has been developed using bioluminescent resonance energy transfer technology; fluorescent protein probes for acetyl-CoA have been designed using bacterial PanZ protein and cpGFP, enabling visualization and quantitative detection at the living cell level. While the developed acetyl-CoA protein probes have solved some of these problems, they still suffer from unsuitable affinity, small response amplitude, and inability to be applied in mammalian cells. Therefore, there is an urgent need to develop new detection methods to achieve simple, rapid, highly specific, real-time, localized, quantitative, and high-throughput detection of acetyl-CoA both intracellularly and in vitro. Summary of the Invention

[0004] The purpose of this invention is to provide probes and methods for real-time, high-throughput, and quantitative detection of acetyl-CoA inside and outside cells.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] The first aspect of this invention provides a variant of an acetyl-CoA binding protein, wherein:

[0007] (1) Having the sequence shown in SEQ ID NO:1 and having mutations at one, two, three or more sites selected from the following: I19, R21, T77, K85, G87, P133, P134, said mutations including amino acid modifications, substitutions or deletions.

[0008] (2) is a sequence that has at least 70% sequence identity with the sequence of (1) and has the mutation described in (1) and retains the ability to bind to acetyl-CoA.

[0009] In one or more embodiments, the mutation includes mutations at sites selected from any of the following groups: (1) P133, P134 and I19, (2) P133, P134 and R21, (3) P133, P134 and T77, (4) P133, P134 and K85, (5) P133, P134 and G87.

[0010] In one or more embodiments, I19 mutates to L; in one or more embodiments, R21 mutates to M or D; in one or more embodiments, T77 mutates to V; in one or more embodiments, K85 mutates to N; in one or more embodiments, G87 mutates to A; in one or more embodiments, P133 mutates to Y; in one or more embodiments, P134 mutates to D.

[0011] In one or more embodiments, the mutation comprises mutations selected from any of the following groups: (1) P133Y, P134D and I19L, (2) P133Y, P134D and R21M, (3) P133Y, P134D and R21D, (4) P133Y, P134D and T77V, (5) P133Y, P134D and K85N, (6) P133Y, P134D and G87A.

[0012] A second aspect of the present invention provides an optical probe comprising an acetyl-CoA-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the acetyl-CoA-sensitive polypeptide. The acetyl-CoA-sensitive polypeptide is divided into a first part and a second part by the optically active polypeptide.

[0013] In one or more embodiments, the acetyl-CoA optical probe includes an acetyl-CoA-sensitive polypeptide B and an optically active polypeptide A, wherein the optically active polypeptide A is located within the sequence of the acetyl-CoA-sensitive polypeptide, and the acetyl-CoA-sensitive polypeptide B is divided into a first part B1 and a second part B2 to form a probe structure of type B1-A-B2.

[0014] In one or more embodiments, the optically active polypeptide is located at one or more sites of the acetyl-CoA-sensitive polypeptide selected from the following: 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 20 / 27, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 21 / 27, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 22 / 27, 23 / 24, 23 / 25, 23 / 26, 23 / 27, 37 / 38, 37 / 39, 38 / 39. 46 / 47, 46 / 48, 47 / 48, 59 / 60, 59 / 61, 59 / 62, 59 / 63, 59 / 64, 59 / 65, 59 / 66, 59 / 67, 59 / 68, 59 / 69, 60 / 61, 60 / 62, 60 / 63, 60 / 64, 60 / 65, 60 / 66, 60 / 67, 60 / 68, 60 / 69, 61 / 62, 61 / 63, 61 / 64, 61 / 65, 61 / 66, 61 / 67, 61 / 68, 61 / 69, 62 / 63, 62 / 64, 62 / 6 5, 62 / 66, 62 / 67, 62 / 68, 62 / 69, 63 / 64, 63 / 65, 63 / 66, 63 / 67, 63 / 68, 63 / 69, 64 / 65, 64 / 66, 64 / 67, 64 / 68, 64 / 69, 65 / 66, 65 / 67, 65 / 68, 65 / 69, 66 / 67, 66 / 68, 66 / 69, 67 / 68, 67 / 69, 78 / 79, 78 / 80, 78 / 81, 78 / 82, 78 / 83, 78 / 84, 79 / 80, 79 / 81, 79 / 82, 79 / 83, 79 / 84, 80 / 81, 80 / 82, 80 / 83, 80 / 84, 81 / 82, 81 / 83, 81 / 84, 82 / 83, 82 / 84, 83 / 84, 102 / 103, 102 / 104, 103 / 104, 112 / 113, 122 / 123, 132 / 133, 132 / 134, 132 / 135, 132 / 136, 133 / 134, 133 / 135, 133 / 136, 134 / 135, 134 / 136, and 135 / 136. The numbers correspond to the full length of the acetyl-CoA-sensitive polypeptide.

[0015] In one or more embodiments, the acetyl-CoA-sensitive polypeptide is an acetyl-CoA-binding protein or a functional variant thereof.

[0016] In one or more embodiments, the acetyl-CoA-sensitive polypeptide has:

[0017] (1) The sequence shown in SEQ ID NO:1, or a sequence that has at least 70% sequence identity with it and retains acetyl-CoA binding activity.

[0018] (2) The sequence of the acetyl-CoA binding protein variant described in any embodiment of the first aspect of this document, or

[0019] (3) has at least 70% sequence identity with the sequence described in (2) and has the mutation described in (2) and retains the sequence sensitive to acetyl-CoA.

[0020] 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 proteins (such as cpYFP shown in SEQ ID NO:2), orange fluorescent proteins (such as cpmOrange shown in SEQ ID NO:3), red fluorescent proteins (such as mKate shown in SEQ ID NO:4 or 8, or mcherry shown in SEQ ID NO:5), green fluorescent proteins (such as cpGFP shown in SEQ ID NO:6), blue fluorescent proteins (such as cpBFP shown in SEQ ID NO:7), and apple red fluorescent proteins (such as cpmApple shown in SEQ ID NO:9). Preferably, the optically active polypeptide is cpYFP, ​​cpGFP, cpBFP, or cpmApple. More preferably, the optically active polypeptide is cpYFP. In one or more embodiments, the fluorescent protein has any of the sequences shown in SEQ ID NO:2-9, and preferably, the fluorescent protein has any of the sequences shown in SEQ ID NO:2, 6, 7, and 9.

[0021] In one or more embodiments, the optically active polypeptide has: (1) any of the sequences shown in SEQ ID NO:2-9, (2) the sequence shown in SEQ ID NO:2 and a mutation at the Y1 site, the mutation including modification, substitution or deletion of an amino acid; preferably, the mutation is Y1V, or (3) a variant sequence that has at least 70% sequence identity with (1) or (2) and retains the function of the fluorescent protein.

[0022] In one or more embodiments, the sequence of the acetyl-CoA-sensitive polypeptide is as shown in SEQ ID NO:1, and the optically active polypeptide is as shown in any one of SEQ ID NO:2, 6, 7, and 9, wherein the optically active polypeptide is located at any one or more sites selected from the following positions of the acetyl-CoA-sensitive polypeptide: 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 20 / 27, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 21 / 27, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 22 / 27, 23 / 24, 23 / 25, 23 / 26, 23 / 27, 37 / 38, 37 / 39, 38 / 39, 46 / 47, 46 / 48, 47 / 48, 59 / 60, 59 / 61, 59 / 62, 59 / 63, 59 / 64, 59 / 65, 59 / 66, 59 / 67, 59 / 68, 59 / 69, 60 / 61, 60 / 62, 60 / 63, 60 / 64, 60 / 65, 60 / 66, 60 / 67, 60 / 68, 60 / 69, 61 / 62, 61 / 63, 61 / 64, 61 / 65, 61 / 6 6, 61 / 67, 61 / 68, 61 / 69, 62 / 63, 62 / 64, 62 / 65, 62 / 66, 62 / 67, 62 / 68, 62 / 69, 63 / 64, 63 / 65, 63 / 66, 63 / 67, 63 / 68, 63 / 69, 64 / 65, 64 / 66, 64 / 67, 64 / 68, 64 / 69, 65 / 66, 65 / 67, 65 / 68, 65 / 69, 66 / 67, 66 / 68, 66 / 69, 67 / 68, 67 / 69 , 78 / 79, 78 / 80, 78 / 81, 78 / 82, 78 / 83, 78 / 84, 79 / 80, 79 / 81, 79 / 82, 79 / 83, 79 / 84, 80 / 81, 80 / 82, 80 / 83, 80 / 84, 81 / 82, 81 / 83, 81 / 84, 82 / 83, 82 / 84, 83 / 84, 102 / 103, 102 / 104, 103 / 104, 112 / 113, 122 / 123, 132 / 133, 247920

[0023] 132 / 134, 132 / 135, 132 / 136, 133 / 134, 133 / 135, 133 / 136, 134 / 135, 134 / 136, and 135 / 136. The numbers correspond to the full length of the acetyl-CoA-sensitive polypeptide.

[0024] In one or more embodiments, the acetyl-CoA-sensitive polypeptide is as shown in SEQ ID NO:1, and the optically active polypeptide is as shown in any one of SEQ ID NO:2, 6, 7, 9, or a variant thereof having the following mutation at the amino acid position corresponding to the first amino acid of SEQ ID NO:2: Y1V. The optically active polypeptide is located at any one or more of the following sites selected from the acetyl-CoA-sensitive polypeptide: 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 20 / 27, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 21 / 27, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 22 / 27, 23 / 24, 23 / 25, 23 / 26, 23 / 27, 37 / 38. , 37 / 39, 38 / 39, 46 / 47, 46 / 48, 47 / 48, 59 / 60, 59 / 61, 59 / 62, 59 / 63, 59 / 64, 59 / 65, 59 / 66, 59 / 67, 59 / 68, 59 / 69, 60 / 61, 60 / 62, 60 / 63, 60 / 64, 60 / 65, 60 / 66, 60 / 67, 60 / 68, 60 / 69, 61 / 62, 61 / 63, 61 / 64, 61 / 65, 61 / 66, 61 / 67, 61 / 68, 61 / 69, 62 / 63, 6 2 / 64, 62 / 65, 62 / 66, 62 / 67, 62 / 68, 62 / 69, 63 / 64, 63 / 65, 63 / 66, 63 / 67, 63 / 68, 63 / 69, 64 / 65, 64 / 66, 64 / 67, 64 / 68, 64 / 69, 65 / 66, 65 / 67, 65 / 68, 65 / 69, 66 / 67, 66 / 68, 66 / 69, 67 / 68, 67 / 69, 78 / 79, 78 / 80, 78 / 81, 78 / 82, 78 / 83, 78 / 84, 79 / 80, 79 / 81, 79 / 82, 79 / 83, 79 / 84, 80 / 81, 80 / 82, 80 / 83, 80 / 84, 81 / 82, 81 / 83, 81 / 84, 82 / 83, 82 / 84, 83 / 84, 102 / 103, 102 / 104, 103 / 104, 112 / 113, 122 / 123, 132 / 133, 132 / 134, 132 / 135, 132 / 136, 133 / 134, 133 / 135, 133 / 136, 134 / 135, 134 / 136, and 135 / 136. The numbers correspond to the full length of the acetyl-CoA-sensitive polypeptide.

[0025] In one or more embodiments, the optical probe contains an acetyl-CoA-sensitive polypeptide as shown in SEQ ID NO:1, and has one or more of the following mutations: I19, R21, T77, K85, G87, P133, P134, and an optically active polypeptide as shown in SEQ ID NO:2, 6, 7, 9, or the mutation corresponding to SEQ ID NO:1. The first amino acid of NO:2 has the following variant with mutation: Y1V, optically active polypeptide located at amino acid positions 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 20 / 27, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 21 / 27, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 22 / 27, 23 / 24, 23 / 25, 23 / 26, 23 / 27, 37 / 38, 37 / 39, 38 / 3 9, 46 / 47, 46 / 48, 47 / 48, 59 / 60, 59 / 61, 59 / 62, 59 / 63, 59 / 64, 59 / 65, 59 / 66, 59 / 67, 59 / 68, 59 / 69, 60 / 61, 60 / 62, 60 / 63, 60 / 64, 60 / 65, 60 / 66, 60 / 67, 60 / 68, 60 / 69, 61 / 62, 61 / 63, 61 / 64, 61 / 65, 61 / 66, 61 / 67, 61 / 68, 61 / 69, 62 / 63, 62 / 64, 62 / 65, 62 / 66, 62 / 67, 62 / 68, 62 / 69, 63 / 64, 63 / 65, 63 / 66, 63 / 67, 63 / 68, 63 / 69, 64 / 65, 64 / 66, 64 / 67, 64 / 68, 64 / 69, 65 / 66, 65 / 67, 65 / 68, 65 / 69, 66 / 67, 66 / 68, 66 / 69, 67 / 68, 67 / 69, 78 / 79, 78 / 80, 78 / 81, 78 / 82, 78 / 83, 78 / 84, 79 / 80, 79 / 81, 7 9 / 82, 79 / 83, 79 / 84, 80 / 81, 80 / 82, 80 / 83, 80 / 84, 81 / 82, 81 / 83, 81 / 84, 82 / 83, 82 / 84, 83 / 84, 102 / 103, 102 / 104, 103 / 104, 112 / 113, 122 / 123, 132 / 133, 132 / 134, 132 / 135, 132 / 136, 133 / 134, 133 / 135, 133 / 136, 134 / 135, 134 / 136, and 135 / 136. The numbers correspond to the full length of the acetyl-CoA-sensitive polypeptide.Preferably, the mutation of the acetyl-CoA-sensitive polypeptide includes mutations selected from any of the following groups: (1) P133Y, P134D and I19L, (2) P133Y, P134D and R21M, (3) P133Y, P134D and R21D, (4) P133Y, P134D and T77V, (5) P133Y, P134D and K85N, (6) P133Y, P134D and G87A.

[0026] In one or more embodiments, the optical probe contains an acetyl-CoA-sensitive polypeptide as shown in SEQ ID NO:1, and an optically active polypeptide as shown in any one of SEQ ID NO:2, 6, 7, and 9, wherein the optically active polypeptide is located at position 134 / 135 of the acetyl-CoA-sensitive polypeptide, and the optical probe has the following mutations: (1) P133Y, P134D, I19L of the acetyl-CoA-sensitive polypeptide and Y1V of the optically active polypeptide; (2) P133Y, P134D, R21M of the acetyl-CoA-sensitive polypeptide and Y1V of the optically active polypeptide; (3) acetyl-CoA-sensitive... (4) Acetyl-CoA-sensitive peptides P133Y, P134D, R21D and optically active peptide Y1V, (5) Acetyl-CoA-sensitive peptides P133Y, P134D, K85N and optically active peptide Y1V, (6) Acetyl-CoA-sensitive peptides P133Y, P134D, G87A and optically active peptide Y1V.

[0027] In one or more embodiments, the optical probe is as shown in any of SEQ ID NO:11-16.

[0028] In one or more embodiments, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linkers of this invention can be any amino acid sequence of any length. In one or more embodiments, the optically active polypeptide flanking the linker comprises a linker of no more than 5 amino acids, for example, linkers of 0, 1, 2, 3, or 4 amino acids. In one or more embodiments, the linker flanking the optically active polypeptide comprises amino acid Y. In one or more embodiments, linker Y is located at the N-terminus and / or C-terminus of the optically active polypeptide. In one or more embodiments, the optical probe is configured as follows: a first portion B1 of an acetyl-CoA-sensitive polypeptide, a first linker Y1, an optically active polypeptide A, a second linker Y2, and a second portion B2 of the acetyl-CoA-sensitive polypeptide. In one embodiment, the optical probe of this invention does not comprise a linker.

[0029] In one or more embodiments, the optical probe of the present invention further includes a localization sequence for positioning the probe to a specific organelle, such as a cell. Preferred organelles are subcellular organelles, more preferably the cytoplasm, nucleus, and mitochondria.

[0030] In one or more embodiments, the optically active polypeptide is cpYFP, ​​located at any one or more sites selected from the following: 22 / 25, 46 / 48, 59 / 69, 60 / 68, 62 / 66, 133 / 135, 134 / 135, and 134 / 136 of the acetyl-CoA-sensitive polypeptide. The acetyl-CoA-sensitive polypeptide (1) is as shown in SEQ ID NO:1, or (2) is a variant of the acetyl-CoA-binding protein described in any embodiment of the first aspect herein;

[0031] In one or more embodiments, the optically active polypeptide is cpGFP, located at one or more sites selected from the following sites of the acetyl-CoA-sensitive polypeptide: 22 / 25, 22 / 26, 46 / 48, 59 / 69, 60 / 67, 62 / 67, 133 / 135, and 134 / 136. The acetyl-CoA-sensitive polypeptide (1) is as shown in SEQ ID NO:1, or (2) is a variant of the acetyl-CoA-binding protein described in any embodiment of the first aspect herein;

[0032] In one or more embodiments, the optically active polypeptide is cpBFP located at one or more sites selected from the following: 22 / 26, 38 / 39, 62 / 67, 133 / 136, and 134 / 136. The acetyl-CoA-sensitive polypeptide (1) is as shown in SEQ ID NO:1, or (2) is a variant of the acetyl-CoA-binding protein described in any embodiment of the first aspect herein;

[0033] In one or more embodiments, the optically active polypeptide is cpmApple, located at one or more sites selected from the following: sites 46 / 47, 59 / 69, 60 / 67, 62 / 66, 133 / 135, and 134 / 136 of the acetyl-CoA-sensitive polypeptide. The acetyl-CoA-sensitive polypeptide (1) is as shown in SEQ ID NO:1, or (2) is a variant of the acetyl-CoA-binding protein described in any embodiment of the first aspect herein; [Supplementary content, corresponding to specific embodiments 2-5]

[0034] Another aspect of the present invention provides a fusion polypeptide comprising the optical probe described in any embodiment herein and other polypeptides, said other polypeptides including a localization sequence, a label for easy purification, or a label for an immunoreaction. In some embodiments, the optical probe described herein further comprises other polypeptides fused thereto. These other polypeptides do not affect the properties of the optical probe. In some embodiments, the other polypeptides are located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptides include a localization sequence (e.g., a polypeptide that localizes the optical probe to different organelles or sub-organelles), a label for easy purification, or a label for an immunoreaction (e.g., immunoblotting). A linker may be present between the optical probe and other polypeptides in the fusion polypeptide described herein.

[0035] Another aspect of the present invention provides a nucleic acid molecule comprising: (1) the coding sequence of an acetyl-CoA binding protein variant, optical probe, or fusion polypeptide as described in any embodiment herein, or (2) a complementary sequence to (1), or (3) a fragment of (1) or (2). The fragment is a primer.

[0036] The present invention also relates to variants of the aforementioned nucleic acid molecules, including fragments, analogs, derivatives, soluble fragments and variants encoding the nucleic acid sequences or complementary sequences thereof.

[0037] This invention also provides nucleic acid constructs comprising the nucleic acid molecules described herein. The nucleic acid sequence encodes a protein variant, optical probe, or fusion peptide as described in any embodiment of this invention.

[0038] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.

[0039] In one or more embodiments, the nucleic acid molecule is operatively linked to an expression control sequence.

[0040] In some implementations, the expression vector is selected from prokaryotic expression vectors, eukaryotic expression vectors, and viral vectors.

[0041] In another aspect, the present invention provides a host cell that: (1) contains, expresses, or secretes the optical probe or fusion polypeptide described in any embodiment of the present invention; (2) contains the nucleic acid molecule described in any embodiment of the present invention; or (3) contains the nucleic acid construct described in any embodiment of the present invention. The host cell is preferably *Escherichia coli*.

[0042] In another aspect, the present invention provides a detection kit comprising the optical probe, fusion peptide, nucleic acid molecule, nucleic acid construct, or host cell described herein. Optionally, the detection kit may also include other reagents required for detecting acetyl-CoA using the optical probe.

[0043] In one or more embodiments, the test kit further comprises one or more reagents selected from the following: buffer solution, culture medium, acetyl-CoA standard.

[0044] The present invention provides a method for preparing the optical probe described herein, comprising: providing a host cell expressing the optical probe or fusion polypeptide described herein, culturing the host cell under conditions of cell expression, and isolating the optical probe or fusion polypeptide.

[0045] In one or more embodiments, the method includes the following steps: 1) incorporating a nucleic acid molecule encoding the optical probe or fusion polypeptide described herein into an expression vector; 2) transferring the expression vector into a host cell; 3) culturing the host cell under conditions suitable for expression of the expression vector; and 4) isolating the optical probe or fusion polypeptide.

[0046] Another aspect of the present invention provides a method for detecting acetyl-CoA in a sample, comprising: contacting the sample with the optical probe or fusion peptide or host cell described herein, and detecting changes in the optically active peptide. The detection can be performed in vivo, in vitro, subcellular, or in situ. The sample may be, for example, blood.

[0047] This article also provides a method for quantifying acetyl-CoA in a sample, comprising: contacting the optical probe or fusion peptide or host cell described herein with the sample, detecting optical changes in the optically active peptide, and quantifying acetyl-CoA in the sample based on the optical changes in the optically active peptide.

[0048] Another aspect of the present invention provides a method for screening compounds (e.g., drugs), comprising: contacting the optical probe or fusion peptide or host cell described herein with a candidate compound in a system containing acetyl-CoA; detecting optical changes in the optically active peptide; and screening the compound based on the optical changes in the optically active peptide. The method can screen compounds in high throughput.

[0049] In one or more embodiments, the host cells described herein are contacted with the candidate compound in a system containing acetyl-CoA, and optical changes in the optically active peptide indicate whether the candidate compound can regulate the uptake of acetyl-CoA by the cells.

[0050] Another aspect of the present invention provides a method for intracellular and / or extracellular localization of the acetyl-CoA, comprising: contacting the acetyl-CoA-containing system with the optical probe or the host cell, and detecting optical changes in the optically active polypeptide.

[0051] In one or more embodiments, the system is a solution system, a cellular system, or a subcellular system.

[0052] Another aspect of the present invention provides the use of the acetyl-CoA optical probes, fusion peptides, or host cells described herein in detecting acetyl-CoA in samples, screening compounds, or intracellular and / or extracellular localization of acetyl-CoA. In one or more embodiments, the localization is real-time localization.

[0053] Another aspect of the present invention provides the use of the acetyl-CoA optical probe or fusion peptide or polynucleotide or nucleic acid construct described herein in the preparation of a kit for detecting acetyl-CoA in a sample, screening compounds, or intracellular and / or extracellular localization of acetyl-CoA. Attached Figure Description

[0054] Figure 1 This is an SDS-PAGE image of the exemplary acetyl-CoA optical probe described in Example 1;

[0055] Figure 2 This is a graph showing the response of the exemplary acetyl-CoA optical probe containing cpYFP and acetyl-CoA binding protein to acetyl-CoA as described in Example 2.

[0056] Figure 3 This is a graph showing the response changes of the acetyl-CoA optical probe containing cpGFP and acetyl-CoA binding protein to acetyl-CoA as described in Example 3.

[0057] Figure 4 This is a graph showing the response of the exemplary acetyl-CoA optical probe containing cpBFP and acetyl-CoA binding protein to acetyl-CoA as described in Example 4.

[0058] Figure 5 This is a graph showing the response of the exemplary acetyl-CoA optical probe containing cpmApple and acetyl-CoA binding protein to acetyl-CoA as described in Example 5.

[0059] Figure 6 The fluorescence spectrum of the exemplary acetyl-CoA optical probe described in Example 7 is shown in Figure 7.

[0060] Figure 7 The titration curves of the exemplary acetyl-CoA probe described in Example 7 to different concentrations of acetyl-CoA are shown.

[0061] Figure 8 This is a bar chart showing the specificity of the exemplary acetyl-CoA optical probe described in Example 7 for the detection of acetyl-CoA analogs.

[0062] Figure 9 This is a photograph showing the subcellular organelle localization of the exemplary acetyl-CoA optical probe described in Example 8 in mammalian cells.

[0063] Figure 10 This is a schematic diagram illustrating the dynamic monitoring of acetyl-CoA concentration in the cytoplasm of mammalian cells using an exemplary acetyl-CoA optical probe as described in Example 8.

[0064] Figure 11 This is a dot plot of the exemplary acetyl-CoA optical probe described in Example 9 for high-throughput compound screening at the live cell level;

[0065] Figure 12 This is a bar chart showing the quantification of acetyl-CoA in mouse and human blood using the exemplary acetyl-CoA optical probe described in Example 10. Detailed Implementation

[0066] When a value or range is given, the term “about” as used herein means that the value or range is within 20%, 10%, and 5% of the given value or range.

[0067] The terms “comprising,” “including,” and their equivalents as used herein include the meanings of “containing” and “composed of,” for example, a composition “comprising” X may consist of only X or may contain other substances, such as X+Y.

[0068] As used herein, the terms "acetyl-CoA-sensitive peptide" or "acetyl-CoA-responsive peptide" refer to a peptide that responds to acetyl-CoA, and the response includes any response to chemical, biological, electrical, or physiological parameters of the peptide in relation to the interaction with the sensitive peptide. Responses include small changes, such as changes in the orientation of amino acids or peptide fragments of the peptide, and changes in the primary, secondary, or tertiary structure of the peptide, including, for example, changes in protonation, electrochemical potential, and / or conformation. "Conformation" is the three-dimensional arrangement of the primary, secondary, and tertiary structures of a molecule containing side groups; a conformational change occurs when the three-dimensional structure of the molecule changes. Examples of conformational changes include a change from an α-helix to a β-sheet or vice versa. It is understood that a detectable change need not be a conformational change, as long as the fluorescence of the fluorescent protein moiety is altered. The acetyl-CoA-sensitive peptides described herein may also include their functional variants. Functional variants of acetyl-CoA-sensitive peptides include, but are not limited to, variants that can interact with acetyl-CoA to undergo the same or similar changes as the parental acetyl-CoA-sensitive peptide.

[0069] As used in this invention, the term "optical probe" refers to an acetyl-CoA-sensitive polypeptide fused to an optically active polypeptide (e.g., a fluorescent protein), which is operatively inserted into the acetyl-CoA-sensitive polypeptide (e.g., an acetyl-CoA-binding protein). The acetyl-CoA-binding protein can sense changes in acetyl-CoA concentration, and its spatial conformation changes during dynamic changes in acetyl-CoA concentration. The inventors have discovered that when an optically active polypeptide is fused to an acetyl-CoA-sensitive polypeptide (e.g., an acetyl-CoA-binding protein), the conformational change resulting from the acetyl-CoA-sensitive polypeptide's specific binding to physiological concentrations of acetyl-CoA causes a conformational change in the optically active polypeptide (e.g., a fluorescent protein), thereby altering the optical properties of the optically active polypeptide. By plotting standard curves using fluorescence of the fluorescent protein measured at different acetyl-CoA concentrations, the presence and / or level of acetyl-CoA can be detected and analyzed. The acetyl-CoA-sensitive polypeptide described in this invention includes, but is not limited to, acetyl-CoA-binding protein GAT or GNATs protein mutants with more than 90% homology to it. An exemplary GAT protein is shown in SEQ ID NO:1. The exemplary acetyl-CoA binding protein GAT described in this invention is derived from Bacillus licheniformis, which can sense changes in acetyl-CoA concentration. During dynamic changes in acetyl-CoA concentration, the spatial conformation of the acetyl-CoA binding protein also changes. When describing the optical probe of this invention (e.g., when describing insertion sites or mutation sites), the amino acid residue numbers are all referenced to SEQ ID NO:1.

[0070] A protein-based "optically active peptide" is a peptide capable of emitting fluorescence. Fluorescence is an optical property of an optically active peptide, which can be used as a means of detecting the responsiveness of the optical probes of the present invention. As used herein, the term "fluorescence property" refers to the molar extinction coefficient at an appropriate excitation wavelength, fluorescence quantum efficiency, shape of the excitation or emission spectrum, maximum excitation wavelength and maximum emission wavelength, amplitude of excitation at two different wavelengths, ratio of emission amplitude at two different wavelengths, excited-state lifetime, or fluorescence anisotropy. A measurable difference in any of these properties between active and inactive states is sufficient to determine 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 fluorescence property, for example, the amount of fluorescence at a specific wavelength or the integral of fluorescence over the emission spectrum. Preferably, the protein substrate is selected to have fluorescence properties that are easily distinguishable between inactive and activated conformational states. The optically active peptides described herein may also include their functional variants. Functional variants of optically active peptides include, but are not limited to, variants that can undergo the same or similar fluorescence property changes as the parent optically active peptide.

[0071] In this article, "response fold" refers to the normalized fluorescence ratio. The greater the deviation of the probe's response fold from 1 (whether it increases or decreases), the greater the change in the probe's response to the substrate relative to the control, or the greater its responsiveness. For example, in this application, the normalized ratio is measured by detecting the change in the ratio of fluorescence intensity at 528nm emission after excitation at 420nm to fluorescence intensity at 528nm emission after excitation at 485nm. 420 / 485 The response factor is calculated as follows:

[0072] Fluorescence signal values ​​were corrected by subtracting the detection signal values ​​from cells that did not express the probe protein. pH-sensitive interference was eliminated by dividing the change in the ratio of probe fluorescence intensity in parallel experimental groups by the change in the ratio of control fluorescence intensity to obtain corrected data.

[0073] F = Fsampte - F B B LK

[0074]

[0075]

[0076]

[0077] F represents fluorescence intensity, and Fsample represents the total fluorescence intensity of the sample expressing the fluorescent probe. BLK This represents the background fluorescence intensity of samples without expressed fluorescent probes. F 485 F represents the fluorescence intensity of a fluorescent protein sample excited at 485 nm and emitted at 528 nm. 420 This indicates the fluorescence intensity of the fluorescent protein sample excited at 420 nm and emitted at 528 nm. The Normalized Ratio represents the ratio of the fluorescence intensity of the probe to acetyl-CoA. The Normalized Ratio control represents the ratio of the fluorescence intensity of the pH control probe to acetyl-CoA. The pH-corrected Normalized Ratio is the fold change in probe or response after pH correction. pHNormalized Ratio 420 / 485 The greater the deviation from 1 (whether it increases or decreases), the greater the change factor or response factor of the probe.

[0078] "Connector" or "linking region" refers to an amino acid or nucleotide sequence that links two parts in a polypeptide, protein, or nucleic acid of the present invention. Exemplarily, in the present invention, the number of amino acids at the amino terminus of the linking region between the acetyl-CoA-sensitive polypeptide and the optically active polypeptide is selected to be 0-3, and the number of amino acids at the carboxyl terminus is selected to be 0-2. When the recombinant optical probe is used as a basic unit to link with a functional protein, it can be fused to the amino acid or carboxyl terminus of the recombinant optical probe. The connector sequence can be a short peptide chain composed of one or more flexible amino acids, such as Y.

[0079] As used herein, the terms "chromophore," "fluorophore," and "fluorescent protein" are synonymous, referring to proteins that emit fluorescence under excitation light. Fluorescent proteins are fundamental detection methods in the field of bioscience. Examples include the commonly used green fluorescent protein GFP and its cyclically rearranged derivatives such as blue fluorescent protein (cpBFP), green fluorescent protein (cpGFP), and yellow fluorescent protein (cpYFP); and the commonly used red fluorescent protein RFP, and its cyclically rearranged derivatives such as cpmApple, cpmOrange, and cpmKate. The sequences of exemplary fluorescent proteins are shown in any of SEQ ID NO:2-9. Preferably, the sequences of exemplary fluorescent proteins are shown in any of SEQ ID NO:2, 6, 7, and 9.

[0080] Green fluorescent protein (GFP) was originally extracted from the bioluminescent jellyfish *Aequorea Victoria*. It consists of 238 amino acids and has a molecular weight of approximately 26 kDa. GFP has a unique barrel-shaped structure formed by 12 β-sheet chains, encapsulating a chromophore tripeptide (Ser65-Tyr66-Gly67). In the presence of oxygen, it spontaneously forms a chromophore structure of p-hydroxybenzyl imidazolinone, producing fluorescence. GFP fluorescence does not require cofactors and is very stable, making it an excellent imaging tool. GFP has two excitation peaks: a main peak at 395 nm produces emission light at 508 nm, while an excitation peak at 475 nm produces emission light at 503 nm. An exemplary cpGFP is shown in SEQ ID NO:6.

[0081] Yellow fluorescent protein (YFP) is derived from green fluorescent protein (GFP), and its amino acid sequence shares over 90% homology with GFP. The key difference between YFP and GFP lies in the mutation of amino acid position 203 from threonine to tyrosine (T203Y). Compared to the original AvGFP, the main excitation wavelength of YFP is red-shifted to 514 nm, while the emission wavelength changes to 527 nm. Based on this, a site-directed mutation (S65T) at amino acid position 65 of YFP yields the fluorescence-enhanced yellow fluorescent protein (EYFP). cpYFP is obtained by linking the original N-terminus and C-terminus of GFP with a flexible short peptide chain. A new N-terminus and C-terminus are created near the chromophore of the original GFP. Amino acids 145–238 of the original protein are used as the N-terminus of the new protein, and amino acids 1–144 of the original protein are used as the C-terminus. The two fragments are linked by 5–9 flexible short peptide chains. In this invention, the proximal chromophore is preferably located at amino acids Y144 and N145; the flexible short peptide chain is preferably VDGGSGGTG or GGSGG. An exemplary cpYFP sequence is shown in SEQ ID NO:2.

[0082] Red fluorescent protein (RFP) was initially extracted from marine corals. Wild RFP is an oligomeric protein, which is not conducive to fusion expression in organisms. Subsequently, red fluorescent proteins of different color bands were derived from RFP, the most commonly used being mCherry and mKate. An example of mCherry is shown in SEQ ID NO:4 or 8. An example of mCherry is shown in SEQ ID NO:5.

[0083] In other embodiments, the fluorescent protein may also be one or more of the following: blue fluorescent protein cpBFP with an amino acid sequence as shown in SEQ ID NO:7, orange fluorescent protein cpmOrange with an amino acid sequence as shown in SEQ ID NO:3, and apple red fluorescent protein cpmApple with an amino acid sequence as shown in SEQ ID NO:9.

[0084] The acetyl-CoA optical probe of this invention comprises an acetyl-CoA-sensitive polypeptide B, such as acetyl-CoA binding protein or a variant thereof, and an optically active polypeptide A, such as fluorescent protein or a variant thereof. The optically active polypeptide A is inserted into the acetyl-CoA-sensitive polypeptide B, dividing B into two parts, B1 and B2, forming a B1-A-B2 probe structure; the interaction between the acetyl-CoA-sensitive polypeptide B and acetyl-CoA results in an enhanced optical signal from the optically active polypeptide A.

[0085] In the optical probe of the present invention, the optically active polypeptide can be located at any position of the acetyl-CoA-sensitive polypeptide. In one embodiment, the optically active polypeptide is located at any position of the acetyl-CoA-sensitive polypeptide in the NC direction. Exemplarily, the optically active polypeptide is located at any one or more of the following sites on the acetyl-CoA-sensitive polypeptide: 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 20 / 27, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 21 / 27, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 22 / 27, 23 / 24, 23 / 25, 23 / 26, 23 / 27, 37 / 38, 37 / 39, 38 / 39, 46 / 47. 46 / 48, 47 / 48, 59 / 60, 59 / 61, 59 / 62, 59 / 63, 59 / 64, 59 / 65, 59 / 66, 59 / 67, 59 / 68, 59 / 69, 60 / 61, 60 / 62, 60 / 63, 60 / 64, 60 / 65, 60 / 66, 60 / 67, 60 / 68, 60 / 69, 61 / 62, 61 / 63, 61 / 64, 61 / 65, 61 / 66, 61 / 67, 61 / 68, 61 / 69, 62 / 63, 62 / 64, 62 / 65, 62 / 66, 62 / 67, 62 / 68, 62 / 69, 63 / 64, 63 / 65, 63 / 66, 63 / 67, 63 / 68, 63 / 69, 64 / 65, 64 / 66, 64 / 67, 64 / 68, 64 / 69, 65 / 66, 65 / 67, 65 / 68, 65 / 69, 66 / 67, 66 / 68, 66 / 69, 67 / 68, 67 / 69, 78 / 79, 78 / 80, 78 / 81, 78 / 82, 78 / 83, 78 / 84, 79 / 80, 79 / 81, 79 / 82, Locations 79 / 83, 79 / 84, 80 / 81, 80 / 82, 80 / 83, 80 / 84, 81 / 82, 81 / 83, 81 / 84, 82 / 83, 82 / 84, 83 / 84, 102 / 103, 102 / 104, 103 / 104, 112 / 113, 122 / 123, 132 / 133, 132 / 134, 132 / 135, 132 / 136, 133 / 134, 133 / 135, 133 / 136, 134 / 135, 134 / 136, and / or 135 / 136. In a preferred embodiment, the optical probe is as shown in SEQ ID NO:10.

[0086] In this paper, at sites represented in the "X / Y" format, the optically active polypeptide has portions of acetyl-CoA-sensitive polypeptide at both ends. The N-terminus of the optically active polypeptide consists of the N-terminal starting amino acid (e.g., any amino acid from position 1 to 12) to the Xth amino acid of the acetyl-CoA-sensitive polypeptide sequence, and the C-terminus consists of the Yth amino acid to the C-terminal ending amino acid (e.g., any amino acid from position Y to position 112). If the two numbers in the "X / Y" format are consecutive integers, it indicates that the optically active polypeptide is located between the amino acids represented by those numbers. For example, the insertion site 134 / 135 indicates that the optically active polypeptide is located between amino acids 134 and 135 of the acetyl-CoA-sensitive polypeptide. If the two numbers in the site represented in the form of "X / Y" are not consecutive integers and X is less than Y, it indicates that the optically active polypeptide replaces the amino acid between the amino acids indicated by the number. For example, insertion site 133 / 135 indicates that the optically active polypeptide replaces amino acid 134 of the acetyl-CoA sensitive polypeptide, and insertion site 132 / 136 indicates that the optically active polypeptide replaces amino acids 133-135 of the acetyl-CoA sensitive polypeptide.

[0087] In one or more embodiments, the optical probe comprises, from the N-terminus to the C-terminus, residues 1-X of SEQ ID NO:1, an optically active polypeptide or a variant thereof represented by any one of SEQ ID NO:2, 6, 7, and 9, and SEQ ID NO:1. The residue at position Y-146 of NO:1, where X and Y are selected from any of the following groups: (1) X is 20, Y is 21, (2) X is 20, Y is 22, (3) X is 20, Y is 23, (4) X is 20, Y is 24, (5) X is 20, Y is 25, (6) X is 20, Y is 26, (7) X is 20, Y is 27, (8) X is 21, Y is 22, (9) X is 21, Y is 23, (10) X is 21, Y is 24, (11) X is 21, Y is 25, (12) X is 21, Y is 26, (13) X is 21, Y is 27, (14) X is 22, Y is 23, 247920

[0088] (15) X is 22, Y is 24. (16) X is 22, Y is 25. (17) X is 22, Y is 26. (18) X is 22, Y is 27. (19) X is 23, Y is 24. (20) X is 23, Y is 25. (21) X is 23, Y is 26. (22) X is 23, Y is 27. (23) X is 37, Y is 3. 8, (24) X is 37, Y is 39, (25) X is 38, Y is 39, (26) X is 46, Y is 47, (27) X is 46, Y is 48, (28) X is 47, Y is 48, (29) X is 59, Y is 60, (30) X is 59, Y is 61, (31) X is 59, Y is 62, (32) X is 59, Y It is 63, (33)X is 59, Y is 64, (34)X is 59, Y is 65, (35)X is 59, Y is 66, (36)X is 59, Y is 67, (37)X is 59, Y is 68, (38)X is 59, Y is 69, (39)X is 60, Y is 61, (40)X is 60, Y is 62, (41)X is 60 Y is 63, (42) X is 60, Y is 64, (43) X is 60, Y is 65, (44) X is 60, Y is 66, (45) X is 60, Y is 67, (46) X is 60, Y is 68, (47) X is 60, Y is 69, (48) X is 61, Y is 62, (49) X is 61, Y is 63, (50) X is 6 1. Y is 64, (51) X is 61, Y is 65, (52) X is 61, Y is 66, (53) X is 61, Y is 67, (54) X is 61, Y is 68, (55) X is 61, Y is 69, (56) X is 62, Y is 63, (57) X is 62, Y is 64, (58) X is 62, Y is 65, (59) X X is 62, Y is 66, (60) X is 62, Y is 67, (61) X is 62, Y is 68, (62) X is 62, Y is 69, (63) X is 63, Y is 64, (64) X is 63, Y is 65, (65) X is 63, Y is 66, (66) X is 63, Y is 67, (67) X is 63, Y is 68, (68) (69) X is 63, Y is 69, (69) X is 64, Y is 65, (70) X is 64, Y is 66, (71) X is 64, Y is 67, (72) X is 64, Y is 68, (73) X is 64, Y is 69, (74) X is 65, Y is 66, (75) X is 65, Y is 67, (76) X is 65, Y is 68, ( 77) X is 65, Y is 69; (78) X is 66, Y is 67; (79) X is 66, Y is 68; (80) X is 66, Y is 69; (81) X is 67, Y is 68; (82) X is 67, Y is 69; (83) X is 78, Y is 79; (84) X is 78, Y is 80; (85) X is 78, Y is 81.(86) X is 78, Y is 82, (87) X is 78, Y is 83, (88) X is 78, Y is 84, (89) X is 79, Y is 80, (90) X is 79, Y is 81, (91) X is 79, Y is 82, (92) X is 79, Y is 83, (93) X is 79, Y is 84, (94) X is 80, Y is 81, ( 95) X is 80, Y is 82, (96) X is 80, Y is 83, (97) X is 80, Y is 84, (98) X is 81, Y is 82, (99) X is 81, Y is 83, (100) X is 81, Y is 84, (101) X is 82, Y is 83, (102) X is 82, Y is 84, (103) X is 83, Y is 84 (104) X is 102, Y is 103; (105) X is 102, Y is 104; (106) X is 103, Y is 104; (107) X is 112, Y is 113; (108) X is 122, Y is 123; (109) X is 132, Y is 133; (110) X is 132, Y is 134; (111) X is 1 32, Y is 135, (112) X is 132, Y is 136, (113) X is 133, Y is 134, (114) X is 133, Y is 135, (115) X is 133, Y is 136, (116) X is 134, Y is 135, (117) X is 134, Y is 136, (118) X is 135, Y is 136. An exemplary optical probe sequence is shown in SEQ ID NO:10, which is a probe formed by an optically active polypeptide at position 134 / 135 of an acetyl-CoA-sensitive polypeptide. The sequences of the remaining probes of this invention can be readily determined from the description herein.

[0089] When referring to a polypeptide or protein, the term "variant" or "mutant" as used in this invention includes variants that have the same function as the polypeptide or protein but have a different sequence. These variants include, but are not limited to, deletions, insertions, and / or substitutions of one or more amino acids (typically 1-30, preferably 1-20, more preferably 1-10, most preferably 1-5) in the sequence of the polypeptide or protein, and sequences obtained by adding one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) to its carboxyl terminus and / or amino terminus. Variants of polypeptides or proteins may include: homologous sequences, conserved variants, allelic variants, natural mutants, and induced mutants. These variants may also comprise polypeptides or proteins with 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 polypeptide or protein. The goal is to avoid being limited by theory, where changes to amino acid residues do not alter the overall conformation and function of the polypeptide or protein—a phenomenon known as functionally conserved mutation. For example, in this field, substitution with amino acids of similar or identical properties typically does not change the function of the polypeptide or protein. In this field, amino acids with similar properties often refer to families of amino acids with similar side chains, which are well-defined. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, adding one or more amino acids to the amino terminus and / or carboxyl terminus generally does not alter the function of a polypeptide or protein. Conserved amino acid substitutions for many common, known non-genetically encoded amino acids are known in the art. Conserved substitutions for other non-coding amino acids can be determined based on a comparison of their physical properties with those of their genetically encoded amino acids.

[0090] In two or more polypeptide or nucleic acid sequences, the term "identity" or "percentage of identity" refers to the similarity of two or more sequences or subsequences, or the similarity of a certain percentage of amino acid residues or nucleotides in a specified region, when compared and matched for maximum correspondence using methods known in the art, such as sequence comparison algorithms, through manual alignment and visual inspection (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity, within a comparison window or specified region). For example, preferred algorithms suitable for determining the percentage of sequence identity and the percentage of sequence similarity are the BLAST and BLAST 2.0 algorithms, see Altschul et al. (1977) Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol. 215:403, respectively.

[0091] As is known to those skilled in the art, gene cloning often requires the design of suitable restriction enzyme sites, which inevitably introduces one or more irrelevant residues at the end of the expressed polypeptide or protein, without affecting the activity of the target polypeptide or protein. Similarly, to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside host cells, or facilitate the purification of recombinant proteins, it is often necessary to add amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein. These include, but are not limited to, suitable adaptor peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E-binding proteins, protein A, tags such as 6His or Flag, or proteolytic enzyme sites of factor Xa, thrombin, or enterokinase. An exemplary GAT protein is the full-length amino acid sequence shown in SEQ ID NO:1, which retains its binding function to acetyl-CoA and does not affect the optical property changes of the inserted optically active polypeptide in response to acetyl-CoA binding.

[0092] The optical probe of the present invention may comprise a mutated acetyl-CoA-sensitive polypeptide, wherein a mutated acetyl-CoA-binding protein variant of SEQ ID NO:1 or a truncated variant thereof exhibits a different binding activity from acetyl-CoA at sites selected from the following: I19, R21, T77, K85, G87, P133, P134. The amino acid mutation includes modification, substitution, or deletion of amino acids. In a preferred embodiment, the mutation of the acetyl-CoA-binding protein variant includes mutations at sites selected from any of the following groups: (1) P133, P134, and I19; (2) P133, P134, and R21; (3) P133, P134, and T77; (4) P133, P134, and K85; (5) P133, P134, and G87.

[0093] In one or more embodiments, as an example, in SEQ ID NO:1 or a truncated variant thereof, I19 mutates to L. In one or more embodiments, R21 mutates to M or D. In one or more embodiments, T77 mutates to V. In one or more embodiments, K85 mutates to N. In one or more embodiments, G87 mutates to A. In one or more embodiments, P133 mutates to Y. In one or more embodiments, P134 mutates to D.

[0094] In one or more embodiments, the mutation comprises mutations selected from any of the following groups: (1) P133Y, P134D, and I19L; (2) P133Y, P134D, and R21M; (3) P133Y, P134D, and R21D; (4) P133Y, P134D, and T77V; (5) P133Y, P134D, and K85N; (6) P133Y, P134D, and G87A. The present invention provides acetyl-CoA binding protein variants having these mutations and optical probes comprising such acetyl-CoA binding protein variants as acetyl-CoA-sensitive peptides.

[0095] The optical probe of the present invention may comprise a mutated optically active polypeptide. In some embodiments, the mutated optically active polypeptide has a mutation at the Y1 site, said mutation including modification, substitution, or deletion of an amino acid; in one or more embodiments, said mutation is Y1V. In one or more embodiments, the optical probe comprises any of the amino acid sequences SEQ ID NO:10-16 or variants thereof. In one or more embodiments, the optical probe provided by the present invention comprises a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with any of the amino acid sequences SEQ ID NO:10-16. In a preferred embodiment, the optical probe provided by the present invention comprises a sequence substantially similar to or identical to any of the amino acid sequences SEQ ID NO:10-16.

[0096] In one or more embodiments, the optically active polypeptide has the sequence shown in SEQ ID NO:2 or a variant thereof having the following mutation at the first amino acid position: 1V, the optically active polypeptide being located at one or more sites selected from the following acetyl-CoA sensitive polypeptide: 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 20 / 27, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 21 / 27, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 22 / 27, 23 / 24, 23 / 25, 23 / 26, 23 / 27, 37 / 38. 37 / 39, 38 / 39, 46 / 47, 46 / 48, 47 / 48, 59 / 60, 59 / 61, 59 / 62, 59 / 63, 59 / 64, 59 / 65, 59 / 66, 59 / 67, 59 / 68, 59 / 69, 60 / 61, 60 / 62, 60 / 63, 60 / 64, 60 / 65, 60 / 66, 60 / 67, 60 / 68, 60 / 69, 61 / 62, 61 / 63, 61 / 64, 61 / 65, 61 / 66, 61 / 67, 61 / 68, 61 / 69, 62 / 63, 62 / 64, 62 / 65, 62 / 66, 62 / 67, 62 / 68, 62 / 69, 63 / 64, 63 / 65, 63 / 66, 63 / 67, 63 / 68, 63 / 69, 64 / 65, 64 / 66, 64 / 67, 64 / 68, 64 / 69, 65 / 66, 65 / 67, 65 / 68, 65 / 69, 66 / 67, 66 / 68, 66 / 69, 67 / 68, 67 / 69, 78 / 79, 78 / 80, 78 / 81, 78 / 82, 78 / 83, 78 / 84, 79 / 80, 79 / 81 , 79 / 82, 79 / 83, 79 / 84, 80 / 81, 80 / 82, 80 / 83, 80 / 84, 81 / 82, 81 / 83, 81 / 84, 82 / 83, 82 / 84, 83 / 84, 102 / 103, 102 / 104, 103 / 104, 112 / 113, 122 / 123, 132 / 133, 132 / 134, 132 / 135, 132 / 136, 133 / 134, 133 / 135, 133 / 136, 134 / 135, 134 / 136 and / or 135 / 136.

[0097] In some embodiments, the optically active polypeptide is located at position 134 / 135 of the acetyl-CoA-sensitive polypeptide, the acetyl-CoA-sensitive polypeptide having the sequence shown in SEQ ID NO:1 or a variant having at least 70% sequence identity with it and retaining acetyl-CoA binding activity, the optically active polypeptide having the sequence shown in any one of SEQ ID NO:2, 6, 7, 9, and the acetyl-CoA variant having a mutation selected from any one of the following: (1) P133Y, P134D, and I19L; (2) P133Y, P134D, and R21M; (3) P133Y, P134D, and R21D; (4) P133Y, P134D, and T77V; (5) P133Y, P134D, and K85N; (6) P133Y, P134D, and G87A and optionally the optically active polypeptide is mutated to (7) Y1V.

[0098] Exemplarily, the optical probe provided by the present invention comprises any of the amino acid sequences SEQ ID NO:10-16 or variations thereof. In one embodiment, the optical probe provided by the present invention comprises a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with any of the amino acid sequences SEQ ID NO:10-16. In a preferred embodiment, the optical probe provided by the present invention comprises a sequence substantially similar to or identical to the amino acid sequences SEQ ID NO:10-16.

[0099] As used herein, the terms “functional variant,” “derivative,” and “analyte” refer to a protein that substantially retains the same biological function or activity as the original polypeptide or protein (e.g., GAT protein or fluorescent protein). Functional variants, derivatives, or analogs of the polypeptides or proteins (e.g., GAT protein or fluorescent protein) of the present invention may be (i) proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by fusing a mature protein with another compound (e.g., a compound that extends the protein's half-life, such as polyethylene glycol); or (iv) proteins formed by fusing an additional amino acid sequence to this protein sequence (e.g., a secreted sequence or a sequence used to purify this protein or a proteogenic sequence, or a fusion protein formed with an antigen IgG fragment). Based on the teachings herein, these functional variants, derivatives, and analogs are within the scope well known to those skilled in the art.

[0100] The difference between the analogue and the original polypeptide or protein can be a difference in amino acid sequence, a difference in modification that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis through radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biology techniques.

[0101] The analogues also include those having residues different from naturally occurring L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β- or γ-amino acids). It should be understood that the acetyl-CoA-sensitive polypeptides of the present invention are not limited to the representative proteins, variants, derivatives, and analogues listed above. Modifications (generally without altering the primary structure) include chemically derived forms of proteins, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during protein synthesis and processing or further processing steps. This modification can be accomplished by exposing the protein to glycosylating enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Proteins modified to improve their resistance to proteolysis or optimize their solubility are also included.

[0102] The present invention also provides a method for preparing the above-mentioned acetyl-CoA optical probe, comprising the following steps: 1) incorporating the nucleic acid sequence encoding the acetyl-CoA optical probe described herein into an expression vector; 2) transferring the expression vector into a host cell; 3) culturing the host cell under conditions suitable for expression of the expression vector; and 4) isolating the acetyl-CoA optical probe.

[0103] As used herein, the term "nucleic acid" or "nucleotide" can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. When referring to nucleic acids, 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 a substitution of a nucleic acid, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the protein it encodes. The nucleic acids of this invention may comprise a nucleotide sequence with 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 described nucleic acid sequence. This invention also relates to nucleic acid fragments that hybridize with the sequences described above. As used herein, a “nucleic acid fragment” contains at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR).

[0104] The full-length sequence or fragments of the optical probe or fusion protein of this invention can generally be obtained by PCR amplification, artificial synthesis, or recombinant methods. For PCR amplification, primers can be designed according to the nucleotide sequence disclosed in this invention, and a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequence. When the nucleotide sequence is greater than 2500 bp, it is preferable to perform 2 to 6 PCR amplifications, and then splice the fragments from each amplification in the correct order. This invention does not impose any special limitations on the PCR amplification procedure and system; conventional PCR amplification procedures and systems in the art can be used. Recombinant methods can also be used to obtain the relevant sequence in large quantities. This typically involves cloning it into a vector, transforming it into cells, and then isolating and purifying the relevant polypeptide or protein from the proliferated host cells using conventional methods. Furthermore, artificial synthesis methods can be used to synthesize the relevant sequence, especially when the fragment length is short. In this invention, when the nucleotide sequence of the optical probe is less than 2500 bp, artificial synthesis methods can be used. The artificial synthesis method is a conventional DNA artificial synthesis method in the art, without other special requirements. Typically, long sequences are obtained by first synthesizing multiple small fragments and then ligating them. Currently, the DNA sequence encoding the protein of this invention (or its functional variants, derivatives, or analogues) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art. Mutations can be introduced into the protein sequence of this invention using methods such as mutagenic PCR or chemical synthesis.

[0105] After obtaining the nucleotide sequence encoding an optical probe, this invention incorporates the nucleotide sequence encoding the optical probe into an expression vector to obtain a recombinant expression vector. The terms "expression vector" and "recombinant vector" used herein are used interchangeably and refer to prokaryotic or eukaryotic vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors. These vectors can replicate and stably express in a host cell. An important characteristic of these recombinant vectors is that they typically contain an expression control sequence. The term "expression control sequence" used herein refers to an element that can be operatively linked to the target gene to regulate the transcription, translation, and expression of the target gene. This can be an origin of replication, promoter, marker gene, or translation control element, including enhancers, operons, terminators, ribosome binding sites, etc. The choice of expression control sequence depends on the host cell used. Recombinant vectors applicable in this invention include, but are not limited to, bacterial plasmids. In recombinant expression vectors, "operative linking" refers to the connection of the target nucleotide sequence to a regulatory sequence in a manner that allows the expression of the nucleotide sequence. Those skilled in the art are familiar with methods for constructing expression vectors containing the coding sequence of the fusion protein of this invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be effectively ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: the *E. coli* lac or trp promoter; the *λ* phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, the retroviral LTR, and other known promoters that control gene expression 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 be a commercially available pCDF vector, with no other special requirements. Exemplarily, the nucleotide sequence encoding the optical probe and the expression vector are double-digested with BamHI and XhoI, respectively, and then the digestion products are ligated to obtain the recombinant expression vector. This invention does not specifically limit the specific steps and parameters of digestion and ligation; conventional steps and parameters in the art can be used.

[0106] After obtaining the recombinant expression vector, the vector is transformed into a host cell to produce a protein or peptide including a fusion protein. This transfer process can be performed using conventional techniques well known to those skilled in the art, such as transformation or transfection. The host cell described in this invention refers to a cell capable of receiving and accommodating recombinant DNA molecules, serving as the site for recombinant gene amplification. Ideally, the recipient cell should meet the conditions of easy acquisition and proliferation. The "host cell" of this invention can include prokaryotic and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells, and mammalian cells. Specifically, it can be bacterial cells of *Escherichia coli*, *Streptomyces*, and *Salmonella typhimurium*, fungal cells such as yeast, plant cells, insect cells of *Drosophila S2* or *Sf9*, animal cells such as CHO, COS, HEK293, HeLa cells, or Bowes melanoma cells, etc., including but not limited to the aforementioned host cells. The host cell is preferably a variety of cells that are conducive to gene product expression or fermentation production, such cells are well known and commonly used in the art. An exemplary host cell used in the embodiments of this invention is *Escherichia coli* strain BL21-DE3. Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0107] The method for transferring DNA to host cells described in this invention is a conventional method in the art, including calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipid transfection, native competent cells, chemically mediated transfer, or electroporation. When the host is a prokaryote such as *Escherichia coli*, the preferred method is the CaCl2 or MgCl2 method, and the steps used are well known in the art. When the host cell is a eukaryotic cell, the following DNA transfection methods can be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0108] This invention involves transferring an expression vector into host cells, followed by amplification and expression culture of the host cells to isolate an acetyl-CoA optical probe. The host cell amplification and expression culture can be performed using conventional methods. Depending on the type of host cells used, the culture medium can be any conventional medium. Culture is carried out under conditions suitable for host cell growth.

[0109] In this invention, the optical probe is expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated or purified using various separation methods based on its physical, chemical, and other properties. This invention does not specifically limit the method for separating the acetyl-CoA fluorescent protein; conventional methods for separating fusion proteins in the art can be used. These methods are well known to those skilled in the art and include, but are not limited to, conventional refolding, salting out, centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof. In one embodiment, the optical probe is separated using His-tagged affinity chromatography.

[0110] This invention also provides the application of the acetyl-CoA optical probe in real-time localization, quantitative detection, and high-throughput compound screening of acetyl-CoA. In one aspect, the acetyl-CoA optical probe is preferably linked to signal peptides at different sites within the cell, transferred into the cell, and its real-time localization of acetyl-CoA is achieved by detecting the intensity of fluorescence signals within the cell; quantification of acetyl-CoA is then performed using a standard acetyl-CoA titration curve. The standard acetyl-CoA titration curve of this invention is plotted based on the fluorescence signals obtained by the acetyl-CoA optical probe at different concentrations of acetyl-CoA. The acetyl-CoA optical probe of this invention is directly transferred into the cell, eliminating the need for time-consuming sample processing during real-time localization and quantitative detection of acetyl-CoA, thus improving accuracy. In high-throughput compound screening, the acetyl-CoA optical probe of this invention adds different compounds to the cell culture medium and measures changes in acetyl-CoA content to screen for compounds that affect changes in acetyl-CoA content. The application of the acetyl-CoA optical probe described in this invention in real-time localization, quantitative detection of acetyl-CoA, and high-throughput compound screening is not for diagnostic or therapeutic purposes and does not involve the diagnosis or treatment of diseases.

[0111] In this document, concentrations, contents, percentages, and other values ​​are expressed in range form. It should also be understood that this range form is used for convenience and brevity only, and should be flexibly interpreted to include the values ​​explicitly mentioned at the upper and lower limits of the range, as well as all individual values ​​or subranges included within that range.

[0112] Example

[0113] The following detailed description of the acetyl-CoA optical probe provided by the present invention, in conjunction with specific embodiments, should not be construed as limiting the scope of protection of the present invention.

[0114] I. Experimental Materials and Reagents

[0115] The embodiments primarily employ conventional genetic engineering molecular biology cloning methods, cell culture, and imaging methods, which are well-known to those skilled in the art. Examples include: Jane Rothcomems et al.'s *Molecular Cloning: A Laboratory Manual* (3rd edition, August 2002, Science Press, Beijing); Fereschney et al.'s *Animal Cell Culture: A Basic Technique Guide* (5th edition, translated by Zhang Jingbo, Xu Cunshuan et al.); and J.S. Bonnie Fesnon, M. Dassault et al.'s *A Concise Laboratory Manual of Cell Biology* (translated by Zhang Jingbo et al.). Those skilled in the art can readily implement this invention by making minor modifications and variations based on the following embodiments, and all such modifications and variations fall within the scope of the claims of this application.

[0116] The pCDF-cpYFP and pCDF-acetyl-CoA binding protein plasmids used in the examples were constructed by the Protein Laboratory of East China University of Science and Technology, and the pCDF plasmid vectors were purchased from Invitrogen. All primers used for PCR were synthesized, purified, and identified correctly by mass spectrometry by Shanghai Jierui Biotechnology Co., Ltd. The expression plasmids constructed in the examples were all sequenced by BGI Genomics and J. Lee Sequencing. The Taq DNA polymerase used in each example was purchased from Dongsheng Biotechnology, the pfu DNA polymerase from Tiangen Biotech (Beijing) Co., Ltd., and the PrimeSTAR DNA polymerase from TaKaRa. Corresponding polymerase buffers and dNTPs were included with each purchase. Restriction endonucleases such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, and SpeI, as well as T4 ligase and T4 phosphorylase (T4 PNK), were purchased from Fermentas, and corresponding buffers were included with each purchase. The Lip2000 transfection kit was purchased from Invitrogen. Acetyl-CoA and other amino acids were purchased from Sigma-Aldrich. Unless otherwise stated, inorganic salts and other chemical reagents were purchased from Sigma-Aldrich. HEPES salt, ampicillin (Amp), and puromycin were purchased from Amersco. The 96-well detection blackboard and the 384-well fluorescence detection blackboard were purchased from WHB.

[0117] The DNA purification kits used in these examples were purchased from BBI (Canada), and the general plasmid extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd. The cloned strain Mach1 was purchased from Invitrogen. Nickel affinity chromatography columns and desalting column packing materials were both from GE Healthcare.

[0118] The main instruments used in the examples include: Biotek Synergy 2 multi-functional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge 22R benchtop high-speed refrigerated centrifuge (Beckman, USA), PCR amplifier (Biometra, Germany), ultrasonic disruptor (Ningbo Xinzhi Co., Ltd.), nucleic acid electrophoresis apparatus (Shenneng Bocai Co., Ltd.), fluorescence spectrophotometer (Varian, USA), CO2 constant temperature cell incubator (SANYO), and inverted fluorescence microscope (Nikon, Japan).

[0119] II. Molecular Biology Methods and Cellular Experimental Methods

[0120] II.1 Polymerase Chain Reaction (PCR):

[0121] 1. PCR amplification of the target fragment:

[0122] This method is mainly used for gene fragment amplification and colony PCR identification of positive clones. The PCR amplification reaction system 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, sterile ultrapure water (ddH2O) 41.5-42 μL, total volume 50 μL. The PCR amplification program is as follows: denaturation at 95℃ for 2-10 minutes, 30 cycles (94-96℃ for 30-45 seconds, 50-65℃ for 30-45 seconds, 72℃ for a certain time (600 bp / min)), extension at 72℃ for 10 minutes.

[0123] 2. Long fragment (>2500bp) amplification PCR:

[0124] The long-fragment amplification used in this invention is mainly a reverse PCR amplification vector, a technique used in the following examples to obtain site-directed mutagenesis. Reverse PCR primers are designed at the mutation site, with one primer containing the mutated nucleotide sequence at its 5' end. The amplified product then contains the corresponding mutation site. The long-fragment amplification PCR reaction system 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, sterile ultrapure water (ddH2O) 33.5 μL, total volume 50 μL. The PCR amplification program is as follows: denaturation at 95℃ for 5 minutes, 30 cycles (98℃ for 10 seconds, 50-68℃ for 5-15 seconds, 72℃ for a certain time (1000bp / min)), extension at 72℃ for 10 minutes; or denaturation at 95℃ for 5 minutes, 30 cycles (98℃ for 10 seconds, 68℃ for a certain time (1000bp / min)), extension at 72℃ for 10 minutes.

[0125] II.2 Endonuclease digestion reaction:

[0126] The double enzyme digestion system for the plasmid vector is as follows: 20 μL plasmid vector (approximately 1.5 μg), 5 μL 10× buffer, 1-2 μL restriction endonuclease, and 1-2 μL of restriction endonuclease, then add sterile ultrapure water to a total volume of 50 μL. Reaction conditions: 37℃, 1-7 hours.

[0127] II.3 Phosphorylation of DNA fragments at the 5' end

[0128] Plasmids or genomes extracted from microorganisms contain phosphate groups at their ends, while PCR products do not. Therefore, a phosphate addition reaction is required at the 5' end of the PCR product. Only DNA molecules with phosphate groups at their ends can undergo ligation. The phosphorylation reaction system is as follows: 5-8 μL of PCR product DNA sequence, 1 μL of 10×T4 ligase buffer, 1 μL of T4 polynucleotide kinase (T4 PNK), and 0-3 μL of sterile ultrapure water, for a total volume of 10 μL. The reaction conditions are 37°C for 30 minutes to 2 hours, followed by inactivation at 72°C for 20 minutes. T4 PNK is an abbreviation for T4 polynucleotide kinase, used for the addition reaction of the 5' phosphate group of DNA molecules.

[0129] II.4 Ligation reaction between target fragment and vector

[0130] The methods for connecting different fragments and carriers vary, and this invention uses three connection methods.

[0131] 1. Blunt-end junctions of blunt-ended short fragments and linearized vectors

[0132] The principle of this method is that the blunt-end product obtained by PCR is phosphorylated at the 5' end of a DNA fragment using T4 PNK, and then ligated with a linearized vector using PEG4000 and T4 DNA ligase to obtain a recombinant plasmid. The homologous recombination ligation system is as follows: 4 μL of T4 PNK-treated DNA fragment, 4 μL of linearized vector fragment, 1 μL of PEG4000, 1 μL of 10×T4 ligase buffer, and 1 μL of T4 DNA ligase, for a total of 10 μL. The reaction conditions are 22℃ for 30 minutes.

[0133] 2. Ligation of DNA fragments with sticky ends and vector fragments with sticky ends.

[0134] DNA fragments digested by restriction endonucleases typically produce prominent sticky ends, which can then be ligated to vector fragments containing sequence complementarity to form recombinant plasmids. The ligation reaction system is as follows: 1-7 μL of the digested PCR product DNA fragment, 0.5-7 μL of the digested plasmid, 1 μL of 10×T4 ligase buffer, 1 μL of T4 DNA ligase, and sterile ultrapure water to a total volume of 10 μL. The reaction conditions are 16℃ for 4-8 hours. The mass ratio of the PCR product fragment to the vector double-digested product is approximately between 2:1 and 6:1.

[0135] 3. Ligation reaction involving the self-circularization of DNA fragment products phosphorylated at the 5' end following site-directed mutagenesis using reverse PCR.

[0136] The 5' phosphorylated DNA fragment was ligated to the 3' and 5' ends of the linearized vector via a self-circularization ligation reaction to obtain a recombinant plasmid. The self-circularization ligation reaction system was as follows: 10 μL phosphorylation reaction mixture, 0.5 μL T4 ligase (5 U / μL), total volume 10.5 μL. Reaction conditions: 16℃, 4–16 hours.

[0137] II.5 Preparation and Transformation of Competent Cells

[0138] Preparation of competent cells:

[0139] 1. Pick a single colony (e.g., Mach1) and inoculate it into 5 mL of LB medium. Incubate overnight at 37°C with a shaker.

[0140] 2. Transfer 0.5-1 mL of the overnight culture to 50 mL of LB medium and incubate at 37°C and 220 rpm for 3 to 5 hours, until OD (dose-free survival) is achieved. 600 It reached 0.5.

[0141] 3. Pre-cool the cells in an ice bath for 2 hours.

[0142] Centrifuge at 4.4℃ and 4000rpm for 10 minutes.

[0143] 5. Discard the supernatant, resuspend the cells in 5 mL of pre-cooled buffer, and add resuspending buffer to a final volume of 50 mL after homogenization.

[0144] 6. Ice bath for 45 minutes.

[0145] Centrifuge at 7.4℃ and 4000rpm for 10 minutes, then resuspend the bacteria in 5mL of ice-cold storage buffer.

[0146] 8. Place 100 μL of bacterial culture in each EP tube and store at -80°C or in liquid nitrogen.

[0147] Resuspension buffer: CaCl2 (100mM), MgCl2 (70mM), NaAc (40mM)

[0148] Storage buffer: 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10×CaCl2 (1 M), 1 mL 10×MgCl2 (700 mM), 1 mL 10×NaAc (400 mM), 4.6 mL ddH2O

[0149] Transformation of competent cells:

[0150] 1. Take 100 μL of competent cells and thaw them on an ice bath.

[0151] 2. Add an appropriate volume of ligation product, gently mix by pipetting, and incubate on ice for 30 minutes. The volume of ligation product added is usually less than 1 / 10 of the competent cell volume.

[0152] 3. Place the bacterial solution in a 42°C water bath for 90 seconds to heat shock, then quickly transfer it to an ice bath and place it for 5 minutes.

[0153] 4. Add 500 μL LB and incubate at 200 rpm for 1 hour on a constant temperature shaker at 37℃.

[0154] 5. Centrifuge the bacterial culture at 4000 rpm for 3 minutes, and keep 200 μL of supernatant. Spread the bacterial cells evenly on the surface of an agar plate containing appropriate antibiotics. Incubate the plate upside down in a 37°C incubator overnight.

[0155] II.6 Protein Expression, Purification, and Fluorescence Detection

[0156] 1. Transform the expression vector (e.g., the pCDF-based acetyl-CoA optical probe expression vector) into BL21(DE3) cells, incubate upside down overnight, pick clones from the plate into 250ml Erlenmeyer flasks, place them on a 37℃ shaker, and incubate at 220rpm until OD=0.4-0.8. Add 1 / 1000 (v / v) of IPTG (1M) and induce expression at 18℃ for 24-36 hours.

[0157] 2. After induction of expression, centrifuge at 4000 rpm for 30 minutes to collect the bacteria. Resuspend the bacterial pellet in 50 mM phosphate buffer and sonicate until the bacterial cells are clear. Centrifuge at 9600 rpm at 4°C for 20 minutes.

[0158] 3. The supernatant from centrifugation was purified by a self-assembled nickel affinity chromatography column to obtain the protein. The protein after nickel affinity chromatography was then purified by a self-assembled desalting column to obtain the protein dissolved in 100mM HEPES buffer (pH 7.4).

[0159] 4. After SDS-PAGE identification of the purified protein, the probe was diluted with assay buffer (100mM HEPES, 100mM NaCl, pH 7.4) to a final concentration of 0.2-5μM. Acetyl-CoA was prepared into a stock solution with a final concentration of 50mM using assay buffer (100mM HEPES, 100mM NaCl, pH 7.4).

[0160] 5. Take 100 μl of 1 μM protein solution, incubate at 37℃ for 10 minutes, add acetyl-CoA for titration, and measure the fluorescence intensity of the protein at 528 nm emission after excitation by 420 nm light and at 528 nm emission after excitation by 485 nm light. The fluorescence excitation and emission measurements of the samples were performed using a multifunctional fluorescent microplate reader.

[0161] 6. Take 100 μl of 1 μM protein solution, incubate at 37℃ for 10 minutes, add acetyl-CoA, and measure the absorption and fluorescence spectra of the protein. The absorption and fluorescence spectra of the samples were measured using a spectrophotometer and a fluorescence spectrophotometer.

[0162] II.7 Transfection and Fluorescence Detection of Mammalian Cells

[0163] 1. The pCDNA3.1+-based acetyl-CoA optical probe plasmid was transfected into HeLa cells using Lipofectamine 2000 (Invitrogen) transfection reagent and cultured in a 37°C, 5% CO2 cell culture incubator. Fluorescence detection was performed 24–36 h after the exogenous gene was fully expressed.

[0164] 2. After the expression was induced, the adherent HEK293 cells were washed three times with PBS and placed in HBSS solution for fluorescence microscopy and microplate reader detection.

[0165] Example 1: Acetyl-CoA binding protein particles

[0166] The GAT(1-146) gene (SEQ ID NO:1) from Bacillus licheniformis was amplified by PCR. The PCR product was recovered after gel electrophoresis and digested with BamHI and XhoI enzymes. The pCDF vector was also double-digested with the corresponding enzymes. After ligation with T4 DNA ligase, the product was used to transform DH5α cells. The transformed DH5α cells were plated on LB agar plates (streptomycin 100 μg / mL) and incubated overnight at 37°C. Plasmids were extracted from the grown DH5α transformants and identified by PCR. Positive plasmids, after being correctly sequenced, were used for subsequent plasmid construction.

[0167] Example 2: Expression and detection of cpYFP optical probes at different insertion sites

[0168] In this embodiment, the following sites were selected for insertion into cpYFP based on pCDF-GAT to obtain the corresponding pCDF-GAT-cpYFP plasmids: 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 20 / 27, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 21 / 27, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 22 / 27, 23 / 24, 23 / 25, 23 / 26, 23 / 27, 37 / 38, 37 / 39, 38 / 39, 46 / 47, 46 / 48, 47 / 48, 59 / 60, 59 / 61, 59 / 62, 59 / 63, 59 / 64, 59 / 65, 59 / 66, 59 / 67, 59 / 68, 59 / 69, 60 / 61, 60 / 62, 60 / 63, 60 / 64, 60 / 65, 60 / 66, 60 / 67, 60 / 68, 60 / 69, 61 / 62, 6 1 / 63, 61 / 64, 61 / 65, 61 / 66, 61 / 67, 61 / 68, 61 / 69, 62 / 63, 62 / 64, 62 / 65, 62 / 66, 62 / 67, 62 / 68, 62 / 69, 63 / 64, 63 / 65, 63 / 66, 63 / 67, 63 / 68, 63 / 69, 64 / 65, 64 / 66, 64 / 67, 64 / 68, 64 / 69, 65 / 66, 65 / 67, 65 / 68, 65 / 6 9, 66 / 67, 66 / 68, 66 / 69, 67 / 68, 67 / 69, 78 / 79, 78 / 80, 78 / 81, 78 / 82, 78 / 83, 78 / 84, 79 / 80, 79 / 81, 79 / 82, 79 / 83, 79 / 84, 80 / 81, 80 / 82, 80 / 83, 80 / 84, 81 / 82, 81 / 83, 81 / 84, 82 / 83, 82 / 84, 83 / 84, 102 / 103, 247920

[0169] 102 / 104, 103 / 104, 112 / 113, 122 / 123, 132 / 133, 132 / 134, 132 / 135, 132 / 136, 133 / 134, 133 / 135, 133 / 136, 134 / 135 (i.e., SEQ ID NO:10), 134 / 136 and / or 135 / 136.

[0170] The cpYFP DNA fragment was generated using PCR, and a homologous sequence from the cpYFP terminal was introduced at the 5' end using primers. PCR amplification produced a linearized pCDF-GlnK1 vector, whose 5' and 3' ends contained sequences (15 bp–25 bp) completely identical to those at the cpYFP terminals. The linearized pCDF-GAT and cpYFP fragments underwent homologous recombination using the Hieff Clone Enzyme. The product was transformed into DH5α, and the transformed DH5α was plated on LB agar plates (streptomycin 50 μg / mL) and incubated overnight at 37°C. Positive clones identified by PCR were subjected to plasmid extraction and sequencing. Sequencing was performed by J. Lee or BGI Genomics.

[0171] After successful sequencing, the recombinant plasmid was transformed into BL21(DE3) to induce expression, and the protein was purified. SDS-PAGE electrophoresis showed a size around 44 kDa. This size is consistent with the size of the pCDF-cpYFP-GAT fusion protein containing the purified His-tag. Results are as follows... Figure 1 As shown.

[0172] Acetyl-CoA response screening was performed using the supernatant of fragmented *E. coli* expressing the GAT-cpYFP fusion protein. The detection signal of the fusion fluorescent protein containing 100 μM acetyl-CoA was divided by the detection signal of the fusion fluorescent protein without acetyl-CoA. Results are as follows: Figure 2 As shown, the detection results indicate that optical probes with an acetyl-CoA response greater than 1.3 times or less than 0.75 times compared to the control were inserted at sites 22 / 25, 46 / 48, 59 / 69, 60 / 68, 62 / 66, 133 / 135, 134 / 135, and 134 / 136.

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

[0174] Following the method in Example 2, cpYFP was replaced with cpGFP to construct an acetyl-CoA green fluorescent protein probe. Figure 3 As shown, the detection results indicate that optical probes with an acetyl-CoA response greater than 1.3 times or less than 0.75 times compared to the control were inserted at sites 22 / 25, 22 / 26, 46 / 48, 59 / 69, 60 / 67, 62 / 67, 133 / 135, and 134 / 136.

[0175] Example 4: Expression and detection of cpBFP optical probes at different insertion sites

[0176] Following the method in Example 2, cpYFP was replaced with cpBFP to construct an acetyl-CoA blue fluorescent protein probe. Figure 4 As shown, the detection results indicate that optical probes with an acetyl-CoA response greater than 1.3 times or less than 0.75 times compared to the control were inserted at sites 22 / 26, 38 / 39, 62 / 67, 133 / 136, and 134 / 136.

[0177] Example 5: Expression and detection of cpmApple optical probes at different insertion sites

[0178] Following the method in Example 2, cpYFP was replaced with cpmApple to construct a red fluorescent probe for acetyl-CoA. Figure 5 As shown, the detection results indicate that optical probes with an acetyl-CoA response greater than 1.2 times or less than 0.8 times compared to the control were inserted at sites 46 / 47, 59 / 69, 60 / 67, 62 / 66, 133 / 135, and 134 / 136.

[0179] Example 6: Expression and detection of mutated cpYFP optical probe

[0180] Based on the optical probes obtained in Example 2, which were inserted at sites 134 / 135, the probes were linearized by reverse PCR. The sequences of the mutation sites were introduced into the primers, and the resulting PCR products were subjected to homologous recombination using Hieff Clone Enzyme to establish a mutant library. The recombinant plasmid of the mutant library was transformed into BL21(DE3) to induce expression. The response to acetyl-CoA was screened using the supernatant of E. coli expressing the probe protein. The detection signal of the fusion fluorescent protein containing 100 μM acetyl-CoA was divided by the detection signal of the fusion fluorescent protein without acetyl-CoA. The results are shown in Table 1. The detection results show that the optical probes with a response to acetyl-CoA more than 4 times are listed below.

[0181] Table 1. Sequences of the mutated optical probes

[0182] serial number Insertion site mutation <![CDATA[R 420 / 485 ]]> sequence I3 134 / 135 GAT-P133Y&P134D&I19L&cpYFP Y1V 4.12 SEQ ID NO:11 T8 134 / 135 GAT-P133Y&P134D&R21M&cpYFP Y1V 4.39 SEQ ID NO:12 T9 134 / 135 GAT-P133Y&P134D&R21D&cpYFP Y1V 4.13 SEQ ID NO:13 F2 134 / 135 GAT-P133Y&P134D&T77V&cpYFP Y1V 5.11 SEQ ID NO:14 C19 134 / 135 GAT-P133Y&P134D&K85N&cpYFP Y1V 4.02 SEQ ID NO:15 G1 134 / 135 GAT-P133Y&P134D&G87A&cpYFP Y1V 4.37 SEQ ID NO:16

[0183] Example 7: Performance of optical probe mutants

[0184] For example, purified acetyl-CoA optical probes GAT-P133Y&P134D&R21M&cpYFP Y1V were treated with 0 mM and 500 μM acetyl-CoA for 10 minutes, respectively, and then fluorescence spectra were detected using a fluorescence spectrophotometer. For excitation spectrum determination: the emission wavelength was fixed at 540 nm, and the excitation spectrum from 350 to 505 nm was recorded, read every 1 nm. The results showed that the probe had two excitation peaks at 420 nm and 500 nm, as shown in the figure. Figure 6As shown in (A). For emission spectrum determination, with fixed excitation wavelengths of 420 nm and 485 nm, the emission spectra from 500 to 600 nm were recorded, with readings every 1 nm. The results showed that the probe's emission peak at 420 nm excitation was 512 nm, and the peak at 485 nm excitation was 519 nm. After adding 500 μM acetyl-CoA, the fluorescence intensity of the probe under 420 nm excitation remained almost unchanged; however, under 485 nm excitation, the fluorescence intensity decreased to seven times that without acetyl-CoA. Figure 6 As shown in (B) and (C).

[0185] The acetyl-CoA optical probes listed in Table 1 of Example 6 were used for acetyl-CoA detection at concentration gradients (0-500 μM). After treating the probes for 10 minutes, 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 measured. The probe titration results are as follows: Figure 7 As shown, the results indicate that different mutants have different affinities for acetyl-CoA.

[0186] The probes in the examples in Table 1 were subjected to specificity testing, and their reactivity with acetyl-CoA analogs was detected. The results showed that they had good specificity. Figure 8 As shown.

[0187] Example 8: Subcellular organelle localization of optical probes and performance of optical probes within subcellular organelles

[0188] In this embodiment, different localization signal peptides were fused with the optical probes GAT-P133Y, P134D, R21M, and cpYFPY1V to localize the optical probes to different organelles. HEK293 cells were transfected with plasmids fused with different localization signal peptides for 36 hours, washed with PBS, and placed in HBSS solution for fluorescence detection using an inverted fluorescence microscope under the FITC channel. Results are as follows: Figure 9 As shown, the acetyl-CoA optical probe can be localized to subcellular organelles, including the cytoplasm, outer membrane, nucleus, endoplasmic reticulum, mitochondria, and nuclear exclusion region, by fusing with different specific localization signal peptides. Fluorescence is observed in different subcellular structures, and the distribution and intensity of the fluorescence vary.

[0189] HEK293 cells were transfected with a cytoplasmic optical probe plasmid for 36 hours. After washing with PBS, the cells were placed in solutions supplemented with 10 μM or 30 μM BMS-303141. The ratio of fluorescence intensity at 420 nm excitation to 528 nm emission and vice versa was measured over a 30-minute timeframe. BMS-303141 is an inhibitor of ATP-citrate lyase, which can reduce intracellular acetyl-CoA levels. Results are as follows: Figure 10 As shown, the R of the sample with 10 μM BMS-303141 added... 420 / 485 The concentration gradually decreased, with a minimum decrease of 6%; after adding 30 μM BMS-303141 and detecting for 30 minutes, the R value of the sample was... 420 / 485 It gradually decreased, with the lowest decrease reaching 45%.

[0190] Example 9: High-throughput compound screening in living cells based on optical probes

[0191] In this embodiment, we used HeLa cells expressing GAT-P133Y&P134D&R21M&cpYFP Y1V in the cytoplasm for high-throughput compound screening.

[0192] Transfected HeLa cells were washed with PBS, treated with HBSS solution (without acetyl-CoA) for 1 hour, and then treated with 10 μM of the compound for 1 hour. Acetyl-CoA was added to each sample. The ratio of fluorescence intensity at 420 nm excitation to 528 nm emission and the ratio of fluorescence intensity at 485 nm excitation to 528 nm emission were recorded using a microplate reader. Samples without any compound treatment were used as controls for standardization. Results are as follows: Figure 11 As shown. Of the 2000 compounds used, the vast majority had minimal effect on acetyl-CoA entry into cells. Three compounds increased the cells' ability to take up acetyl-CoA, while two others significantly reduced it.

[0193] Example 10: Quantitative detection of acetyl-CoA in blood using an optical probe

[0194] In this study, purified GAT-P133Y&P134D&R21M&cpYFP Y1V was used to analyze acetyl-CoA in the blood supernatant of mice and humans.

[0195] After mixing GAT-P133Y, P134D, R21M, and cpYFP Y1V with diluted blood supernatant and treating for 10 minutes, the ratio of fluorescence intensity at 420nm excitation and 528nm emission to fluorescence intensity at 485nm excitation and 528nm emission was detected using an ELISA reader. The results are as follows: Figure 12 As shown, the acetyl-CoA content in mouse blood is around 700 nM, while the acetyl-CoA content in human blood is around 1.6 μM.

[0196] As can be seen from the above embodiments, the acetyl-CoA optical probe provided by the present invention has a relatively small protein molecular weight and is easy to mature. It exhibits large fluorescence dynamic changes, good specificity, and can be expressed in cells through gene manipulation. It can be used to locate and quantify acetyl-CoA in and out of cells in real time, and can also be used for high-throughput compound screening.

[0197] Other implementation plans

[0198] This specification describes many embodiments. However, it should be understood that various modifications that may be learned by those skilled in the art upon reading this specification without departing from the spirit and scope of the invention should also be included within the scope of the appended claims.

[0199] Partial sequence

[0200] SEQ ID NO:1GAT(1-146)

[0201] MIEVKPINAEDTYEIRHRILRPNQPLEACMYETDLLGGAFHLGGYYRGKLISIASFHKAEHSELEGEEQYQLRGM

[0202] ATLEGYREQKAGSTLIRHAEELLRKKGADLLWCNARTSVSGYYEKLGFSEQGEVYDIPPIGPHILMYKKLT

[0203] SEQ ID NO:2cpYFP

[0204] YNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDH

[0205] MVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL

[0206] KLICTTGKLPVPWPPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGD

[0207] TLVNRIELKGIDFKEDGNILGHKLEYN

[0208] SEQ ID NO:3cpmOrange

[0209] VSERMYPEDGVLKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEG

[0210] RHPTGGRDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTK

[0211] GGPLPFAWDILSPQFTYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLR

[0212] GTNFPPDGPVMQKKTMGWEA

[0213] SEQ ID NO:4cpmKate

[0214] MGGRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHKLNGGTGGSMVSKGEE

[0215] LIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIP

[0216] DFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLYPA

[0217] DGGLEGRSDMALKLVGGGHLICNLKTTYRSKK

[0218] SEQ ID NO:5mCherry

[0219] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYG

[0220] SKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTM

[0221] GWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQY

[0222] ERAEGRHSTGGMDELYK

[0223] SEQ ID NO:6cpGFP

[0224] NVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLE

[0225] FVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTT

[0226] GKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE

[0227] LKGIDFKEDGNILGHKLEYN

[0228] SEQ ID NO:7cpBFP

[0229] NVYIKADKQKNGIKANFKIRHNIEGGGVQLAYHYQQNTPIGDGGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLE

[0230] FVTAAGITLGMDELYKGGTGGSESMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT

[0231] TGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPGGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRI

[0232] ELKGIDFKEDGNILGHKLEYN

[0233] SEQ ID NO:8mKate

[0234] MSELITENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFMYGSKTFINHTQ

[0235] GIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLY

[0236] PADGGLEGRADMALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLIKEADKETYVEQHEVAVAR

[0237] YCDLPSKLGHKLN

[0238] SEQ ID NO:9cpmApple

[0239] VSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEG

[0240] RHSTGGMDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGEPYEAFQTAKLKVTK

[0241] GGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLR

[0242] GTNFPPDGPVMQKKTMGWEA

[0243] SEQ ID NO:10GAT-134 / 135-cpYFP

[0244] MIEVKPINAEDTYEIRHRILRPNQPLEACMYETDLLGGAFHLGGYYRGKLISIASFHKAEHSELEGEEQYQLRGM

[0245] ATLEGYREQKAGSTLIRHAEELLRKKGADLLWCNARTSVSGYYEKLGFSEQGEVYDIPPYNSDNVYIMADKQK

[0246] NGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLG

[0247] MDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPT

[0248] LVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE

[0249] DGNILGHKLEYNIGPHILMYKKLT

[0250] SEQ ID NO:11GAT-134 / 135-cpYFP(GAT-P133Y&P134D&I19L&cpYFP Y1V)

[0251] MIEVKPINAEDTYEIRHRLLRPNQPLEACMYETDLLGGAFHLGGYYRGKLISIASFHKAEHSELEGEEQYQLRGM

[0252] ATLEGYREQKAGSTLIRHAEELLRKKGADLLWCNARTSVSGYYEKLGFSEQGEVYDIYDVNSDNVYIMADKQK

[0253] NGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLG

[0254] MDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPT

[0255] LVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE

[0256] DGNILGHKLEYNIGPHILMYKKLT

[0257] SEQ ID NO:12 GAT-134 / 135-cpYFP(GAT-P133Y&P134D&R21M&cpYFP Y1V)

[0258] MIEVKPINAEDTYEIRHRILMPNQPLEACMYETDLLGGAFHLGGYYRGKLISIASFHKAEHSELEGEEQYQLRGM

[0259] ATLEGYREQKAGSTLIRHAEELLRKKGADLLWCNARTSVSGYYEKLGFSEQGEVYDIYDVNSDNVYIMADKQK

[0260] NGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLG

[0261] MDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPT

[0262] LVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE

[0263] DGNILGHKLEYNIGPHILMYKKLT

[0264] SEQ ID NO:13 GAT-134 / 135-cpYFP(GAT-P133Y&P134D&R21D&cpYFP Y1V)

[0265] MIEVKPINAEDTYEIRHRILDPNQPLEACMYETDLLGGAFHLGGYYRGKLISIASFHKAEHSELEGEEQYQLRGM

[0266] ATLEGYREQKAGSTLIRHAEELLRKKGADLLWCNARTSVSGYYEKLGFSEQGEVYDIYDVNSDNVYIMADKQK

[0267] NGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLG

[0268] MDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPT

[0269] LVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE

[0270] DGNILGHKLEYNIGPHILMYKKLT

[0271] SEQ ID NO:14 GAT-134 / 135-cpYFP(GAT-P133Y&P134D&T77V&cpYFP Y1V)

[0272] MIEVKPINAEDTYEIRHRILRPNQPLEACMYETDLLGGAFHLGGYYRGKLISIASFHKAEHSELEGEEQYQLRGM

[0273] AVLEGYREQKAGSTLIRHAEELLRKKGADLLWCNARTSVSGYYEKLGFSEQGEVYDIYDVNSDNVYIMADKQ

[0274] KNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITL

[0275] GMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWP

[0276] TLVTTLGYGLKFCARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE

[0277] DGNILGHKLEYNIGPHILMYKKLT

[0278] SEQ ID NO:15 GAT-134 / 135-cpYFP(GAT-P133Y&P134D&K85N&cpYFP Y1V)

[0279] MIEVKPINAEDTYEIRHRILRPNQPLEACMYETDLLGGAFHLGGYYRGKLISIASFHKAEHSELEGEEQYQLRGM

[0280] ATLEGYREQNAGSTLIRHAEELLRKKGADLLWCNARTSVSGYYEKLGFSEQGEVYDIYDVNSDNVYIMADKQK

[0281] NGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLG

[0282] MDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPT

[0283] LVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE

[0284] DGNILGHKLEYNIGPHILMYKKLT

[0285] SEQ ID NO:16 GAT-134 / 135-cpYFP(GAT-P133Y&P134D&G87A&cpYFP Y1V)

[0286] MIEVKPINAEDTYEIRHRILRPNQPLEACMYETDLLGGAFHLGGYYRGKLISIASFHKAEHSELEGEEQYQLRGM

[0287] ATLEGYREQKAASTLIRHAEELLRKKGADLLWCNARTSVSGYYEKLGFSEQGEVYDIYDVNSDNVYIMADKQK

[0288] NGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLG

[0289] MDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPT

[0290] LVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE

[0291] DGNILGHKLEYNIGPHILMYKKLT

Claims

1. A variant of an acetyl-CoA binding protein, wherein: (1) Having the sequence shown in SEQ ID NO:1 and having mutations at one, two, three or more sites selected from the following: I19, R21, T77, K85, G87, P133 and P134, said mutations including amino acid modifications, substitutions or deletions. (2) is a sequence that has at least 70% sequence identity with the sequence of (1) and has the mutation described in (1) while retaining the ability to bind to acetyl-CoA. Preferably, the mutation includes mutations at sites selected from any of the following groups: (a) P133, P134 and I19, (b) P133, P134 and R21, (c) P133, P134 and T77, (d) P133, P134 and K85, (e) P133, P134 and G87; More preferably, the I19 mutation is L; the R21 mutation is M or D; the T77 mutation is V; the K85 mutation is N; the G87 mutation is A; the P133 mutation is Y; and the P134 mutation is D. More preferably, the mutation includes mutations selected from any of the following groups: (i) P133Y, P134D and I19L, (ii) P133Y, P134D and R21M, (iii) P133Y, P134D and R21D, (iv) P133Y, P134D and T77V, (v) P133Y, P134D and K85N, (vi) P133Y, P134D and G87A.

2. An optical probe comprising an acetyl-CoA-sensitive polypeptide and an optically active polypeptide, wherein, The acetyl-CoA-sensitive polypeptide is: (1) the sequence shown in SEQ ID NO:1, or a sequence having at least 70% sequence identity with it and retaining acetyl-CoA binding activity; (2) the sequence of the acetyl-CoA-binding protein variant of claim 1; or (3) a sequence having at least 70% sequence identity with the sequence of (2) and having the mutation of (2) and retaining sensitivity to acetyl-CoA. The optically active polypeptide is a fluorescent protein or a variant thereof. The optically active polypeptide is located at one or more of the following sites on the acetyl-CoA-sensitive polypeptide: 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 20 / 27, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 21 / 27, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 22 / 27, 23 / 24, 23 / 25, 23 / 26, 23 / 27, 37 / 38, 37 / 39, 38 / 39, 46 / 47, 46 / 48 , 47 / 48, 59 / 60, 59 / 61, 59 / 62, 59 / 63, 59 / 64, 59 / 65, 59 / 66, 59 / 67, 59 / 68, 59 / 69, 60 / 61, 60 / 62, 60 / 63, 60 / 64, 60 / 65, 60 / 66, 60 / 67, 60 / 68, 60 / 69, 61 / 62, 61 / 63, 61 / 64, 61 / 65, 61 / 66, 61 / 67, 61 / 68, 61 / 69, 62 / 63, 62 / 64, 62 / 65, 62 / 66, 62 / 67, 62 / 68, 62 / 69, 63 / 64, 63 / 65, 63 / 66, 63 / 67, 63 / 68, 63 / 69, 64 / 65, 64 / 66, 64 / 67, 64 / 68, 64 / 69, 65 / 66, 65 / 67, 65 / 68, 65 / 69, 66 / 67, 66 / 68, 66 / 69, 67 / 68, 67 / 69, 78 / 79, 78 / 80, 78 / 81, 78 / 82, 78 / 83, 78 / 84, 79 / 80, 79 / 81, 79 / 82, 7 9 / 83, 79 / 84, 80 / 81, 80 / 82, 80 / 83, 80 / 84, 81 / 82, 81 / 83, 81 / 84, 82 / 83, 82 / 84, 83 / 84, 102 / 103, 102 / 104, 103 / 104, 112 / 113, 122 / 123, 132 / 133, 132 / 134, 132 / 135, 132 / 136, 133 / 134, 133 / 135, 133 / 136, 134 / 135, 134 / 136 and / or 135 / 136, The variant of the fluorescent protein contains a mutation at the Y1 site. Preferably, the optically active polypeptide is selected from any one of the following: cpYFP, ​​cpGFP, cpBFP, and cpmApple.

3. The optical probe as described in claim 2, characterized in that, The fluorescent protein is as shown in any one of SEQ ID NO:2-9, wherein the Y1 variant of the fluorescent protein is mutated to V; Preferably, the fluorescent protein is as shown in any one of SEQ ID NO:2, 6, 7, and 9.

4. A fusion polypeptide comprising the optical probe of claim 2 or 3 and other polypeptides, said other polypeptides including a localization sequence, a tag for easy purification, or a tag for an immune response.

5. A nucleic acid molecule comprising the following sequence: (a) A multinucleotide sequence encoding the optical probe of claim 2 or 3, or the fusion polypeptide of claim 4, or (b)(a) complementary sequences.

6. Nucleic acid constructs, including the nucleic acid molecule of claim 6. Preferably, the nucleic acid construct is an expression vector.

7. A host cell, wherein the host cell: (1) Containing, expressing or secreting the optical probe of claim 2 or 3 or the fusion polypeptide of claim 4; (2) Contains the nucleic acid molecule as described in claim 5; or (3) It includes the nucleic acid construct of claim 6.

8. A test kit comprising: (1) The optical probe of claim 2 or 3 or the fusion polypeptide of claim 4. (2) The nucleic acid molecule according to claim 5, (3) The nucleic acid construct according to claim 6, (4) The host cell according to claim 7, The test kit may optionally include other reagents required for the detection of acetyl-CoA using an optical probe. Preferably, the test kit further comprises one or more reagents selected from the following: buffer solution, culture medium, and acetyl-CoA standard.

9. A method for preparing the optical probe of claim 2 or 3 or the fusion polypeptide of claim 4, comprising: The host cells of claim 7 are cultured under conditions in which the optical probe is expressed, and the optical probe is isolated from the culture.

10. The use of the optical probe of claim 2 or 3, the fusion polypeptide of claim 4, the nucleic acid molecule of claim 5, the nucleic acid construct of claim 6, and / or the host cell of claim 7 in detecting acetyl-CoA in a sample, screening compounds, or intracellular and / or extracellular localization of acetyl-CoA.

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