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WO2025232798A1 - Sonde optique de myo-inositol ainsi que son procédé de préparation et son utilisation - Google Patents

Sonde optique de myo-inositol ainsi que son procédé de préparation et son utilisation

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Publication number
WO2025232798A1
WO2025232798A1 PCT/CN2025/093250 CN2025093250W WO2025232798A1 WO 2025232798 A1 WO2025232798 A1 WO 2025232798A1 CN 2025093250 W CN2025093250 W CN 2025093250W WO 2025232798 A1 WO2025232798 A1 WO 2025232798A1
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WO
WIPO (PCT)
Prior art keywords
inositol
sensitive
optically active
polypeptide
optical probe
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/093250
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English (en)
Chinese (zh)
Inventor
杨弋
赵玉政
李写
汪永明
戴杭
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
East China University of Science and Technology
Original Assignee
East China University of Science and Technology
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Filing date
Publication date
Application filed by East China University of Science and Technology filed Critical East China University of Science and Technology
Publication of WO2025232798A1 publication Critical patent/WO2025232798A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • This invention relates to the field of optical probe technology, and in particular to an inositol optical probe, its preparation method, and its application.
  • MI Myo-Iositol
  • IP3 inositol triphosphate
  • PIP2/PIP3 phosphatidylinositol phospholipids
  • inositol is essential for the smooth functioning of a wide range of cellular functions, including cell growth and survival, peripheral nerve development and function, osteogenic processes, and cell proliferation.
  • HPLC high-performance liquid chromatography
  • LC-MS liquid chromatography-mass spectrometry
  • Isotope labeling and enzyme activity detection methods cannot monitor changes in inositol within living cells in real time. Therefore, there is an urgent need to develop genetically encoded fluorescent probes that can monitor the dynamic changes of inositol within living cells in situ.
  • the purpose of this invention is to provide probes and methods for real-time, high-throughput, and quantitative detection of inositol inside and outside cells.
  • the present invention provides the following technical solution:
  • the first aspect of this invention provides an inositol-binding protein variant, wherein:
  • (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 inositol.
  • the inositol-binding protein variant has the sequence shown in SEQ ID NO: 1 or a functional fragment thereof after removing amino acids 2-45, and: (a) has a mutation at one, two or more sites selected from the group consisting of P313, F314, and/or, (b) has a mutation at one, two, three, four or more sites selected from the group consisting of D62, H137, Q180, L235, D260, Q280, P313, F314.
  • the mutation includes a mutation at a site selected from any of the following groups: (1) P313 and F314; or, the mutation includes a mutation at a site selected from any of the following groups: (2) P313, F314 and D260, (3) P313, F314, D260 and L235, (4) P313, F314 and L235, (5) P313, F314 and Q280, (6) P313, F314, D260 and Q280.
  • L235 mutates to G, Y, A, or I.
  • D260 mutates to L, V, W, S, F, or Y.
  • Q280 mutates to S, G, or Y.
  • P313 mutates to A, L, P, R, S, F, G, Y, C, or V.
  • F314 mutates to I, C, L, V, W, H, or M.
  • the mutation comprises mutations selected from any of the following groups: (1) P313A and F314I, (2) P313L and F314C, (3) P313P and F314L, (4) P313A and F314L, (5) P313L and F314V, (6) P313L and F314I, (7) P313R and F314I, (8) P313S and F314V, (9) P313F and F314C, (10) P313G and F314C, (11) P313R and F3 14V, (12) P313Y and F314C, (13) P313Y and F314L, (14) P313F and F314L, (15) P313C and F314W, (16) P313V and F314H, (17) P313Y and F314V, (18) P313F and F314M, (19) P313A, F314L and D260L, (20) P313A
  • an inositol optical probe comprising an inositol-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the inositol-sensitive polypeptide.
  • the inositol-sensitive polypeptide is divided into a first part and a second part by the optically active polypeptide.
  • the inositol optical probe includes an inositol-sensitive polypeptide B and an optically active polypeptide A, wherein the optically active polypeptide A is located within the sequence of the inositol-sensitive polypeptide B, dividing the inositol-sensitive polypeptide B into a first part B1 and a second part B2, forming a probe structure of type B1-A-B2.
  • the optically active polypeptide is located at any one or more sites of the inositol-sensitive polypeptide selected from the following: 153/153, 153/154, 154/153, 154/154, 279/279, 279/280, 279/281, 280/279, 280/280, 280/281, 281/279, 281/280, 281/281, 312/312, 312/313, 312/314, 313/312, 313/313, 313/314, 314/312, 314/313, 314/314.
  • the inositol-sensitive polypeptide is an inositol-binding protein or a functional variant thereof.
  • the inositol-sensitive peptide has:
  • (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 inositol.
  • 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.
  • the fluorescent protein is selected from yellow fluorescent protein, green fluorescent protein, blue fluorescent protein, and red fluorescent protein. In one embodiment, the fluorescent protein has the sequence shown in any of SEQ ID NO: 2-5.
  • the functional variant of the fluorescent protein has a mutation at amino acid positions 1-3 or 244-246, preferably 1 or 246.
  • the functional variant of the fluorescent protein includes a mutation at amino acid position 1 of the fluorescent protein to T, S, N or G, and/or a mutation at amino acid position 246 of the fluorescent protein to F or C.
  • a functional variant of the fluorescent protein has the sequence shown in SEQ ID NO: 2 and a mutation at the Y1 site.
  • the mutation is Y1R, Y1S, Y1N, or Y1G, and/or
  • a functional variant of the fluorescent protein has the sequence shown in SEQ ID NO: 2 and a mutation at the N246 position.
  • the mutation is N246F or N246C.
  • the fluorescent protein has the sequence shown in SEQ ID NO: 2 or a variant having any of the following mutations at its first amino acid position: Y1R, Y1S, Y1N or Y1G, and/or a variant having any of the following mutations at its 246th amino acid position: N246F or N246C, wherein the optically active polypeptide is located at position 314/313 of the inositol-sensitive polypeptide.
  • 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: Y1R, Y1S, Y1N or Y1G, and/or a variant having a mutation at its 246th amino acid position of any of the following groups: N246F or N246C, wherein the optically active polypeptide is located at position 313/313 of the inositol-sensitive polypeptide.
  • 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.
  • the optically active polypeptide flanking the linker comprises a linker of no more than 5 amino acids, such as linkers of 0, 1, 2, 3, or 4 amino acids.
  • the linker flanking the optically active polypeptide comprises amino acid Y.
  • linker Y is located at the N-terminus and/or C-terminus of the optically active polypeptide.
  • the optical probe is as follows: a first portion B1 of the inositol-sensitive polypeptide, Y, optically active polypeptide A, and a second portion B2 of the inositol-sensitive polypeptide.
  • the optical probe of this invention does not comprise a linker.
  • the optical probe of the present invention further includes a positioning sequence for positioning the probe to a specific organelle, such as a cell.
  • the sequence of the inositol-sensitive polypeptide is as shown in SEQ ID NO: 1 or its functional fragment after removing amino acids 2-45
  • the optically active polypeptide is as shown in any one of SEQ ID NO: 2-5
  • the optically active polypeptide is located at any one or more sites selected from the following sites of the inositol-sensitive polypeptide: 153/153, 153/154, 154/153, 154/ 154, 279/279, 279/280, 279/281, 280/279, 280/280, 280/281, 281/279, 281/280, 281/281, 312/312, 312/313, 312/314, 313/312, 313/313, 313/314, 314/312, 314/313, 314/314.
  • the site numbers are in accordance with the sequence SEQ ID NO: 1.
  • the inositol-sensitive polypeptide is as shown in SEQ ID NO: 1 or a functional fragment thereof after truncating amino acids 2-45
  • 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 corresponding to the first or 246th amino acid of SEQ ID NO: 2: Y1R, Y1S, Y1N, Y1G, N246F or N246C.
  • optically active peptide is located at any one or more of the following sites in the inositol-sensitive peptide: 153/153, 153/154, 154/153, 154/154, 279/279, 279/280, 279/281, 280/279, 280/280, 280/281, 281/279, 281/280, 281/281, 312/312, 312/313, 312/314, 313/312, 313/313, 313/314, 314/312, 314/313, 314/314.
  • the optical probe comprises an inositol-sensitive polypeptide as shown in SEQ ID NO: 1 or a functional fragment thereof after removing amino acids 2-45, and having one or more of the following mutations: D62, H137, Q180, L235, D260, Q280, P313, F314.
  • the optically active polypeptide comprises an inositol-sensitive polypeptide 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 or 246th amino acid of SEQ ID NO: 2: Y1R, Y1S, Y1N, Y1G, N246F, or N246C, wherein the optically active polypeptide is located at 153/153, 153/154, or 154/153 of the inositol-sensitive polypeptide.
  • the mutations in the inositol-sensitive peptide include mutations selected from any of the following groups: (1) P313A and F314I, (2) P313L and F314C, (3) P313P and F314L, (4) P313A and F314L, (5) P313L and F314V, (6) P313L and F314I, (7) P313R and F314I, (8) P313S and F314V, (9) P313F and F314C, (10) P313G and F314C, (11) P313R and F314I.
  • the optical probe comprises an inositol-sensitive polypeptide as shown in SEQ ID NO: 1 or a functional fragment thereof after removing amino acids 2-45, an optically active polypeptide as shown in SEQ ID NO: 2-5, the optically active polypeptide being located at position 313/313 of the inositol-sensitive polypeptide, and the optical probe having the following mutations: (1) P313A and F314I of the inositol-sensitive polypeptide, (2) P313L and F31 of the inositol-sensitive polypeptide.
  • the optical probe contains an inositol-sensitive polypeptide as shown in SEQ ID NO: 1 or a functional fragment thereof after removing amino acids 2-45, an optically active polypeptide as shown in SEQ ID NO: 2-5, the optically active polypeptide being located at position 314/313 of the inositol-sensitive polypeptide, and the optical probe having the following mutations: (37) P313Y and F314C of the inositol-sensitive polypeptide, (38) P313Y and F314L of the inositol-sensitive polypeptide, (39) P313F and F314L of the inositol-sensitive polypeptide, (40) ...
  • the optical probe is as shown in SEQ ID NO: 7-8.
  • a fusion polypeptide comprising the optical probe described herein and other polypeptides.
  • the other polypeptides are located at the N-terminus and/or C-terminus of the optical probe.
  • the other polypeptides include a localization sequence (e.g., a polypeptide that localizes the optical probe to a different organelle or sub-organelle), a tag for easy purification, or a tag for use in an immunoreaction (e.g., immunoblotting).
  • a linker may be present between the optical probe and the other polypeptides in the fusion polypeptide described herein.
  • 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.
  • 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.
  • 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.
  • the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.
  • the nucleic acid molecule is operatively linked to an expression control sequence.
  • the expression vector is selected from prokaryotic expression vectors, eukaryotic expression vectors, and viral vectors.
  • 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*.
  • Another aspect of the present invention provides an inositol detection kit, comprising the optical probes described herein or fusion peptides or polynucleotides or optical probes prepared as described herein.
  • the kit further comprises one or more reagents selected from the following: buffer solutions, culture media, and inositol standards.
  • 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.
  • the method includes the following steps: 1) incorporating a nucleic acid molecule encoding the inositol 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 inositol optical probe.
  • Another aspect of the present invention provides a method for detecting inositol 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.
  • This article also provides a method for quantifying inositol in a sample, comprising: contacting the sample with the optical probe or fusion peptide or host cell described herein, detecting optical changes in the optically active peptide, and quantifying inositol in the sample based on the optical changes in the optically active peptide.
  • 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 an inositol-containing system, 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.
  • the host cells described herein are contacted with the candidate compound in an inositol-containing system, and optical changes in the optically active peptide indicate whether the candidate compound can regulate cellular uptake of inositol.
  • Another aspect of the present invention provides a method for intracellular and/or extracellular localization of the inositol, comprising: contacting the inositol-containing system with the optical probe or the host cell, and detecting optical changes in the optically active peptide.
  • the system is a solution system, a cellular system, or a subcellular system.
  • Another aspect of the present invention provides the use of the inositol optical probes or fusion peptides or host cells described herein in the intracellular and/or extracellular detection of inositol, screening compounds, or inositol in a sample.
  • the localization is real-time localization.
  • Another aspect of the present invention provides the use of the inositol optical probes or fusion peptides or polynucleotides or nucleic acid constructs or host cells described herein in the preparation of kits for detecting inositol in samples, screening compounds or intracellular and/or extracellular localization of inositol.
  • the beneficial effects of this invention are as follows:
  • the inositol optical probe provided by this invention is easy to mature, exhibits large fluorescence dynamic changes, and has good specificity. Furthermore, it can be expressed in cells through gene manipulation, enabling real-time, high-throughput, and quantitative detection of inositol both inside and outside cells, eliminating time-consuming sample processing steps.
  • Experimental results show that the inositol optical probe provided by this application achieves a response to inositol that is more than 5 times that of the control. It can also perform localization, qualitative, and quantitative detection of inositol in subcellular structures such as the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, lysosomes, and Golgi apparatus. Additionally, it allows for high-throughput compound screening and quantitative detection of inositol in blood.
  • Figure 1 shows an SDS-PAGE image of an exemplary inositol optical probe
  • Figure 2 shows the fluorescence spectral properties of an exemplary inositol optical probe
  • Figure 3 shows the titration curves of different concentrations of inositol using an exemplary inositol optical probe
  • Figure 4 shows a photograph of the subcellular organelle localization of an exemplary inositol optical probe in a mammalian cell
  • Figure 5 is a schematic diagram of the dynamic monitoring of inositol concentration in the cytoplasm of mammalian cells using an exemplary inositol optical probe.
  • Figure 6 is a dot plot of an exemplary inositol optical probe used for high-throughput compound screening at the live cell level
  • Figure 7 is a bar chart showing the quantification of inositol in mouse and human blood using an exemplary inositol optical probe.
  • composition “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.
  • the term "inositol-sensitive peptide” refers to a peptide that responds to inositol, and the response includes any response to chemical, biological, electrical, or physiological parameters of the peptide in relation to the interaction with the sensitive peptide.
  • Responses include small changes, such as changes in the orientation of amino acids or peptide fragments of the peptide, and changes in the primary, secondary, or tertiary structure of the peptide, including, for example, changes in protonation, electrochemical potential, and/or conformation.
  • Conformation is the three-dimensional arrangement of the primary, secondary, and tertiary structures of a molecule containing side groups; a conformational change occurs when the three-dimensional structure of the molecule changes.
  • conformational changes include a change from an ⁇ -helix to a ⁇ -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 inositol-sensitive peptides described herein may also include their functional variants. Functional variants of inositol-sensitive peptides include, but are not limited to, variants that can interact with inositol to undergo the same or similar changes as the parental inositol-sensitive peptide.
  • the inositol-sensitive polypeptides described in this invention include, but are not limited to, the inositol-binding protein ACEI_1806 (PDB: 4RU1, sequence as shown in SEQ ID NO: 1) derived from the cellulolytic bacterium *Acidothermus cellulolyticus*, or variants thereof with more than 90% homology.
  • Inositol-binding proteins can sense changes in inositol concentration, and their spatial conformation changes during dynamic changes in inositol concentration.
  • Truncated variants of 4RU1 can also be used in this invention, such as the functional fragment of SEQ ID NO: 1 after removing amino acids 2-45.
  • the amino acid residue numbers of the inositol-sensitive polypeptides mentioned are all referenced to SEQ ID NO: 1.
  • optical probe refers to an inositol-sensitive peptide fused to an optically active peptide.
  • inositol-sensitive peptides such as inositol-binding proteins
  • optically active peptides e.g., fluorescent proteins
  • an optically active polypeptide e.g., a fluorescent protein
  • an optically active polypeptide is operatively inserted into an inositol-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.
  • 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.
  • 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 polypeptides include, but are not limited to, variants that can undergo the same or similar fluorescence property changes as the parent optically active polypeptide.
  • 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.
  • 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.
  • the fluorescent protein in the optical probe also includes functional variants with mutations, including but not limited to fluorescent proteins with mutations at the first and/or 246th amino acids corresponding to SEQ ID NO: 2.
  • the mutation at the first position is preferably P, N, S, T, A, or E; the mutation at the 246th position is preferably F or C.
  • the functional variant of the fluorescent protein has any of the sequences shown in SEQ ID NO: 2-5 and has a mutation selected from any group of the following at the first amino acid corresponding to SEQ ID NO: 2: Y1P; Y1N; Y1S; Y1T; Y1A, or Y1E.
  • the functional variant of the fluorescent protein has any of the sequences shown in SEQ ID NO: 2-5 and has a mutation selected from any group of the following at the 246th amino acid corresponding to SEQ ID NO: 2: N246F or N246C. In some embodiments, the functional variant of the fluorescent protein has the sequence shown in any of SEQ ID NO: 2-5 and has mutations selected from any of the following groups at amino acid positions 1 and 246 corresponding to SEQ ID NO: 2: Y1P; Y1N; Y1S; Y1T; Y1A; Y1E; N246F; N246C. 1S indicates a mutation of S at amino acid position 1, and so on.
  • the optically active polypeptide is in the N-C direction of the inositol-sensitive polypeptide, specifically residues 153/153, 153/154, 154/153, 154/154, 279/279, 279/280, 279/281, 280/279, 280/280, 280/281, 281/279, 281/280, 281/281, 312/312, 312/313, 312/314, 313/312, 313/313, 313/314, 314/312, 314/313, 314/314, or substitutes thereof, with the number corresponding to the full length of the inositol-sensitive polypeptide.
  • the optically active polypeptide has partial inositol-sensitive polypeptides at both ends.
  • the N-terminus of the optically active polypeptide is the N-terminal starting amino acid (e.g., any amino acid from position 1 to 153) to the Xth amino acid of the inositol-sensitive polypeptide sequence
  • the C-terminus of the optically active polypeptide is the Yth amino acid to the C-terminal ending amino acid (e.g., any amino acid from position Y to 333) of the inositol-sensitive polypeptide sequence.
  • insertion site 153/154 indicates that the optically active polypeptide is located between amino acids 153 and 154 of the inositol-sensitive polypeptide.
  • insertion site 312/314 indicates that the optically active polypeptide replaces amino acid 313 of the inositol-sensitive polypeptide.
  • X is greater than Y in the "X/Y" format, it indicates that the inositol-sensitive polypeptide portion located at the N-terminus of the optically active polypeptide terminates at the Xth amino acid in the inositol-sensitive polypeptide sequence, while the inositol-sensitive polypeptide portion located at the C-terminus of the optically active polypeptide terminates at the Yth amino acid in the inositol-sensitive polypeptide sequence.
  • An acid-based insertion site indicates that the N-terminus of the optically active polypeptide is fused with an N-terminal starting amino acid (e.g., any amino acid from position 1 to 333) to amino acid 154 of the inositol-sensitive polypeptide sequence, and the C-terminus of the optically active polypeptide is fused with an amino acid from position 153 to the C-terminal ending amino acid (e.g., amino acid 333) of the inositol-sensitive polypeptide sequence, with an exemplary structure of: (amino acids 1 to 154 of the inositol-sensitive polypeptide sequence) - (optically active polypeptide) - (amino acids 153 to 333 of the inositol-sensitive polypeptide sequence); if the two numbers in the site represented in the form of "X/Y" are not consecutive integers and X equals Y, it indicates that the optically active polypeptide is inserted between the N-terminal starting amino acid (e.
  • the optically active polypeptide represented by SEQ ID NO: 2, 3, 4 or 5 is located at the functional fragment of SEQ ID NO: 1 or the fragment after removing amino acids 2-45, and the inositol-sensitive polypeptide is selected from any one or more of the following sites: 153/153, 153/154, 154/153, 154/154, 279/279, 279/280, 279/281, 280/279, 280/280, 280/281, 281/279, 281/280, 281/281, 312/312, 312/313, 312/314, 313/312, 313/313, 313/314, 314/312, 314/313, 314/314.
  • the optical probe comprises, from the N-terminus to the C-terminus, residues 1, 46-X of SEQ ID NO: 1, an optically active polypeptide or a variant thereof shown in any one of SEQ ID NO: 2-5, and residues Y-333 of SEQ ID NO: 1, wherein X and Y are selected from any one of the following groups: (1) X is 153, Y is 153, (2) X is 153, Y is 154, (3) X is 154, Y is 153, (4) X is 154, 154, (5) X is 279, Y is 279, (6) X is 279, Y is 280, (7) X is 279, Y is 281, (8) X is 280, Y is 280, (9) X is 279, Y is 281, (10) X is 280, Y is 280, (11) X is 280, Y is 280, (12) X is 279, Y is 281,
  • Y is 279, (9) X is 280, Y is 280, (10) X is 280, Y is 281, (11) X is 281, Y is 279, (12) X is 281, Y is 280, (13) X is 281, Y is 281, (14) X is 312, Y is 312, (15) X is 312, Y is 313, (16) X is 312, Y is 314, (17) X is 313, Y is 312, (18) X is 313, Y is 313, (19) X is 313, Y is 314, (20) X is 314, Y is 312, (21) X is 314, Y is 313, (22) X is 314, Y is 314.
  • the optical probe comprises, from N-terminus to C-terminus, the N-terminal sequence of the inositol-sensitive polypeptide shown at residues 1, 46-313 of SEQ ID NO: 1, the optically active polypeptide or a variant thereof shown at SEQ ID NO: 2, and the C-terminal sequence of the inositol-sensitive polypeptide shown at residues 313-333 of SEQ ID NO: 1, and the optical probe has the following mutations: (1) P313A and F314I of the C-terminal sequence of the inositol-sensitive polypeptide, (2) P313L and F314C of the inositol-sensitive polypeptide, (3) P313P and F314L of the C-terminal sequence of the inositol-sensitive polypeptide, (4) P313A and F314L of the C-terminal sequence of the inositol-sensitive polypeptide, (5) P313L and F314V of the C-
  • the optical probe comprises, from N-terminus to C-terminus, the N-terminal sequence of the inositol-sensitive polypeptide represented by residues 1, 46-313 of SEQ ID NO: 1, the optically active polypeptide or a variant thereof represented by SEQ ID NO: 2, and the C-terminal sequence of the inositol-sensitive polypeptide represented by residues 313-333 of SEQ ID NO: 1, and the optical probe has mutations including P313A and F314L mutations in the C-terminal sequence of the inositol-sensitive polypeptide and mutations selected from any one of the following groups: (16) inositol-sensitive polypeptide (17) D260L of inositol-sensitive peptide, (18) D260V of inositol-sensitive peptide, and 1S of optically active peptide, (29) D260L and L235G of inositol-sensitive peptide, and 1S of optically active peptide
  • Inositol-sensitive peptide 1S (25) Inositol-sensitive peptide D260F, and optically active peptide 1T
  • Inositol-sensitive peptide D260Y and optically active peptide 1T
  • Inositol-sensitive peptide L235Y (28) Inositol-sensitive peptide L235A, and optically active peptide 1S
  • Inositol-sensitive peptide L235I Inositol-sensitive peptide L235I, and optically active peptide 1T
  • Inositol-sensitive peptide Q280S (31) Inositol-sensitive peptide Q280G, and optically active peptide 1S, (2 ...
  • the optical probe comprises, from N-terminus to C-terminus, the N-terminal sequence of the inositol-sensitive polypeptide shown at residues 1, 46-314 of SEQ ID NO: 1, the optically active polypeptide or a variant thereof shown at SEQ ID NO: 2, and the C-terminal sequence of the inositol-sensitive polypeptide shown at residues 313-333 of SEQ ID NO: 1, and the optical probe has the following mutations: (1) P313Y and F314C of the C-terminal sequence of the inositol-sensitive polypeptide, (2) P313Y and F314L of the C-terminal sequence of the inositol-sensitive polypeptide, (3) P313F and F314L of the C-terminal sequence of the inositol-sensitive polypeptide, (4) inositol (5) P313C and F314W of the C-terminal sequence of the inositol-sensitive peptide,
  • 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.
  • 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:
  • 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.
  • F represents fluorescence intensity.
  • F ⁇ sub> sample ⁇ /sub> represents the total fluorescence intensity of the sample expressing the fluorescent probe
  • F ⁇ sub>BLK ⁇ /sub> represents the background fluorescence intensity of the sample not expressing the fluorescent probe
  • F ⁇ sub> cpYFP ⁇ /sub> represents the fluorescence intensity of the sample used as a pH control.
  • F ⁇ sub>485 ⁇ /sub> represents the fluorescence intensity emitted at 528 nm when the fluorescent protein sample is excited at 485 nm
  • F ⁇ sub>420 ⁇ /sub> represents the fluorescence intensity emitted at 528 nm when the fluorescent protein sample is excited at 420 nm.
  • Ratiosensor represents the fluorescence intensity ratio of the probe
  • Ratio cpYFP represents the fluorescence intensity ratio of the corresponding probe to the pH control fluorescent protein.
  • the Normalized Ratio 420/485 is the fold change of the probe or the response multiple. The greater the deviation of the Normalized Ratio 485/420 from 1 (whether it increases or decreases), the greater the fold change of the probe or the response multiple.
  • variants include 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.
  • 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.
  • variants may also comprise polypeptides or proteins with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the polypeptide or protein.
  • the goal is to avoid being limited by theory and to change amino acid residues without altering the overall conformation and function of the polypeptide or protein—that is, functionally conserved mutations.
  • substitution with amino acids of similar or identical properties typically does not change the function of the polypeptide or protein.
  • amino acids with similar properties often refer to families of amino acids with similar side chains, which are well-defined.
  • amino acids with basic side chains e.g., lysine, inositol, 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, inositol, phenylalanine, methionine, inositol
  • amino acids with ⁇ -branched side chains e.g., threonine, valine, isoleucine
  • amino acids with aromatic side chains e.g., tyrosine, phenylalanine, inositol, histidine
  • amino acids For example, adding one or more amino acids to the amino terminus and/or carboxyl terminus generally does not alter the function of a polypeptide or protein.
  • conserved amino acid substitutions for many common, known non-genetically encoded amino acids are known in the art.
  • conserveed 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.
  • Connector or “linking region” refers to an amino acid or nucleotide sequence that links two parts in the polypeptide, protein, or nucleic acid of the present invention.
  • the number of amino acids at the amino terminus of the linking region between the inositol-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.
  • inositol-binding protein variants with mutations at the following sites exhibit different binding activities than inositol: SEQ ID NO: 1 or functional fragments thereof with amino acids truncated from positions 2-45, namely D62, H137, Q180, L235, D260, Q280, P313, and F314.
  • the amino acid mutations include modifications, substitutions, or deletions of amino acids.
  • This invention provides inositol-binding protein variants having these mutations and optical probes comprising such inositol-binding protein variants as inositol-sensitive peptides.
  • the inositol-sensitive peptide in the optical probe is an inositol-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.
  • the inositol-sensitive polypeptide in the optical probe is as shown in SEQ ID NO: 1 or its functional fragment after removing amino acids 2-45
  • the optically active polypeptide is as shown in SEQ ID NO: 2, located at 153/153, 153/154, 154/153, 154/154, 279/279, 279/280, 279/
  • the mutations of the optical probe at sites 281, 280/279, 280/280, 280/281, 281/279, 281/280, 281/281, 312/312, 312/313, 312/314, 313/312, 313/313, 313/314, 314/312, 314/313, and 314/314 are shown in any row of Tables 5 and 6.
  • the sequence of the inositol-sensitive polypeptide is as shown in SEQ ID NO: 1 or its functional fragment after removing amino acids 2-45
  • the optically active polypeptide is as shown in any one of SEQ ID NO: 2-5
  • the optically active polypeptide is located at any one or more sites selected from the following sites of the inositol-sensitive polypeptide: 153/153, 153/154, 154/153, 15 4/154, 279/279, 279/280, 279/281, 280/279, 280/280, 280/281, 281/279, 281/280, 281/281, 312/312, 312/313, 312/314, 313/312, 313/313, 313/314, 314/312, 314/313, 314/314.
  • the optical probe comprises an inositol-sensitive polypeptide as shown in SEQ ID NO: 1 or a functional fragment thereof after removing amino acids 2-45, an optically active polypeptide as shown in SEQ ID NO: 2-5, the optically active polypeptide being located at position 313/313 of the inositol-sensitive polypeptide, and the optical probe having the following mutations: (1) P313A and F314I of the inositol-sensitive polypeptide, (2) P313L and F31 of the inositol-sensitive polypeptide.
  • the optical probe contains an inositol-sensitive polypeptide as shown in SEQ ID NO: 1 or a functional fragment thereof after removing amino acids 2-45, and an optically active polypeptide as shown in SEQ ID NO: 2-5, the optically active polypeptide being located at position 314/313 of the inositol-sensitive polypeptide, and the optical probe having the following mutations: (1) P313Y and F314C of the inositol-sensitive polypeptide, (2) P313Y and F314L of the inositol-sensitive polypeptide, (3) P313F and F314L of the inositol-sensitive polypeptide, (4) ...
  • Inositol-sensitive peptides P313C and F314W Inositol-sensitive peptides P313V and F314H
  • Inositol-sensitive peptides P313Y and F314V Inositol-sensitive peptides P313F and F314M, and optically active peptides 1T and 246F
  • Inositol-sensitive peptides P313Y and F314C, and optically active peptides 1T and 246F Inositol-sensitive peptides P313Y and F314L, and optically active peptides 1T and 246F.
  • 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).
  • preferred algorithms suitable for determining the percentage of sequence identity and the percentage of sequence similarity are the BLAST and BLAST2.0 algorithms, see Altschul et al. (1977) Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol. 215:403, respectively.
  • 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.
  • 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.
  • Suitable adaptor peptides 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.
  • 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., inositol-binding protein or fluorescent protein).
  • Functional variants, derivatives, or analogs of the polypeptides or proteins (e.g., inositol-binding protein or fluorescent protein) of the present invention may be (i) proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by the fusion of 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 the fusion of an additional amino acid sequence into 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).
  • proteins with one or more conserved or non-conserved amino acid residues preferably conserved amino acid
  • 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 ⁇ - or ⁇ -amino acids).
  • L-amino acids such as D-amino acids
  • non-naturally occurring or synthetic amino acids such as ⁇ - or ⁇ -amino acids.
  • Modifications 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 proteolytic hydrolysis or optimize their solubility are also included.
  • 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 prote
  • the fusion polypeptide of this invention comprises the optical probe described herein and other polypeptides.
  • 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.
  • 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.
  • the subcellular organelles described herein include the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, Golgi apparatus, lysosomes, and peroxisomes.
  • the tags used for purification or for immunoblotting include 6-histidine (6*His), glutathione S-transferase (GST), and Flag.
  • nucleic acid molecules encoding the inositol-sensitive polypeptide or optical probe described herein.
  • nucleic acid 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.
  • variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • nucleotide variants include degenerate variants, substitution variants, deletion variants, and insertion variants.
  • 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.
  • nucleic acid fragments that hybridize with the above-described sequences.
  • 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).
  • the full-length sequence or fragment thereof 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.
  • mutations can be introduced into the protein sequence of this invention through methods such as mutagenic PCR or chemical synthesis.
  • 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 prior to 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.
  • 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.
  • 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.
  • 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.
  • "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.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector may be a commercially available pCDF vector, with no other special requirements.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • This invention involves transferring an expression vector into host cells, followed by amplification and expression culture of the host cells to isolate the inositol optical probe.
  • the host cell amplification and expression culture can be performed using conventional methods.
  • the culture medium can be any conventional medium. Culture is carried out under conditions suitable for host cell growth.
  • the optical probe is expressed intracellularly, on the cell membrane, or secreted extracellularly.
  • 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 inositol fluorescent protein; conventional methods for separating fusion proteins in the art can be used.
  • optical probe is separated using His-tagged affinity chromatography.
  • This invention also provides the application of the inositol optical probe in real-time localization, quantitative detection, and high-throughput compound screening of inositol.
  • the inositol optical probe is preferably linked to signal peptides at different sites within the cell, transferred into the cell, and used to perform real-time localization of inositol by detecting the intensity of fluorescence signals within the cell; quantitative detection of inositol is then performed by combining a standard inositol titration curve with changes in fluorescence signals. Changes in fluorescence signals are displayed, for example, by a standardized fluorescence signal ratio.
  • 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 standard inositol titration curve of this invention is plotted based on the fluorescence signals obtained by the inositol optical probe at different concentrations of inositol.
  • the inositol optical probe of this invention is directly transferred into the cell, eliminating the need for time-consuming sample processing during real-time localization and quantitative detection of inositol, thus improving accuracy.
  • the inositol optical probe of this invention adds different compounds to the cell culture medium and measures changes in inositol content to screen for compounds that affect changes in inositol content.
  • the application of the inositol optical probe described in this invention in real-time localization, quantitative detection of inositol, and high-throughput compound screening is not for diagnostic or therapeutic purposes and does not involve the diagnosis or treatment of diseases.
  • 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 contains other reagents required for the detection of inositol.
  • these other reagents are well known in the art, such as buffers, cell culture media, and inositol standards.
  • Exemplary buffers include, for example, 100 mM HEPES and 100 mM NaCl, pH 7.4.
  • 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 D.W. Russell, translated by Huang Peitang et al. (Molecular Cloning 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. Bonifensenon, M. Dassault et al.'s "A Concise Guide to Cell Biology Laboratory Techniques," translated by Zhang Jingbo et al.
  • the pCDF-cpYFP and pCDF-inositol 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.
  • Inositol and other compounds were purchased from Sigma-Aldrich. Unless otherwise stated, inorganic salts and other chemical reagents were purchased from Sigma-Aldrich. HEPES salt, ampicillin (Amp), and puromycin were purchased from Amersco. The 96-well detection blackboard and the 384-well fluorescence detection blackboard were purchased from Grenier.
  • 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.
  • the main instruments used in the examples include: Biotek Synergy2 multi-functional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge 22R benchtop high-speed refrigerated centrifuge (Beckman, USA), PCR amplifier (Biometra, Germany), ultrasonic disruptor (Ningbo Xinzhi Co., Ltd.), nucleic acid electrophoresis apparatus (Shenneng Bocai Co., Ltd.), fluorescence spectrophotometer (Varian, USA), CO2 incubator (SANYO), and inverted fluorescence microscope (Nikon, Japan).
  • the PCR amplification reaction system is as follows: template sequence 0.5-1 ⁇ L, forward primer (25 ⁇ M) 0.5 ⁇ L, reverse primer (25 ⁇ M) 0.5 ⁇ L, 10 ⁇ pfu buffer 5 ⁇ L, pfu DNA polymerase 0.5 ⁇ L, dNTP (10 mM) 1 ⁇ L, sterile ultrapure water ( ddH2O ) 41.5-42 ⁇ L, total volume 50 ⁇ L.
  • the PCR amplification program is as follows: denaturation at 95°C for 2-10 minutes, 30 cycles (94-96°C for 30-45 seconds, 50-65°C for 30-45 seconds, 72°C for a certain time (600 bp/min)), extension at 72°C for 10 minutes.
  • 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 ⁇ L, forward primer (25 ⁇ M) 0.5 ⁇ L, reverse primer (25 ⁇ M) 0.5 ⁇ L, 5 ⁇ PrimerSTAR buffer 10 ⁇ L, PrimerSTAR DNA polymerase 0.5 ⁇ L, dNTP (2.5 mM) 4 ⁇ L, sterile ultrapure water ( ddH2O ) 33.5 ⁇ L, total volume 50 ⁇ L.
  • the PCR amplification program is as follows: denaturation at 95°C for 5 minutes, 30 cycles (98°C for 10 seconds, 50-68°C for 5-15 seconds, 72°C for a certain time (1000bp/min)), extension at 72°C for 10 minutes; or denaturation at 95°C for 5 minutes, 30 cycles (98°C for 10 seconds, 68°C for a certain time (1000bp/min)), extension at 72°C for 10 minutes.
  • the double enzyme digestion system for the plasmid vector is as follows: 20 ⁇ L plasmid vector (approximately 1.5 ⁇ g), 5 ⁇ L 10 ⁇ buffer, 11-2 ⁇ L restriction endonuclease, 21-2 ⁇ L restriction endonuclease, and bring the total volume to 50 ⁇ L with sterile ultrapure water. Reaction conditions: 37°C, 1-7 hours.
  • 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 ⁇ L of PCR product DNA sequence, 1 ⁇ L of 10 ⁇ T4 ligase buffer, 1 ⁇ L of T4 polynucleotide kinase (T4 PNK), and 0-3 ⁇ L of sterile ultrapure water, for a total volume of 10 ⁇ L. The reaction conditions are 37°C for 30 minutes to 2 hours, followed by inactivation at 72°C for 20 minutes.
  • 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 ⁇ L of T4 PNK-treated DNA fragment, 4 ⁇ L of linearized vector fragment, 1 ⁇ L of PEG4000, 1 ⁇ L of 10 ⁇ T4 ligase buffer, and 1 ⁇ L of T4 DNA ligase, for a total of 10 ⁇ L.
  • the reaction conditions are 22°C for 30 minutes.
  • 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 ⁇ L of the digested PCR product DNA fragment, 0.5-7 ⁇ L of the digested plasmid, 1 ⁇ L of 10 ⁇ T4 ligase buffer, 1 ⁇ L of T4 DNA ligase, and sterile ultrapure water to a total volume of 10 ⁇ L.
  • the reaction conditions are 16°C for 4-8 hours.
  • 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 ⁇ L phosphorylation reaction mixture, 0.5 ⁇ L T4 ligase (5 U/ ⁇ L), total volume 10.5 ⁇ L. Reaction conditions: 16°C, 4–16 hours.
  • Resuspension buffer CaCl2 (100mM), MgCl2 (70mM), NaAc (40mM)
  • 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
  • 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).
  • the probe was diluted with assay buffer (100mM HEPES, 100mM NaCl, pH 7.4) to a final concentration of 0.2-5 ⁇ M.
  • assay buffer 100mM HEPES, 100mM NaCl, pH 7.4
  • the pCDNA3.1+-based inositol optical probe plasmid was transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen) and cultured in a 37°C, 5% CO2 incubator. Fluorescence detection was performed 24–36 h after the exogenous gene was fully expressed.
  • the adherent HEK293 cells were washed three times with PBS and placed in HBSS solution for fluorescence microscopy and microplate reader detection.
  • the ACEI_1806 gene (PDB: 4RU1, SEQ ID NO: 1) from *Acidothermic cellulose* was truncated from its domains 2-45, and the remaining gene was amplified.
  • the PCR product was subjected to gel electrophoresis, and the pCDF vector was simultaneously digested with the corresponding enzymes. Ligation was performed using T4 DNA ligase, and the product was used to transform DH5 ⁇ cells.
  • the transformed DH5 ⁇ cells were plated on LB agar plates (streptomycin 100 ⁇ g/mL) and incubated overnight at 37°C. Plasmids were extracted from the DH5 ⁇ transformants and identified by PCR. Positive plasmids, after being correctly sequenced, were used for subsequent plasmid construction.
  • Example 2 Expression and detection of cpYFP optical probes at different insertion sites
  • the following sites were selected for insertion of cpYFP based on pCDF-4RU1 to obtain the corresponding pCDF-4RU1-cpYFP plasmids: 153/153, 153/154, 154/153, 154/154, 279/279, 279/280, 279/281, 280/279, 280/280, 280/281, 281/279, 281/280, 281/281, 312/312, 312/313, 312/314, 313/312, 313/313, 313/314, 314/312, 314/313, 314/314.
  • the amino acid sequence of 313/313-4RU1-cpYFP is shown in SEQ ID NO: 6.
  • the cpYFP DNA fragment was generated using PCR, and a homologous sequence from the cpYFP terminal was introduced at the 5' end using primers.
  • PCR amplification produced a linearized pCDF-inositol-binding protein vector, whose 5' and 3' ends contained sequences completely identical to those at the cpYFP terminals (15-20 bp).
  • the linearized pCDF-4RU1 and cpYFP fragments underwent homologous recombination using the Hieff Clone Enzyme.
  • the product was transformed into DH5 ⁇ , and the transformed DH5 ⁇ was plated on LB agar plates (streptomycin 100 ⁇ g/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.
  • Inositol response screening was performed using the supernatant of lysed *E. coli* expressing the 4RU1-cpYFP fusion protein.
  • the detection signal of the fusion fluorescent protein containing 1 mM inositol was divided by the detection signal of the fusion fluorescent protein without inositol.
  • Table 1 the detection results showed that optical probes expressing the 4RU1-cpYFP fusion protein showed an inositol response greater than 1.4 times at sites 313/312, 313/313, and 313/314. Among these, sites 313/313 and 314/313 showed an inositol response unaffected by other 4RU1 binding substrates.
  • Example 3 Expression and detection of cpGFP optical probes at different insertion sites
  • cpYFP was replaced with cpGFP to construct an inositol green fluorescent protein fluorescent probe.
  • Table 2 the detection results showed that optical probes expressing the 4RU1-cpYFP fusion protein in the fragmented supernatant responded to inositol more than 1.4 times.
  • Optical probes inserted at sites 313/312, 313/313, and 313/314 were included. Among them, sites 313/313 and 314/313 showed responses to inositol that were not affected by the binding of other 4RU1 substrates.
  • Example 4 Expression and detection of cpBFP optical probes at different insertion sites
  • cpYFP was replaced with cpBFP to construct a blue fluorescent probe for inositol.
  • Table 3 the detection results showed that optical probes expressing the 4RU1-cpYFP fusion protein in the fragmented supernatant responded to inositol more than 1.4 times.
  • Optical probes inserted at sites 313/312 and 313/313 were also found to be responsive to inositol without being affected by other 4RU1 binding substrates.
  • Example 5 Expression and detection of cpmApple optical probes at different insertion sites
  • cpYFP was replaced with cpmApple to construct an inositol red fluorescent protein probe.
  • Table 4 the detection results showed that optical probes expressing the 4RU1-cpYFP fusion protein in the fragmented supernatant responded to inositol more than 1.4 times.
  • Optical probes inserted at sites 313/312, 313/313, and 313/314 were included, among which the response to inositol was not affected by the binding of other 4RU1 substrates at site 313/313.
  • Example 2 For the optical probes obtained in Example 2 that responded to inositol more than 1.3 times and were unaffected by other 4RU1 binding substrates, i.e., those inserted at the 313/313 and 314/313 sites of 4RU1, linearization of the probe was performed via reverse PCR.
  • the linker mutation site sequence was introduced into the primers, and the resulting PCR product underwent homologous recombination using the HieffClone Enzyme to establish a mutant library.
  • the recombinant plasmid of the mutant library was transformed into BL21(DE3) to induce expression.
  • the probe proteins were purified and screened for inositol response.
  • the detection signal of the fusion fluorescent protein containing 10 mM inositol was divided by the detection signal of the fusion fluorescent protein without inositol.
  • the detection results show that the optical probes that responded to inositol more than or equal to 3 times are shown in Table 5.
  • the sequence of 313/313-4RU1-P313A/F314I-cpYFP is shown in SEQ ID NO: 7
  • the sequence of 314/313-4RU1-P313F/F314M-cpYFP-Y1T/246F is shown in SEQ ID NO: 8.
  • the mutations at P313 and F314 in the 4RU1 protein in the table above are located downstream (C-terminus) of the insertion site.
  • Example 7 Expression and detection of cpYFP optical probes with pocket mutations
  • the probe was linearized using reverse PCR, and the sequence of the mutation site was introduced into the primer. Homologous recombination was performed on the obtained PCR product to obtain a plasmid containing the above-mentioned mutation sites.
  • the mutant plasmid was transformed into BL21(DE3) to induce expression.
  • the mutant probe protein was purified and subjected to an inositol response test.
  • the detection signal of the fusion fluorescent protein containing 2 mM inositol was divided by the detection signal of the fusion fluorescent protein without inositol. The detection results show that the optical probes that responded to inositol more than twice as much are listed in Table 6.
  • the mutations at P313 and F314 in the 4RU1 protein in the table above are located downstream (C-terminus) of the insertion site.
  • the two inositol optical probes described in Examples 6 and 7 were treated with 0 mM and 5 mM inositol for 10 minutes, respectively, and then the fluorescence spectra were detected using a fluorescence spectrophotometer.
  • Excitation spectrum determination Excitation spectra were recorded with an excitation range of 370 nm to 510 nm and an emission wavelength of 530 nm, read every 5 nm. The results showed that the probe had two excitation peaks at approximately 410 and 490 nm, as shown in Figure 2.
  • Emission spectra were measured: with fixed excitation wavelengths of 420 nm and 460 nm, emission spectra were recorded in the ranges of 470-600 nm and 490-600 nm, with readings taken every 5 nm. The results are shown in Figure 2.
  • the 46 purified inositol probes described in Examples 6 and 7 were subjected to inositol detection at concentration gradients (0–10 mM). After treating the purified probes for 10 minutes, the changes 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 were measured. The results are shown in Figure 3.
  • the Kd (binding constant) of the 46 inositol optical probes were 402.50, 97.34, 435.70, 518.70, 508.23, 432.78, 398.9, 283.10, 64.18, 107.21, 112.90, 9.23, 10.43, 9.28, 7.88, 8.98, 12.51, 24.15, 24.35, 9.23, 10.43, 315.37, 35, and 35, respectively.
  • Example 9 Subcellular organelle localization of optical probes and performance of optical probes within subcellular organelles
  • different localization signal peptides are fused with the optical probe 313/313-4RU1-F313A/P314L-cpYFP to localize the optical probe to different organelles.
  • Example 10 High-throughput compound screening in living cells based on optical probes
  • HeLa cells with cytoplasmic expression of 313/313-4RU1-P313A/F314L-cpYFP for high-throughput compound screening.
  • Transfected 293 cells were washed with PBS, treated with HBSS solution (inositol-free) for 1 hour, and then treated with 10 ⁇ M of the compound for 1 hour. Inositol 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. The results are shown in Figure 6. Of the 2000 compounds used, the vast majority had minimal effect on inositol uptake into cells. 23 compounds increased cellular uptake of inositol, while 12 compounds significantly reduced cellular uptake.
  • purified Kd 5 ⁇ M 314/313-4RU1-P313C/F314W-cpYFP was used to analyze inositol in the blood supernatant of mice and humans.
  • the inositol 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 methods. It can locate and quantify inositol in real time inside and outside cells and can also perform high-throughput compound screening.

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Abstract

La présente invention concerne une sonde optique de myo-inositol ainsi que son procédé de préparation et son utilisation. Plus particulièrement, l'invention concerne une sonde optique de myo-inositol, comprenant un polypeptide sensible au myo-inositol et un polypeptide optiquement actif, le polypeptide optiquement actif étant situé à l'intérieur de la séquence du polypeptide sensible au myo-inositol.
PCT/CN2025/093250 2024-05-07 2025-05-07 Sonde optique de myo-inositol ainsi que son procédé de préparation et son utilisation Pending WO2025232798A1 (fr)

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