D-2-hydroxyglutaric acid optical probe as well as preparation method and application thereof

Abstract

The invention relates to a D-2-hydroxyglutaric acid optical probe as well as a preparation method and application thereof. Specifically, the invention provides the D-2-hydroxyglutaric acid optical probe, the D-2-hydroxyglutaric acid optical probe comprises a D-2-hydroxyglutaric acid sensitive polypeptide and an optical active polypeptide, and the optical active polypeptide is located in the sequence of the D-2-hydroxyglutaric acid sensitive polypeptide.

Description

Technical Field

[0001] This invention relates to the field of optical probe technology, and in particular to a D-2-hydroxyglutaric acid optical probe, its preparation method, and its application. Background Technology

[0002] D-2-hydroxyglutaric acid (D-2-HG), an abnormal metabolite of the tricarboxylic acid cycle intermediate α-KG, is closely associated with the development of tumors and neurometabolic diseases due to its abnormal accumulation. Therefore, monitoring D-2-HG metabolism is of great significance for studying the molecular mechanisms of related diseases and for precise clinical treatment.

[0003] Currently, the most commonly used methods for detecting D-2-HG include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS / MS) (Akbay E A et al., Genes Development, 2014, 28(5), 479-490), isotope labeling (Heiden MG V et al., Science, 2010, 329(5998), 1492-1499), and enzyme activity detection. HPLC and LC-MS / MS can efficiently and accurately determine changes in D-2-HG content in samples, but they involve the extraction of intracellular metabolites, making the experimental steps cumbersome and time-consuming. Neither isotope labeling nor enzyme activity detection can monitor changes in D-2-HG in living cells in real time. Therefore, there is an urgent need to develop genetically encoded fluorescent probes that can monitor the dynamic changes of D-2-HG in living cells in situ. Summary of the Invention

[0004] The purpose of this invention is to provide a probe and method for real-time, high-throughput, and quantitative detection of D-2-hydroxyglutaric acid 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 a D-2-hydroxyglutaric acid-binding protein, wherein:

[0007] (a) Having the sequence shown in SEQ ID NO:1 and having mutations at one, two, three, four or more sites selected from the following: Y96, R99, D143, N180, S181, R183, Q184, H195, H217. These mutations include amino acid modifications, substitutions, or deletions.

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

[0009] In one or more embodiments, the D-2-hydroxyglutaric acid binding protein variant has the sequence shown in SEQ ID NO:1 and: (a) has a mutation at one, two or more sites selected from the group consisting of N180, S181, R183, Q184, and / or, (b) has a mutation at one, two, three or more sites selected from the group consisting of Y96, R99, D143, N180, S181, R183, Q184, H195, H217.

[0010] In one or more embodiments, the mutation comprises a mutation at a site selected from any of the following groups: (1) N180 and S181, (2) R183 and Q184; or, the mutation comprises a mutation at a site selected from any of the following groups: (3) Y96, (4) D143, (5) H217, (6) N180, S181 and H217, (7) N180, S181 and D143, (8) (9) N180, S181 and R99, (10) N180, S181 and Y96, (11) N180, S181 and H195, (12) R183, Q184 and H217, (13) R183, Q184 and H217, (14) R183, Q184 and R99, (15) R183, Q184 and D143, (16) R183, Q184 and H195.

[0011] In one or more embodiments, Y96 mutates to I, H, P, or C. In one or more embodiments, R99 mutates to F, P, or C. In one or more embodiments, D143 mutates to A or H. In one or more embodiments, N180 mutates to G, K, T, V, I, M, or W. In one or more embodiments, S181 mutates to R, V, Y, A, P, G, T, S, or D. In one or more embodiments, R183 mutates to W, R, K, S, L, or P. In one or more embodiments, Q184 mutates to V, A, I, W, R, P, or L. In one or more embodiments, H195 mutates to D or S. In one or more embodiments, H217 mutates to Y, P, A, C, or F.

[0012] In one or more embodiments, the mutation comprises mutations selected from any of the following groups: (1) N180G and S181R, (2) N180K and S181V, (3) N180T and S181Y, (4) N180V and S181A, (5) N180G and S181P, (6) N180I and S181A, (7) N180M and S181G, (8) N180V and S181P, (9) N180V and S181R, (10) N180W and S181T, (11) N180M and S181 S, (12) N180W and S181D, (13) R183W and Q184V, (14) R183R and Q184A, (15) R183K and Q184V, (16) R183W and Q184I, (17) R183S and Q184W, (18) R183L and Q184R, (19) R183P and Q184P, (20) R183W and Q184L, (21) Y96I, (22) D143A, (23) D143H, (24) H217A, (25) H217F, (26) N180G, S181R and H217Y, (27) N180G, S181R and D143A, (28) N180G, S181R and R99F, (29) N180I, S181A and R99P, (30) N180I, S181A and D143A, (31) N180I, S181A and H217P, (32) N180I, S181A and H217A, (33) N180I, S181A and Y96H, (34) N180I, S181A and H217C (35) N180I, S181A and H195D, (36) N180I, S181A and Y96H, (37) N180I, S181A and Y96P, (38) R183W, Q184L and H217F, (39) R183W, Q184L and R99C, (40) R183W, Q184L and D143A, (41) R183W, Q184L and Y96C, (42) R183W, Q184L and H195S, (43) R183W, Q184L and H217A.

[0013] Another aspect of the present invention provides a D-2-hydroxyglutaric acid optical probe comprising a D-2-hydroxyglutaric acid-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the D-2-hydroxyglutaric acid-sensitive polypeptide. The D-2-hydroxyglutaric acid-sensitive polypeptide is divided into a first part and a second part by the optically active polypeptide.

[0014] In one or more embodiments, the D-2-hydroxyglutaric acid optical probe includes a D-2-hydroxyglutaric acid-sensitive polypeptide B and an optically active polypeptide A, wherein the optically active polypeptide A is located within the sequence of the D-2-hydroxyglutaric acid-sensitive polypeptide B, and the D-2-hydroxyglutaric acid-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.

[0015] In one or more embodiments, the optically active polypeptide is located at any one or more sites of the D-2-hydroxyglutaric acid-sensitive polypeptide selected from the following sites: 88 / 89, 88 / 90, 87 / 88, 87 / 89, 88 / 89, 89 / 90, 178 / 179, 178 / 180, 178 / 181, 178 / 182, 178 / 183, 179 / 180, 179 / 181, 179 / 182, 179 / 183, 180 / 181, 180 / 182, 180 / 183, 181 / 182, 181 / 183, 182 / 183, 204 / 205, 204 / 206, 204 / 207, 205 / 206, and 206 / 207.

[0016] In one or more embodiments, the D-2-hydroxyglutaric acid sensitive polypeptide is a D-2-hydroxyglutaric acid binding protein or a functional variant thereof.

[0017] In one or more embodiments, the D-2-hydroxyglutaric acid-sensitive polypeptide has:

[0018] (1) The sequence shown in SEQ ID NO:1, or a sequence that has at least 70% sequence identity with them and retains the binding activity to D-2-hydroxyglutaric acid,

[0019] (2) The sequence of the D-2-hydroxyglutaric acid binding protein variant described in any embodiment of the first aspect of this document, or

[0020] (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 D-2-hydroxyglutaric acid.

[0021] In one or more embodiments, the optically active polypeptide is a fluorescent protein or a functional variant thereof, wherein the functional variant of the fluorescent protein has a mutation within 3 amino acids at the linker to the optically active polypeptide.

[0022] In one embodiment, the fluorescent protein is selected from yellow fluorescent protein, green fluorescent protein, blue fluorescent protein, and apple red fluorescent protein. In one embodiment, the fluorescent protein has any of the sequences shown in SEQ ID NO:2-5.

[0023] In one or more embodiments, the functional variant of the fluorescent protein has a mutation at amino acid positions 1-3, preferably position 1. Preferably, the functional variant of the fluorescent protein includes a mutation at amino acid position 1 of the fluorescent protein to P, N, S, T, A or E.

[0024] In one or more embodiments, a functional variant of the fluorescent protein has the sequence shown in SEQ ID NO:2 and a mutation at the Y1 site. Preferably, the mutation is Y1P, Y1N, Y1S, Y1T, Y1A, or Y1E.

[0025] In one or more embodiments, the fluorescent protein has the sequence shown in SEQ ID NO:2 or a variant having a mutation at its first amino acid position of any of the following groups: Y1P, Y1N, Y1S, Y1T, Y1A or Y1E, and the optically active polypeptide is located at position 181 / 183 of the D-2-hydroxyglutaric acid-sensitive polypeptide.

[0026] In one embodiment, 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 embodiment, 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 embodiment, the linker flanking the optically active polypeptide comprises amino acid Y. In one embodiment, linker Y is located at the N-terminus and / or C-terminus of the optically active polypeptide. In one embodiment, the optical probe is as follows: a first portion B1 of the D-2-hydroxyglutaric acid-sensitive polypeptide, Y, optically active polypeptide A, and a second portion B2 of the D-2-hydroxyglutaric acid-sensitive polypeptide. In one embodiment, the optical probe of this invention does not comprise a linker.

[0027] In one embodiment, the optical probe of the present invention further includes a positioning sequence for positioning the probe to a specific organelle, such as a cell.

[0028] In one or more embodiments, the sequence of the D-2-hydroxyglutaric acid-sensitive polypeptide is shown in SEQ ID NO:1, and the optically active polypeptide is shown in SEQ ID NO:1. As shown in any one of NO:2-5, the optically active polypeptide is located at any one or more of the following sites of the D-2-hydroxyglutaric acid-sensitive polypeptide: 88 / 89, 88 / 90, 87 / 88, 87 / 89, 88 / 89, 89 / 90, 178 / 179, 178 / 180, 178 / 181, 178 / 182, 178 / 183, 179 / 180, 179 / 181, 179 / 182, 179 / 183, 180 / 181, 180 / 182, 180 / 183, 181 / 182, 181 / 183, 182 / 183, 204 / 205, 204 / 206, 204 / 207, 205 / 206 and 206 / 207.

[0029] In one or more embodiments, the D-2-hydroxyglutaric acid-sensitive polypeptide is as shown in SEQ ID NO:1, and the optically active polypeptide is as shown in SEQ ID NO:2-5 or a variant thereof having a mutation selected from one or more of the following at the amino acid position corresponding to the first amino acid of SEQ ID NO:2: Y1P, Y1N, Y1S, Y1T, Y1A, or Y1E. The optically active polypeptide is located at one or more of the following sites of the D-2-hydroxyglutaric acid-sensitive polypeptide: 88 / 89, 88 / 90, 87 / 88, 87 / 89, 88 / 89, 89 / 90, 178 / 179, 178 / 18. 0, 178 / 181, 178 / 182, 178 / 183, 179 / 180, 179 / 181, 179 / 182, 179 / 183, 180 / 181, 180 / 182, 180 / 183, 181 / 182, 181 / 183, 182 / 183, 204 / 205, 204 / 206, 204 / 207, 205 / 206 and 206 / 207.

[0030] In one or more embodiments, the optical probe comprises a D-2-hydroxyglutaric acid-sensitive polypeptide as shown in SEQ ID NO:1, and having one or more of the following mutations: Y96, R99, D143, N180, S181, R183, Q184, H195, H217; an optically active polypeptide as shown in SEQ ID NO:2-5 or a variant having a mutation selected from one or more of the following at the amino acid position corresponding to the first amino acid of SEQ ID NO:2: Y1P, Y1N, Y1S, Y1T, Y1A, or Y1E; and an optically active polypeptide located at 88 / 89, 88 / 90, 87 / 88, 87 / 89, 88 / 89, 89 / 90, 178 / 179, 178 / 180, 178 / 181 of the D-2-hydroxyglutaric acid-sensitive polypeptide. The loci are 178 / 182, 178 / 183, 179 / 180, 179 / 181, 179 / 182, 179 / 183, 180 / 181, 180 / 182, 180 / 183, 181 / 182, 181 / 183, 182 / 183, 204 / 205, 204 / 206, 204 / 207, 205 / 206, and 206 / 207.Preferably, the mutations in the D-2-hydroxyglutaric acid-sensitive polypeptide include mutations selected from any of the following groups: (1) N180G and S181R, (2) N180K and S181V, (3) N180T and S181Y, (4) N180V and S181A, (5) N180G and S181P, (6) N180I and S181A, (7) N180M and S181G, (8) N180V and S181P, (9) N180V and S181R, (10) N180W and S181T, (11) N180M and S 181S, (12) N180W and S181D, (13) R183W and Q184V, (14) R183R and Q184A, (15) R183K and Q184V, (16) R183W and Q184I, (17) R183S and Q184W, (18) R183L and Q184R, (19) R183P and Q184P, (20) R183W and Q184L, (21) Y96I, (22) D143A, (23) D143H, (24) H217A, (25) H217 F, (26) N180G, S181R and H217Y, (27) N180G, S181R and D143A, (28) N180G, S181R and R99F, (29) N180I, S181A and R99P, (30) N180I, S181A and D143A, (31) N180I, S181A and H217P, (32) N180I, S181A and H217A, (33) N180I, S181A and Y96H, (34) N180I, S181A and H217 C, (35) N180I, S181A and H195D, (36) N180I, S181A and Y96H, (37) N180I, S181A and Y96P, (38) R183W, Q184L and H217F, (39) R183W, Q184L and R99C, (40) R183W, Q184L and D143A, (41) R183W, Q184L and Y96C, (42) R183W, Q184L and H195S, (43) R183W, Q184L and H217A.

[0031] In one or more embodiments, the optical probe comprises a D-2-hydroxyglutaric acid-sensitive polypeptide as shown in SEQ ID NO:1 and an optically active polypeptide as shown in SEQ ID NO:2-5, wherein the optically active polypeptide is located at position 181 / 183 of the D-2-hydroxyglutaric acid-sensitive polypeptide, and the optical probe has the following mutations: (1) N180G and S181R of the D-2-hydroxyglutaric acid-sensitive polypeptide and 1P of the optically active polypeptide; (2) N180K and S181V of the D-2-hydroxyglutaric acid-sensitive polypeptide and 1N of the optically active polypeptide; (3) N180T and S181Y of the D-2-hydroxyglutaric acid-sensitive polypeptide and 1N of the optically active polypeptide; (4) N180V and S181A of the D-2-hydroxyglutaric acid-sensitive polypeptide and 1S of the optically active polypeptide; (5) D-2-hydroxyglutaric acid-sensitive polypeptide. (6) D-2-hydroxyglutarate-sensitive peptides N180G, S181P, and optically active peptide 1P; (7) D-2-hydroxyglutarate-sensitive peptides N180M, S181G, and optically active peptide 1S; (8) D-2-hydroxyglutarate-sensitive peptides N180V, S181P, and optically active peptide 1P; (9) D-2-hydroxyglutarate-sensitive peptides N180V, S181R, and optically active peptide 1T; (10) D-2-hydroxyglutarate-sensitive peptides N180W, S181T, and optically active peptide 1A; (11) D-2- (12) N180M and S181S of D-2-hydroxyglutaric acid sensitive peptides and 1E of optically active peptides; (13) R183W and Q184V of D-2-hydroxyglutaric acid sensitive peptides; (14) R183D and Q184A of D-2-hydroxyglutaric acid sensitive peptides; (15) R183D and Q184V of D-2-hydroxyglutaric acid sensitive peptides; (16) R183W and Q184I of D-2-hydroxyglutaric acid sensitive peptides; (17) R183S and Q184W of D-2-hydroxyglutaric acid sensitive peptides; (18) D-2-hydroxyglutaric acid sensitive peptides. R183L and Q184R of glutaric acid-sensitive peptides, (19) R183P and Q184P of D-2-hydroxyglutaric acid-sensitive peptides, (20) R183W and Q184L of D-2-hydroxyglutaric acid-sensitive peptides, (21) Y96I of D-2-hydroxyglutaric acid-sensitive peptides, (22) D143A of D-2-hydroxyglutaric acid-sensitive peptides, (23) D143H of D-2-hydroxyglutaric acid-sensitive peptides, (24) H217A of D-2-hydroxyglutaric acid-sensitive peptides, (25) H217F of D-2-hydroxyglutaric acid-sensitive peptides, (26) N180G, S181R, and H217Y of D-2-hydroxyglutaric acid-sensitive peptides,And optically active peptide 1P, (27) D-2-hydroxyglutaric acid sensitive peptide N180G, S181R, D143A, and optically active peptide 1P, (28) D-2-hydroxyglutaric acid sensitive peptide N180G, S181R, R99F, and optically active peptide 1P, (29) D-2-hydroxyglutaric acid sensitive peptide N180I, S181A, R99P, and optically active peptide 1T, (30) D-2-hydroxyglutaric acid sensitive peptide N180I, S181A, D143 A, and 1T of optically active peptides, (31) N180I, S181A, H217P of D-2-hydroxyglutaric acid sensitive peptides, and 1T of optically active peptides, (32) N180I, S181A, H217A of D-2-hydroxyglutaric acid sensitive peptides, and 1T of optically active peptides, (33) N180I, S181A, Y96H of D-2-hydroxyglutaric acid sensitive peptides, and 1T of optically active peptides, (34) N180I, S181A, H of D-2-hydroxyglutaric acid sensitive peptides 217C, and 1T of optically active peptides, (35) N180I, S181A, H195D of D-2-hydroxyglutaric acid sensitive peptides, and 1T of optically active peptides, (36) N180I, S181A, Y96H of D-2-hydroxyglutaric acid sensitive peptides, and 1T of optically active peptides, (37) N180I, S181A, Y96P of D-2-hydroxyglutaric acid sensitive peptides, and 1T of optically active peptides, (38) R183W, Q184L of D-2-hydroxyglutaric acid sensitive peptides. And H217F, (39) R183W, Q184L and R99C of D-2-hydroxyglutaric acid sensitive peptides, (40) R183W, Q184L and D143A of D-2-hydroxyglutaric acid sensitive peptides, (41) R183W, Q184L and Y96C of D-2-hydroxyglutaric acid sensitive peptides, (42) R183W, Q184L and H195S of D-2-hydroxyglutaric acid sensitive peptides, (43) R183W, Q184L and H217A of D-2-hydroxyglutaric acid sensitive peptides.

[0032] The mutated sequences in the embodiments of the present invention are shown in SEQ ID NO:7-49.

[0033] Another aspect of the present invention provides a fusion polypeptide comprising the optical probe described herein and other polypeptides. 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 polypeptides for targeting the optical probe to different organelles or subcellular organelles, tags for purification, or tags for immunoblotting.

[0034] Another aspect of the present invention provides a nucleic acid molecule comprising: (a) a coding sequence of a polypeptide or probe as described in any embodiment herein, or (b) a complementary sequence to (a), or (c) a fragment of (a) or (b). The fragment is a primer.

[0035] The present invention also relates to variants of the aforementioned nucleic acid molecules, including fragments, analogs, derivatives, soluble fragments and variants of the present invention encoding the optical probes or fusion proteins of the present invention, or their complementary sequences.

[0036] In another aspect, the present invention also provides nucleic acid constructs comprising the nucleic acid molecules described herein. The nucleic acid sequence encodes the optical probe or fusion polypeptide described herein.

[0037] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.

[0038] In one or more embodiments, the nucleic acid molecule is operatively linked to an expression control sequence.

[0039] In some implementations, the expression vector is selected from prokaryotic expression vectors, eukaryotic expression vectors, and viral vectors.

[0040] In another aspect, the present invention provides a host cell that: (1) expresses 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*.

[0041] In another aspect, the present invention also provides a D-2-hydroxyglutaric acid detection kit, comprising the optical probes described herein or fusion peptides or polynucleotides or optical probes prepared as described herein.

[0042] In one or more embodiments, the kit further comprises one or more reagents selected from the following: buffer solution, culture medium, and D-2-hydroxyglutaric acid standard.

[0043] Another aspect of 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.

[0044] In one or more embodiments, the method includes the following steps: 1) incorporating a nucleic acid molecule encoding the D-2-hydroxyglutaric acid 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 D-2-hydroxyglutaric acid optical probe.

[0045] Another aspect of the present invention provides a method for detecting D-2-hydroxyglutaric acid 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.

[0046] This article also provides a method for quantifying D-2-hydroxyglutaric acid 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 D-2-hydroxyglutaric acid in the sample based on the optical changes in the optically active peptide.

[0047] 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 D-2-hydroxyglutaric acid; 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.

[0048] In one or more embodiments, the host cells described herein are contacted with the candidate compound in a system containing D-2-hydroxyglutaric acid, and optical changes in the optically active peptide indicate whether the candidate compound can regulate the uptake of D-2-hydroxyglutaric acid by the cells.

[0049] Another aspect of the present invention provides a method for intracellular and / or extracellular localization of the D-2-hydroxyglutaric acid, comprising: contacting a system containing D-2-hydroxyglutaric acid with the optical probe or the host cell, and detecting optical changes in the optically active peptide.

[0050] In one or more embodiments, the system is a solution system, a cellular system, or a subcellular system.

[0051] Another aspect of the present invention provides the application of the D-2-hydroxyglutaric acid optical probe, fusion peptide, or host cell described herein in detecting D-2-hydroxyglutaric acid in a sample, screening compounds, or intracellular / extracellular localization of D-2-hydroxyglutaric acid. In one or more embodiments, the localization is real-time localization.

[0052] The beneficial effects of this invention are as follows: The D-2-hydroxyglutaric acid optical probe provided by this invention is easy to mature, exhibits large fluorescence dynamics, and has good specificity. Furthermore, it can be expressed in cells through gene manipulation, enabling real-time, high-throughput, and quantitative detection of D-2-hydroxyglutaric acid both inside and outside cells, eliminating time-consuming sample processing steps. Experimental results show that the D-2-hydroxyglutaric acid optical probe provided by this application achieves a response to D-2-hydroxyglutaric acid that is more than 5 times that of the control. It can also perform localization, qualitative, and quantitative detection of D-2-hydroxyglutaric acid in subcellular structures such as the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, lysosomes, and Golgi apparatus. Moreover, it allows for high-throughput compound screening and quantitative detection of D-2-hydroxyglutaric acid in blood. Attached Figure Description

[0053] Figure 1 An SDS-PAGE image of an exemplary D-2-hydroxyglutaric acid optical probe;

[0054] Figure 2 Here is a fluorescence spectrum of an exemplary D-2-hydroxyglutaric acid optical probe;

[0055] Figure 3 The titration curves of D-2-hydroxyglutaric acid at different concentrations are shown as an example of the D-2-hydroxyglutaric acid optical probe.

[0056] Figure 4 This is a summary of the specificity of the D-2-hydroxyglutaric acid optical probe for the detection of the other three substrates, sugar metabolism intermediates and similar substances;

[0057] Figure 5 An exemplary photograph of the subcellular organelle localization of the D-2-hydroxyglutaric acid optical probe in mammalian cells;

[0058] Figure 6 This is a schematic diagram illustrating the dynamic monitoring of D-2-hydroxyglutaric acid concentration in the cytoplasm of mammalian cells using an exemplary D-2-hydroxyglutaric acid optical probe.

[0059] Figure 7 A dot plot illustrating high-throughput compound screening at the live-cell level using an exemplary D-2-hydroxyglutaric acid optical probe;

[0060] Figure 8 A bar chart illustrating the quantification of D-2-hydroxyglutaric acid in mouse and human blood using an exemplary D-2-hydroxyglutaric acid optical probe. Detailed Implementation

[0061] 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.

[0062] 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.

[0063] As used herein, the term "D-2-hydroxyglutaric acid-sensitive peptide" refers to a peptide that responds to D-2-hydroxyglutaric acid, and the response includes any response to chemical, biological, electrical, or physiological parameters of the peptide related 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 D-2-hydroxyglutaric acid-sensitive peptides described herein may also include their functional variants. Functional variants of D-2-hydroxyglutaric acid-sensitive peptides include, but are not limited to, variants that can interact with D-2-hydroxyglutaric acid to undergo the same or similar changes as the parental D-2-hydroxyglutaric acid-sensitive peptide.

[0064] The D-2-hydroxyglutaric acid-sensitive polypeptides described in this invention include, but are not limited to, the transcription factor DhdR derived from *Achromobacter denitrificans* NBRC 15125, or variants thereof with more than 90% homology. D-2-hydroxyglutaric acid-binding proteins can sense changes in D-2-hydroxyglutaric acid concentration, and their spatial conformation changes during dynamic changes in D-2-hydroxyglutaric acid concentration. Truncated variants of DhdR can also be used in this invention.

[0065] As used herein, the term "optical probe" refers to a D-2-hydroxyglutaric acid-sensitive polypeptide fused to an optically active polypeptide. The inventors discovered that conformational changes resulting from the specific binding of D-2-hydroxyglutaric acid-sensitive polypeptides, such as D-2-hydroxyglutaric acid-binding proteins, to physiological concentrations of D-2-hydroxyglutaric acid induce conformational changes in optically active polypeptides (e.g., fluorescent proteins), thereby altering the optical properties of the optically active polypeptides. By plotting standard curves using fluorescence data of fluorescent proteins measured at different D-2-hydroxyglutaric acid concentrations, the presence and / or level of D-2-hydroxyglutaric acid can be detected and analyzed. When describing the optical probes of this invention (e.g., when describing insertion or mutation sites), all amino acid residue numbers are referenced to SEQ ID NO:1.

[0066] In the optical probes of the present invention, an optically active polypeptide (e.g., a fluorescent protein) is operatively inserted into a D-2-hydroxyglutaric acid-sensitive polypeptide. A protein-based "optically active polypeptide" is a polypeptide capable of emitting fluorescence. Fluorescence is an optical property of an optically active polypeptide that 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 amplitudes 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 for the utility of the fluorescent protein substrate of the present invention in activity assays. 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 polypeptides described herein may also include functional variants thereof. Functional variants of optically active peptides include, but are not limited to, variants that can undergo changes in fluorescence properties that are the same as or similar to those of the parent optically active peptide.

[0067] As used herein, the term "fluorescent protein" refers to a protein that emits 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. For example, cpYFP is shown in SEQ ID NO:2, cpGFP in SEQ ID NO:3, cpBFP in SEQ ID NO:4, and cpmApple in SEQ ID NO:5.

[0068] The fluorescent protein in the optical probe also includes functional variants with mutations, including but not limited to fluorescent proteins with a mutation at the first amino acid corresponding to SEQ ID NO:2. The mutation at position 1 is preferably P, N, S, T, A, or E. In some embodiments, the functional variant of the fluorescent protein has any of the sequences shown in SEQ ID NO:2-5 and has a mutation at the first amino acid corresponding to SEQ ID NO:2 selected from any of the following groups: Y1P; Y1N; Y1S; Y1T; Y1A, or Y1E. 1S indicates a mutation at position 1 of amino acid S, and so on.

[0069] In the optical probe of the present invention, the optically active polypeptide is a D-2-hydroxyglutaric acid sensitive polypeptide located in the NC direction, with residues 88 / 89, 88 / 90, 87 / 88, 87 / 89, 88 / 89, 89 / 90, 178 / 179, 178 / 180, 178 / 181, 178 / 182, 178 / 183, 179 / 180, 179 / 181, 179 / 182, 179 / 183, 180 / 181, 180 / 182, 180 / 183, 181 / 182, 181 / 183, 182 / 183, 204 / 205, 204 / 206, 204 / 207, 205 / 206, and 206 / 207, or substitutes thereof, with the number corresponding to the full length of the D-2-hydroxyglutaric acid sensitive polypeptide. In this document, if the two numbers in a site represented in "X / Y" form are consecutive integers, it indicates that the optically active polypeptide is located between the amino acids indicated by those numbers. For example, insertion site 88 / 89 indicates that the optically active polypeptide is located between amino acids 88 and 89 of the D-2-hydroxyglutaric acid-sensitive polypeptide. If the two numbers in a site represented in "X / Y" form are not consecutive integers, it indicates that the optically active polypeptide replaces the amino acid between the amino acids indicated by those numbers. For example, insertion site 88 / 90 indicates that the optically active polypeptide replaces amino acid 89 of the D-2-hydroxyglutaric acid-sensitive polypeptide. In an exemplary embodiment, the optically active polypeptide represented by SEQ ID NO:2, 3, 4 or 5 is located at any one or more of the following sites of the D-2-hydroxyglutaric acid-sensitive polypeptide represented by SEQ ID NO:1: 88 / 89, 88 / 90, 87 / 88, 87 / 89, 88 / 89, 89 / 90, 178 / 179, 178 / 180, 178 / 181, 178 / 182, 178 / 183, 179 / 180, 179 / 181, 179 / 182, 179 / 183, 180 / 181, 180 / 182, 180 / 183, 181 / 182, 181 / 183, 182 / 183, 204 / 205, 204 / 206, 204 / 207, 205 / 206 and 206 / 207.

[0070] In this article, "response fold" refers to the standardized 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 ability to the substrate relative to the control, or the greater its responsiveness. For example, in this application, the response fold is calculated by detecting the change in the ratio of fluorescence intensity at 528nm emission from 420nm excitation to fluorescence intensity at 528nm emission from 485nm excitation (Normalized Ratio 420 / 485), as detailed below:

[0071] 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 probe detection signals from parallel experimental groups by the control detection signals to obtain corrected data.

[0072] F = F sample -F BLK

[0073]

[0074] F represents fluorescence intensity. sample F represents the total fluorescence intensity of the sample expressing the fluorescent probe. BLK F represents the background fluorescence intensity of samples without expressed fluorescent probes. cpYFP This indicates the fluorescence intensity of the sample used as a pH control. 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 ratiosensor represents the ratio of the probe's fluorescence intensity. cpYFP This represents the ratio of the fluorescence intensity of the corresponding probe to the pH control fluorescent protein. (Normalized Ratio) 420 / 485 It represents the ratio of the probe change or the response. (Normalized Ratio) 485 / 420 The greater the deviation from 1 (whether it increases or decreases), the greater the change factor or response factor of the probe.

[0075] 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. Variants of polypeptides or proteins may include: homologous sequences, conserved variants, allelic variants, natural mutants, and induced mutants. These variants include, but are not limited to: deletions, insertions, and / or substitutions of one or more (typically 1-30, preferably 1-20, more preferably 1-10, most preferably 1-5) amino acids in the sequence of the polypeptide or protein, and sequences obtained by adding one or more (typically up to 20, preferably up to 10, more preferably up to 5) amino acids to its carboxyl terminus and / or amino terminus. 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. We do not wish to be limited by theory; rather, we seek to alter amino acid residues without changing the overall conformation and function of the polypeptide or protein—that is, functionally conserved mutations. For example, in this field, substitution with amino acids of similar or comparable properties typically does not change the function of the polypeptide or protein. In this field, amino acids of similar properties often refer to amino acid families with similar side chains, which are well-defined. These families include amino acids with basic side chains (e.g., lysine, D-2-hydroxyglutarate, 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, D-2-hydroxyglutarate, phenylalanine, methionine, D-2-hydroxyglutarate), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, D-2-hydroxyglutarate, histidine). For example, adding one or more amino acids to the amino terminus and / or carboxyl terminus generally does not alter the function of the 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.

[0076] "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 D-2-hydroxyglutaric acid-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.

[0077] The inventors have discovered that D-2-hydroxyglutaric acid binding protein variants with mutations at sites selected from the following sites exhibit different binding activities than D-2-hydroxyglutaric acid: Y96, R99, D143, N180, S181, R183, Q184, H195, and H217 of SEQ ID NO:1. The amino acid mutations include modifications, substitutions, or deletions of amino acids.

[0078] This invention provides D-2-hydroxyglutarate-binding protein variants having these mutations and optical probes comprising such D-2-hydroxyglutarate-binding protein variants as D-2-hydroxyglutarate-sensitive peptides. Therefore, in one or more embodiments, the D-2-hydroxyglutarate-sensitive peptide in the optical probe is a D-2-hydroxyglutarate-binding protein variant as described in any embodiment herein, and the fluorescent protein in the optical probe is as shown in SEQ ID NO:2-5 or a functional variant thereof.

[0079] In some specific embodiments, the D-2-hydroxyglutaric acid-sensitive polypeptide in the optical probe is shown in SEQ ID NO:1, and the optically active polypeptide is shown in SEQ ID NO:1. As shown in NO:2, the optically active peptides are located at sites 88 / 89, 88 / 90, 87 / 88, 87 / 89, 88 / 89, 89 / 90, 178 / 179, 178 / 180, 178 / 181, 178 / 182, 178 / 183, 179 / 180, 179 / 181, 179 / 182, 179 / 183, 180 / 181, 180 / 182, 180 / 183, 181 / 182, 181 / 183, 182 / 183, 204 / 205, 204 / 206, 204 / 207, 205 / 206, and 206 / 207 of the D-2-hydroxyglutaric acid-sensitive peptide. Mutations of the optical probes are shown in any row of Tables 5 and 6.

[0080] 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.

[0081] 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 certain 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 such as factor Xa, thrombin, or enterokinase.

[0082] As used herein, the terms “functional fragment,” “derivative,” and “analyte” refer to a protein that substantially retains the same biological function or activity as the original polypeptide or protein (e.g., D-2-hydroxyglutarate-binding protein or fluorescent protein). Functional variants, derivatives, or analogs of the polypeptides or proteins of the present invention (e.g., D-2-hydroxyglutarate-binding protein or fluorescent protein) 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. 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 D-2-hydroxyglutaric acid-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.

[0083] The fusion polypeptide of this invention comprises the optical probe described herein and other polypeptides. 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. The other polypeptides may be located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptides include polypeptides for targeting the optical probe to different organelles or subcellular organelles, tags for purification, or tags for immunoblotting. A linker may be present between the optical probe and other polypeptides in the fusion polypeptide described herein.

[0084] The subcellular organelles described herein include the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, Golgi apparatus, lysosomes, and peroxisomes. In some embodiments, the tags used for purification or for immunoblotting include 6-histidine (6*His), glutathione S-transferase (GST), and Flag.

[0085] This invention comprises nucleic acid molecules encoding the D-2-hydroxyglutaric acid sensitive polypeptide or optical probe described herein. The terms "nucleic acid," "nucleotide," "polynucleotide," or "nucleic acid sequence" as used herein can be in DNA or RNA form. 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 is 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 acid of this invention may comprise a nucleotide sequence with at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the nucleic acid sequence described herein. This invention also relates to nucleic acid fragments that hybridize with the above-described sequences. As used herein, a "nucleic acid fragment" contains at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. The nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR).

[0086] The full-length sequence or fragment of the optical probe or fusion protein of this invention can typically be obtained by PCR amplification, artificial synthesis, or recombinant methods. The steps and reagents used in conventional PCR, synthesis, and recombinant methods are known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through methods such as mutagenic PCR or chemical synthesis.

[0087] This invention also relates to nucleic acid constructs containing the polynucleotides described herein, and one or more regulatory sequences operatively linked to these sequences. The polynucleotides described herein can be manipulated in various ways to ensure the expression of the polypeptide or protein. The nucleic acid constructs can be manipulated before insertion into a vector, depending on the expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0088] In some embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a homologous recombination vector. The polynucleotides of the present invention can be cloned into many types of vectors, such as plasmids, phage particles, phage derivatives, animal viruses, and entrapments.

[0089] Typical expression vectors contain expression control sequences that can be used to regulate the expression of a desired nucleic acid sequence, operatively linked to the nucleic acid sequence described herein or its complement. As used herein, the term "expression control sequence" 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. In recombinant expression vectors, "operative linking" refers to the linking of the target nucleotide sequence to the regulatory sequence in a manner that allows the nucleotide sequence to be expressed. 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 techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, the LTR of retroviruses, 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 may 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 EcoRI, 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.

[0090] 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. The host cell is preferably a variety of cells conducive to gene product expression or fermentation production, such cells being well known and commonly used in the art. Specifically, it can be bacterial cells of *Escherichia coli*, *Streptomyces*, *Salmonella typhimurium*, fungal cells such as yeast, plant cells, insect cells of *Drosophila S2* or *Sf9*, animal cells such as CHO, COS, HEK293, HeLa cells, or Bowes melanoma cells, etc. The exemplary host cell used in the embodiments of this invention is *Escherichia coli* strain BL21-DE3. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

[0091] 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.

[0092] This invention involves transforming an expression vector into host cells, followed by amplification and expression culture of the host cells to isolate the D-2-hydroxyglutaric acid 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.

[0093] 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 D-2-hydroxyglutaric acid 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, percolation, 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.

[0094] This invention also provides the application of the D-2-hydroxyglutaric acid optical probe in real-time localization, quantitative detection, and high-throughput compound screening of D-2-hydroxyglutaric acid. In one aspect, the D-2-hydroxyglutaric acid optical probe is preferably linked to signal peptides at different sites within the cell and transferred into the cell. Real-time localization of D-2-hydroxyglutaric acid is achieved by detecting the intensity of fluorescence signals within the cell. Quantitative detection of D-2-hydroxyglutaric acid is then performed by combining a standard D-2-hydroxyglutaric acid titration curve with changes in fluorescence signals. Changes in fluorescence signals are displayed, for example, by a standardized fluorescence signal ratio. In embodiments involving cpYFP, ​​this ratio is the ratio of the 485 nm to 420 nm fluorescence signals of the sample to the corresponding ratio of the control. The D-2-hydroxyglutaric acid standard titration curve of this invention is plotted based on the fluorescence signals of the D-2-hydroxyglutaric acid optical probe at different concentrations of D-2-hydroxyglutaric acid. The D-2-hydroxyglutaric acid optical probe of this invention is directly transferred into cells, eliminating the need for time-consuming sample processing during real-time localization and quantification of D-2-hydroxyglutaric acid, thus improving accuracy. In high-throughput compound screening, different compounds are added to cell culture medium, and changes in D-2-hydroxyglutaric acid levels are measured to identify compounds that influence these changes. The application of the D-2-hydroxyglutaric acid optical probe in real-time localization, quantification, and high-throughput compound screening of D-2-hydroxyglutaric acid in this invention is for non-diagnostic and non-therapeutic purposes and does not involve the diagnosis or treatment of diseases.

[0095] This invention also provides a detection kit comprising the optical probes, nucleic acid molecules, nucleic acid constructs, and / or cells described herein. The kit further comprises other reagents required for the detection of D-2-hydroxyglutaric acid. These other reagents are well known in the art, such as buffers, cell culture media, and D-2-hydroxyglutaric acid standards. Exemplary buffers include, for example, 100 mM HEPES and 100 mM NaCl, pH 7.4.

[0096] 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.

[0097] Example

[0098] The following detailed description of the D-2-hydroxyglutaric acid optical probe provided by the present invention, in conjunction with embodiments, should not be construed as limiting the scope of protection of the present invention.

[0099] I. Experimental Materials and Reagents

[0100] The embodiments mainly 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 Rothcams et al.'s "Molecular Biology Laboratory Reference Manual," J. Sambrook and DW Russell's "Molecular Cloning: A Laboratory Manual" (3rd edition, August 2002, Science Press, Beijing); Fereschney et al.'s "Animal Cell Culture: A Basic Technical Guide" (5th edition), translated by Zhang Jingbo and 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.

[0101] The pCDF-cpYFP and pCDF-D-2-hydroxyglutarate 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. and BGI Genomics. The expression plasmids constructed in the examples were all sequenced, and the sequencing was performed by BGI Genomics and J. Lee Sequencing. The Taq DNA polymerase used in each example was purchased from Dongsheng Biotechnology, the pfu DNA polymerase was purchased from Tiangen Biotech (Beijing) Co., Ltd., and the primeSTAR DNA polymerase was purchased from TaKaRa. The corresponding polymerase buffer and dNTPs were included with the purchase of the three polymerases. Restriction endonucleases such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, and SpeI, T4 ligase, and T4 phosphorylase (T4 PNK) were purchased from Fermentas, and the corresponding buffers were included with the purchase. The Lip2000 transfection kit was purchased from Invitrogen. Compounds such as D-2-hydroxyglutaric acid were purchased from Sigma-Aldrich. Unless otherwise stated, inorganic salts and other chemical reagents were purchased from Sigma-Aldrich. HEPES salts, ampicillin (Amp), and puromycin were purchased from Amersco. The 96-well detection blackboard and the 384-well fluorescence detection blackboard were purchased from Grenier.

[0102] 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.

[0103] 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 Biotechnology Co., Ltd.), nucleic acid electrophoresis apparatus (Shanghai Shenneng Bocai Biotechnology Co., Ltd.), fluorescence spectrophotometer (Varian, USA), CO2 constant temperature cell incubator (SANYO), and inverted fluorescence microscope (Nikon, Japan).

[0104] II. Molecular Biology Methods and Cellular Experimental Methods

[0105] II.1 Polymerase Chain Reaction (PCR):

[0106] 1. PCR amplification of the target fragment:

[0107] 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.

[0108] 2. Long fragment (>2500bp) amplification PCR:

[0109] 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.

[0110] II.2 Endonuclease digestion reaction:

[0111] The double enzyme digestion system for the plasmid vector is as follows: 20 μL plasmid vector (approximately 1.5 μg), 5 μL 10× buffer, 11-2 μL restriction endonuclease, 21-2 μL restriction endonuclease, and bring the total volume to 50 μL with sterile ultrapure water. Reaction conditions: 37℃, 1-7 hours.

[0112] II.3 Phosphorylation of DNA fragments at the 5' end

[0113] 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.

[0114] II.4 Ligation reaction between target fragment and vector

[0115] The methods for connecting different fragments and carriers vary, and this invention uses three connection methods.

[0116] 1. Blunt-end junctions of blunt-ended short fragments and linearized vectors

[0117] 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.

[0118] 2. Ligation of DNA fragments with sticky ends and vector fragments with sticky ends.

[0119] DNA fragments digested with restriction endonucleases typically produce prominent sticky ends, which can then be ligated to vector fragments containing sequence-complementary sticky ends 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°C for 4-8 hours.

[0120] 3. Ligation reaction involving the self-circularization of DNA fragment products phosphorylated at the 5' end following site-directed mutagenesis using reverse PCR.

[0121] 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.

[0122] II.5 Preparation and Transformation of Competent Cells

[0123] Preparation of competent cells:

[0124] 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.

[0125] 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.

[0126] 3. Pre-cool the cells in an ice bath for 2 hours.

[0127] Centrifuge at 4.4℃ and 4000rpm for 10 minutes.

[0128] 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.

[0129] 6. Ice bath for 45 minutes.

[0130] Centrifuge at 7.4℃ and 4000rpm for 10 minutes, then resuspend the bacteria in 5mL of ice-cold storage buffer.

[0131] 8. Place 100 μL of bacterial culture in each EP tube and store at -80°C or in liquid nitrogen.

[0132] Resuspension buffer: CaCl2 (100mM), MgCl2 (70mM), NaAc (40mM)

[0133] 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

[0134] Transformation of competent cells:

[0135] 1. Take 100 μL of competent cells and thaw them on an ice bath.

[0136] 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.

[0137] 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.

[0138] 4. Add 500 μL LB and incubate at 200 rpm for 1 hour on a constant temperature shaker at 37℃.

[0139] 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.

[0140] II.6 Protein Expression, Purification, and Fluorescence Detection

[0141] 1. Transform the expression vector (e.g., the pCDF-based D-2-hydroxyglutaric acid 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 shaker at 37℃ 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.

[0142] 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.

[0143] 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).

[0144] 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. D-2-hydroxyglutaric acid was prepared into a stock solution with a final concentration of 50mM using assay buffer (100mM HEPES, 100mM NaCl, pH 7.4).

[0145] 5. Take 100 μL of 1 μM protein solution, incubate at 37℃ for 10 minutes, add D-2-hydroxyglutaric acid 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.

[0146] 6. Take 100 μL of 1 μM protein solution, incubate at 37℃ for 10 minutes, add D-2-hydroxyglutaric acid, 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.

[0147] II.7 Transfection and Fluorescence Detection of Mammalian Cells

[0148] 1. The pCDNA3.1+-based D-2-hydroxyglutaric acid optical probe plasmid was transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen) 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.

[0149] 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.

[0150] Example 1: D-2-hydroxyglutaric acid binds to protein particles

[0151] The gene from *Achromobacterium denitrifyingus*, with the DNA domains from positions 2-81 of DhdR removed, was amplified by PCR. The PCR product was recovered after gel electrophoresis and digested with HindIII and XhoI enzymes. The pCDF vector was also double-digested with the corresponding enzymes. Ligation was performed using T4 DNA ligase, and 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 successful sequencing, were used for subsequent plasmid construction.

[0152] Example 2: Expression and detection of cpYFP optical probes at different insertion sites

[0153] In this embodiment, the following sites are selected based on pCDF-DhdR to insert cpYFP, ​​resulting in the corresponding pCDF-DhdR-cpYFP plasmids: 88 / 89, 88 / 90, 87 / 88, 87 / 89, 88 / 89, 89 / 90, 178 / 179, 178 / 180, 178 / 181, 178 / 182, 178 / 183, 179 / 180, 179 / 181, 179 / 182, 179 / 183, 180 / 181, 180 / 182, 180 / 183, 181 / 182, 181 / 183, 182 / 183, 204 / 205, 204 / 206, 204 / 207, 205 / 206, and 206 / 207. As an example only, the amino acid sequence of 181 / 183-DhdR-cpYFP is shown in SEQ ID NO:6.

[0154] A DNA fragment of cpYFP was generated using PCR. Simultaneously, a homologous sequence from the cpYFP terminal was introduced at the 5' end using primers. PCR amplification produced a linearized pCDF-D-2-hydroxyglutaric acid binding protein vector, whose 5' and 3' ends contained sequences completely identical to those at the two ends of cpYFP (15 bp–20 bp). The linearized pCDF-DhdR and cpYFP fragments underwent homologous recombination using HieffClone Enzyme. The product was transformed into DH5α, and the transformed DH5α was plated on LB agar plates (streptomycin 100 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 Sequencing.

[0155] 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 50 kDa. This size is consistent with the size of the pCDF-DhdR-cpYFP fusion protein containing the His-tag purified label. Results are as follows... Figure 1 As shown.

[0156] The supernatant of fragmented *E. coli* expressing the DhdR-cpYFP fusion protein was used for screening for D-2-hydroxyglutaric acid (D-2-hydroxyglutaric acid) response. The detection signal of the fusion fluorescent protein containing 1 Mm of D-2-hydroxyglutaric acid was divided by the detection signal of the fusion fluorescent protein without D-2-hydroxyglutaric acid. As shown in Table 1, the detection results showed that optical probes expressing the DhdR-cpYFP fusion protein showed a D-2-hydroxyglutaric acid response greater than 1.2 times at sites 181 / 182, 181 / 183, and 182 / 183. Among these, sites 181 / 183 showed a D-2-hydroxyglutaric acid response unaffected by the binding of other DhdR substrates.

[0157] Table 1

[0158]

[0159] Example 3: Expression and detection of cpGFP optical probes at different insertion sites

[0160] Following the method described in Example 2, cpYFP was replaced with cpGFP to construct a D-2-hydroxyglutaric acid green fluorescent protein probe. As shown in Table 2, the detection results showed that optical probes expressing the DhdR-cpYFP fusion protein in the fragmented supernatant exhibited a response to D-2-hydroxyglutaric acid exceeding 1.2-fold. Optical probes inserted at sites 181 / 182, 181 / 183, and 182 / 183 were included in this study. Among these, sites 181 / 183 showed a response to D-2-hydroxyglutaric acid unaffected by the binding of other DhdR substrates.

[0161] Table 2

[0162]

[0163] Example 4: Expression and detection of cpBFP optical probes at different insertion sites

[0164] Following the method described in Example 2, cpYFP was replaced with cpBFP to construct a blue fluorescent protein probe for D-2-hydroxyglutaric acid. As shown in Table 3, the detection results showed that optical probes expressing the DhdR-cpYFP fusion protein in the fragmented supernatant exhibited a response to D-2-hydroxyglutaric acid exceeding 1.2-fold. Optical probes inserted at sites 181 / 182, 181 / 183, and 182 / 183 were included in this study. Among these, sites 181 / 183 showed a response to D-2-hydroxyglutaric acid unaffected by the binding of other DhdR substrates.

[0165] Table 3

[0166]

[0167] Example 5: Expression and detection of cpmApple optical probes at different insertion sites

[0168] Following the method described in Example 2, cpYFP was replaced with cpmApple to construct a D-2-hydroxyglutaric acid red fluorescent protein probe. As shown in Table 4, the detection results showed that optical probes expressing the DhdR-cpYFP fusion protein in the fragmented supernatant exhibited a response to D-2-hydroxyglutaric acid exceeding 1.2-fold. Optical probes inserted at sites 181 / 182, 181 / 183, and 182 / 183 were included in this study. Among these, sites 181 / 183 showed a response to D-2-hydroxyglutaric acid unaffected by the binding of other DhdR substrates.

[0169] Table 4

[0170]

[0171] Example 6: Expression and detection of linker-mutated cpYFP optical probes

[0172] For the optical probe obtained in Example 2 that responds to D-2-HG more than 1.2 times and is unaffected by other D-2-HG binding substrates, i.e., the optical probe inserted at the 181 / 183 site of DhdR, the probe was linearized by reverse PCR, and the linker mutation site sequence was introduced into the primers. The resulting PCR product was then subjected to homologous recombination under the action of Hieff Clone Enzyme to establish a mutant library. As an example only, the amino acid sequence of 181 / 183-DhdR-N180G / S181R-cpYFP-Y1P is shown in SEQ ID NO:7.

[0173] The recombinant plasmid of the mutant library was transformed into BL21(DE3) to induce expression. The probe proteins were purified and screened for D-2-HG response. The detection signal of the fusion fluorescent protein containing 2 mM D-2-HG was divided by the detection signal of the fusion fluorescent protein without D-2-HG. The detection results show that optical probes with a D-2-HG response greater than or equal to 2 are listed in Table 5.

[0174] Table 5

[0175] Insertion site DhdR cpYFP <![CDATA[R 485 / 420 ]]> Kd(μM) 181 / 183 1.51 535.42 181 / 183 N180G / S181R Y1P 4.51 10.96 181 / 183 N180K / S181V Y1N 3.97 46.52 181 / 183 N180T / S181Y Y1N 3.35 14.84 181 / 183 N180V / S181A Y1S 3.53 5.22 181 / 183 N180G / S181P Y1P 3.75 15.26 181 / 183 N180I / S181A Y1T 4.91 17.32 181 / 183 N180M / S181G Y1S 3.92 72.4 181 / 183 N180V / S181P Y1P 3.75 34.12 181 / 183 N180V / S181R Y1T 3.4 108 181 / 183 N180W / S181T Y1A 4.77 60.53 181 / 183 N180M / S181S Y1E 3.43 32.25 181 / 183 N180W / S181D Y1T 4.54 24.93 181 / 183 R183W / Q184V 4.45 68.44 181 / 183 R183R / Q184A 4.83 66.54 181 / 183 R183K / Q184V 3.05 132.7 181 / 183 R183W / Q184I 5.31 123.9 181 / 183 R183S / Q184W 3.49 88.85 181 / 183 R183L / Q184R 3.47 65.83 181 / 183 R183P / Q184P 4.52 68.5 181 / 183 R183W / Q184L 5.95 109.7

[0176] Example 7: Expression and detection of cpYFP optical probes with pocket mutations

[0177] For the optical probes 181 / 183-DhdR-cpYFP obtained in Example 2 and 181 / 183-DhdR-N180G / S181R-cpYFP-Y1P, 181 / 183-DhdR-N180I / S181A-cpYFP-Y1T and 181 / 183-DhdR-R183W / Q184L-cpYFP obtained in Example 6, amino acid mutations were performed at positions Y96, R99, D143, H195, H217, and R224 in the D-2-hydroxyglutaric acid binding pocket. The probes were linearized using reverse PCR, introducing the mutated sequences into the primers. Homologous recombination was then performed on the resulting PCR products to obtain plasmids containing the mutations at the above sites. The mutant plasmid was transformed into BL21(DE3) to induce expression. The mutant probe protein was purified and subjected to a D-2-hydroxyglutaric acid response test. The detection signal of the fusion fluorescent protein containing 10 mM D-2-hydroxyglutaric acid was divided by the detection signal of the fusion fluorescent protein without D-2-hydroxyglutaric acid. The detection results show that the optical probes that responded to D-2-hydroxyglutaric acid by more than 3 times are listed in Table 6.

[0178] Table 6

[0179] Insertion site DhdR mutation cpYFP mutation R485 / 420 Kd(μM) 181 / 183 Y96I 4.02 21.38 181 / 183 D143A 3.77 11.96 181 / 183 D143H 3.56 106.4 181 / 183 H217A 3.46 136.4 181 / 183 H217F 3.45 238.7 181 / 183 N180G / S181R / H217Y Y1P 4.43 23.56 181 / 183 N180G / S181R / D143A Y1P 3.65 57.29 181 / 183 N180G / S181R / R99F Y1P 5.62 168.9 181 / 183 N180I / S181A / R99P Y1T 3.84 87.57 181 / 183 N180I / S181A / D143A Y1T 4.10 20.91 181 / 183 N180I / S181A / H217P Y1T 5.31 203.3 181 / 183 N180I / S181A / H217A Y1T 3.43 14.89 181 / 183 N180I / S181A / Y96H Y1T 3.78 78.56 181 / 183 N180I / S181A / H217C Y1T 3.38 78.25 181 / 183 N180I / S181A / H195D Y1T 3.51 42.97 181 / 183 N180I / S181A / Y96H Y1T 3.82 84.21 181 / 183 N180I / S181A / Y96P Y1T 5.01 32.53 181 / 183 R183W / Q184L / H217F 4.09 56.55 181 / 183 R183W / Q184L / R99C 3.54 18.83 181 / 183 R183W / Q184L / D143A 4.00 59.21 181 / 183 R183W / Q184L / Y96C 3.95 42.57 181 / 183 R183W / Q184L / H195S 3.89 57.23 181 / 183 R183W / Q184L / H217A 5.8 664.43

[0180] Example 8. Performance of optical probe mutants

[0181] For example, the two D-2-hydroxyglutaric acid optical probes described in Examples 6 and 7 were treated with 0 mM and 5 mM D-2-hydroxyglutaric acid for 10 minutes, respectively, and then the fluorescence spectra were detected using a fluorescence spectrophotometer.

[0182] Excitation spectrum determination: Excitation spectra were recorded with an excitation range of 400 nm to 500 nm and an emission wavelength of 530 nm, read every 5 nm. The results showed that the probe exhibited two excitation peaks at approximately 420 and 490 nm, as... Figure 2 As shown.

[0183] Emission spectrum determination: With fixed excitation wavelengths of 420 nm and 490 nm, emission spectra were recorded at 500-600 nm and 500-600 nm, read every 5 nm. Results are as follows: Figure 2 As shown.

[0184] The 44 purified D-2-hydroxyglutaric acid probes described in Examples 6 and 7 were subjected to D-2-hydroxyglutaric acid detection at concentration gradients (0–10 mM). After treating the purified 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 results are as follows: Figure 3As shown, the Kd (binding constant) of the 44 D-2-hydroxyglutaric acid optical probes are 535.42, 10.96, 46.52, 14.84, 5.22, 15.26, 17.32, 72.4, 34.12, 108, 60.53, 32.25, 24.93, 68.44, 66.54, 132.7, 123.9, 88.85, 65.83, 68.5, and 68, respectively. 0.5, 109.7, 21.38, 11.96, 106.4, 136.4, 238.7, 23.56, 57.29, 168.9, 87.57, 20.91, 203.3, 14.89, 78.56, 78.25, 42.97, 84.21, 32.53, 56.55, 18.83, 59.21, 42.57, 57.23 and 664.43 μM.

[0185] Forty-four D-2-hydroxyglutaric acid optical probes were tested for reactivity with three DhdR binding substrates (L-2-HG, 2-HG, and α-KG) at 10 mM. Simultaneously, one D-2-hydroxyglutaric acid optical probe was tested for reactivity with sugar metabolism intermediates such as glutaric acid, pyruvate, and citrate at 10 mM. The results showed that it possessed good specificity. Figure 4 As shown.

[0186] Example 9: Subcellular organelle localization of optical probes and performance of optical probes within subcellular organelles

[0187] In this embodiment, different localization signal peptides are fused with the optical probe 181 / 183-DhdR-R183W / Q184L / H217A-cpYFP to localize the optical probe to different organelles.

[0188] After transfecting 293 cells with optical probe plasmids fused with different localization signal peptides for 36 hours, the cells were 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 5 As shown, the D-2-hydroxyglutaric acid optical probe can be localized to subcellular organelles, including the cytoplasm, nucleus, mitochondria, and nuclear exclusion structures, by fusing with different specific localization signal peptides. Fluorescence was observed in all different subcellular structures, and the distribution and intensity of the fluorescence varied.

[0189] Thirty-six hours after transfecting 293 cells with a cytoplasmic optical probe plasmid, the cells were washed with PBS and placed in HBSS solution. The ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to fluorescence intensity at 485 nm excitation and 528 nm emission was detected over a 30-minute period. The results are as follows: Figure 6As shown, 1 mM D-2-HG was added, and the assay continued for 30 minutes. The 485 / 420 of the sample with added D-2-HG gradually increased, reaching a maximum of 7 times the initial value.

[0190] Example 10: High-throughput compound screening based on optical probes in living cells

[0191] In this embodiment, we used HeLa cells with cytoplasmic expression of 181 / 183-DhdR-R183W / Q184L / H217A-cpYFP for high-throughput compound screening.

[0192] Transfected 293 cells were washed with PBS, treated with HBSS solution (without D-2-hydroxyglutaric acid) for 1 hour, and then treated with 10 μM of the compound for 1 hour. D-2-hydroxyglutaric acid 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 7 As shown in the figure, among the 2000 compounds used, the vast majority had minimal effect on the entry of D-2-hydroxyglutaric acid into cells. Twenty-eight compounds were able to increase the cellular uptake of D-2-hydroxyglutaric acid, while another 21 compounds significantly reduced it.

[0193] Example 11: Quantitative detection of D-2-hydroxyglutaric acid in blood using optical probes

[0194] In this embodiment, purified Kd 15 μM 181 / 183-DhdR-R183W / Q184L-cpYFP was used to analyze D-2-HG in the blood supernatant of mice and humans.

[0195] After mixing 181 / 183-DhdR-R183W / Q184L-cpYFP 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 8 As shown, the D-2-HG content in mouse blood is around 79 μM, while the D-2-HG content in human blood is around 58 μM.

[0196] As can be seen from the above embodiments, the D-2-HG 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 locate and quantify D-2-HG in and out of cells in real time and can also perform high-throughput compound screening.

[0197] Other implementation methods

[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] Part of the sequence in this article

[0200] 1>DhdR (1-238 full length)

[0201] MLSKSLTLTEQVARQIAGDIAEGVHSVGAKLPPGRVLAEQYGVSAAVIREATERLRAQGLIQSRQGSGSVVVSRTGAQGFQVSAGLDDREQLASVYELRMELEGGAAALAARRRNATDL AAMAEALAALEANLDHPEQGVEHDIAFHVAIAAATHNRYYQDLLQYLNLQLRLAVSTARTNSRRQEGLTAVVHQEHVAVYDAILAGDPDRARLAATRHLQQAASRLRLDLLSPAARQTS

[0202] 2>cpYFP

[0203] YNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGD VNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

[0204] 3>cpGFP

[0205] NVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICCTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKIDFKEDGNILGHKLEYN

[0206] 4>cpBFP

[0207] NVYIKADKQKNGIKANFKIRHNIEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSESMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPGGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKIDFKEDGNILGHKLEYN

[0208] 5>cpmApple

[0209] VSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHSTGGMDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEA

[0210] 6>181 / 183-DhdR-cpYFP

[0211] MVSAGLDDREQLASVYELRMELEGGAAALAARRRNATDLAAMAEALAALEANLDHPEQGVEHDIAFHVAIAAATHNRYYQDLLQYLNLQLRLAVSTARTNSYNSDNVYIMADKQKNGICANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNRQEGLTAVVHQEHVAVYDAILAGDPDRARLAATRHLQQAASRRLDLLSPAARQTS

[0212] 7>181 / 183-DhdR-N180G / S181R-cpYFP-Y1P

[0213] MVSAGLDDREQLASVYELRMELEGGAAALAARRRNATDLAAMAEALAALEANLDHPEQGVEHDIAFHVAIAAATHNRYYQDLLQYLNLQLRLAVSTARTGRPNSDNVYIMADKQKNGICANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNRQEGLTAVVHQEHVAVYDAILAGDPDRARLAATRHLQQAASRRLDLLSPAARQTS

[0214] 20>181 / 183-DhdR-R183R / Q184A-cpYFP

[0215] MVSAGLDDREQLASVYELRMELEGGAAALAARRRNATDLAAMAEALAALEANLDHPEQGVEHDIAFHVAIAAATHNRYYQDLLQYLNLQLRLAVSTARTRAYNSDNVYIMADKQKNGICANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNRQEGLTAVVHQEHVAVYDAILAGDPDRARLAATRHLQQAASRRLDLLSPAARQTS.

Claims

1. An optical probe comprising a D-2-hydroxyglutaric acid-sensitive polypeptide and an optically active polypeptide, wherein, The D-2-hydroxyglutaric acid-sensitive polypeptide is a DhdR protein or a variant thereof, the optically active polypeptide is a fluorescent protein or a variant thereof, and the optically active polypeptide is located at one or more sites selected from the following: 88 / 89, 88 / 90, 87 / 88, 87 / 89, 88 / 89, 89 / 90, 178 / 179, 178 / 180, 178 / 181, 178 / 182, 178 / 183, 179 / 180, 179 / 181, 179 / 182, 179 / 183, 180 / 181, 180 / 182, 180 / 183, 181 / 182, 181 / 183, 182 / 183, 204 / 205, 204 / 206, 204 / 207, 205 / 206, and 206 / 207, wherein the DhdR protein has the sequence shown in SEQ ID NO:1 or a functional fragment thereof, and variants of the DhdR protein contain mutations selected from the following sites: Y96, R99, D143, N180, S181, R183, Q184, H195, H217, and variants of the fluorescent protein contain a mutation at the Y1 site. Preferably, the optically active polypeptide is selected from any one of the following: cpYFP, ​​cpGFP, cpBFP, and cpmApple.

2. The optical probe as described in claim 1, characterized in that, The fluorescent protein is as shown in any one of SEQ ID NO:2-5, and the Y1 variant of the fluorescent protein is mutated to P, N, S, T, A or E.

3. The optical probe as described in claim 1, characterized in that, Among the variants of the DhdR protein, Y96 is mutated to I, H, P, or C; R99 is mutated to F, P, or C; D143 is mutated to A or H; N180 is mutated to G, K, T, V, I, M, or W; S181 is mutated to R, V, Y, A, P, G, T, S, or D; R183 is mutated to W, R, K, S, L, or P; Q184 is mutated to V, A, I, W, R, P, or L; H195 is mutated to D or S; and H217 is mutated to Y, P, A, C, or F. Preferably, the mutations in the variants of the DhdR protein include mutations selected from any of the following groups: (1) N180G and S181R, (2) N180K and S181V, (3) N180T and S181Y, (4) N180V and S181A, (5) N180G and S181P, (6) N180I and S181A, (7) N180M and S181G, (8) N180V and S181P, (9) N180V and S181R, (10) N180W and S181T, (11) N180M and S1 81S, (12) N180W and S181D, (13) R183W and Q184V, (14) R183R and Q184A, (15) R183K and Q184V, (16) R183W and Q184I, (17) R183S and Q184W, (18) R183L and Q184R, (19) R183P and Q184P, (20) R183W and Q184L, (21) Y96I, (22) D143A, (23) D143H, (24) H217A, (25) H217 F, (26) N180G, S181R and H217Y, (27) N180G, S181R and D143A, (28) N180G, S181R and R99F, (29) N180I, S181A and R99P, (30) N180I, S181A and D143A, (31) N180I, S181A and H217P, (32) N180I, S181A and H217A, (33) N180I, S181A and Y96H, (34) N180I, S181A and H217 C, (35) N180I, S181A and H195D, (36) N180I, S181A and Y96H, (37) N180I, S181A and Y96P, (38) R183W, Q184L and H217F, (39) R183W, Q184L and R99C, (40) R183W, Q184L and D143A, (41) R183W, Q184L and Y96C, (42) R183W, Q184L and H195S, (43) R183W, Q184L and H217A.

4. The optical probe as described in claim 1, characterized in that, The sequence of the D-2-hydroxyglutaric acid-sensitive polypeptide is shown in SEQ ID NO:1, and the optically active polypeptide is shown in SEQ ID NO:

1. As shown in any one of NO:2-5, the optically active polypeptide is located at any one or more of the following sites of the D-2-hydroxyglutaric acid-sensitive polypeptide: 88 / 89, 88 / 90, 87 / 88, 87 / 89, 88 / 89, 89 / 90, 178 / 179, 178 / 180, 178 / 181, 178 / 182, 178 / 183, 179 / 180, 179 / 181, 179 / 182, 179 / 183, 180 / 181, 180 / 182, 180 / 183, 181 / 182, 181 / 183, 182 / 183, 204 / 205, 204 / 206, 204 / 207, 205 / 206 and 206 / 207.

5. A nucleic acid molecule comprising a sequence selected from: (1) Encoding the multinucleotide sequence of the optical probe according to any one of claims 1-4, and (2)(1) complementary sequences.

6. A nucleic acid construct comprising the nucleic acid molecule of claim 5. Preferably, the nucleic acid construct is an expression vector.

7. A host cell, wherein the host cell: (1) The optical probe according to any one of claims 1-4; (2) Contains the nucleic acid molecule as described in claim 5; or (3) It includes the nucleic acid construct of claim 6.

8. A method for preparing the optical probe according to any one of claims 1-4, comprising culturing the host cell according to claim 7, and separating the optical probe from the culture.

9. The use of the optical probe of any one of claims 1-4, the nucleic acid molecule of claim 5, or the nucleic acid construct of claim 6 in a kit for preparing a sample to detect D-2-hydroxyglutaric acid or a screening compound.

10. A test kit comprising... (1) The optical probe according to any one of claims 1-4 or the optical probe prepared by the method of claim 8; (2) The nucleic acid molecule according to claim 5; (3) The nucleic acid construct according to claim 6; or (4) The host cell as described in claim 7; and Other reagents required for the detection of D-2-hydroxyglutaric acid using an optical probe.

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