WO2011021581A1 - Marqueur sélectif pour biopolymère cible - Google Patents

Marqueur sélectif pour biopolymère cible Download PDF

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WO2011021581A1
WO2011021581A1 PCT/JP2010/063754 JP2010063754W WO2011021581A1 WO 2011021581 A1 WO2011021581 A1 WO 2011021581A1 JP 2010063754 W JP2010063754 W JP 2010063754W WO 2011021581 A1 WO2011021581 A1 WO 2011021581A1
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compound
atomic group
group
biopolymer
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Japanese (ja)
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真司 武岡
敏 新井
篤 村田
昌生 高林
美和子 尾崎
スーイン ユン
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学校法人早稲田大学
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    • 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
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

  • the present invention relates to a selective labeling agent for a target biopolymer, and more specifically to a fluorescent labeling agent that selectively emits light only when it binds to a target fusion protein containing a single amino acid repeat sequence.
  • Non-patent Document 1 a fluorescent protein such as a green fluorescent protein to obtain a labeled protein.
  • the green fluorescent protein contains 220 or more amino acids, it affects the higher-order structure and dynamics of the fusion protein, and may not reflect the function of the desired protein.
  • Non-Patent Documents 2 and 3 a method has been developed in which a protein in which a specific tag sequence consisting of two cysteines between two arbitrary amino acids and a specific tag sequence fused with the desired protein is expressed, and then contacted with a specific fluorescent organic arsenic compound for labeling.
  • this method is only suitable for proteins that are localized in the cytoplasm or nucleus since they are only effective when the cysteine residues are completely reduced. Also, malodorous 1,2-dithiol is necessary to reduce the toxicity of arsenic.
  • Non-patent Document 4 a method was developed in which a protein in which a hexamer peptide sequence of histidine was fused with a desired protein was expressed and labeled with nitrilotriacetic acid in the presence of Ni ions.
  • a fluorescent dye compound (chemical formula: 2 ′, 7′-bis (pyridyl-2-) in which a histidine hexamer peptide fusion protein, a ligand that forms a complex in the presence of Zn 2+ ions, and a fluorescent dye are linked.
  • Sulfonamide) -4 ′, 5′-dimethylfluorescein (hereinafter referred to as “HisZiFiT”) has also been developed (Non-patent Document 5, Patent Document 1).
  • the fluorescent dye compounds of these labeling methods emit fluorescence regardless of the presence or absence of complex formation with the target protein, the nonspecific background fluorescence prevents the target protein from being observed.
  • the fluorescent dye compound is injected into the cell, so it is difficult to remove by washing, and it is necessary to solve the problem of nonspecific background fluorescence. High nature.
  • Patent Document 2 a fluorescent dye compound that selectively emits light in association with a fusion protein of histidine hexamer peptide has been developed (Patent Document 2).
  • the fluorescent dye compound cannot emit fluorescence because a ligand and a chromophore of the fluorescent dye form a complex with a metal ion.
  • the chromophore removes the influence of the metal ion, so that it can emit fluorescence.
  • Co ions and Cu ions among the metal ions that form a complex with the fluorescent dye compound described in Patent Document 2 have redox reactivity, which may affect the function of the biopolymer. Therefore, the biopolymer labeling technique that requires these ions is not preferable.
  • the fluorescent dye compound described in Patent Document 2 is limited in usable fluorescent dyes because the chromogenic atomic group itself is involved in complex formation with metal ions.
  • the fluorescent dye compound when labeling a target substance in a living body and in a vesicle, the fluorescent dye compound cannot be sufficiently washed, and thus nonspecific background fluorescence may occur in the living body and in the vesicle. .
  • a complex can be formed with a biopolymer via a metal ion having no redox reactivity, and light can be selectively emitted only when associated with the biopolymer. Non-specificity can be achieved without washing. Therefore, it is necessary to develop a fluorescent dye compound that does not cause background fluorescence based on a principle that can be universally applied to existing fluorescent dyes having various fluorescent properties.
  • the present invention provides a selective fluorescent labeling agent for a target biopolymer.
  • the labeling agent of the present invention comprises a first compound containing a signal transmitting atomic group P and an atomic group X, and a second compound containing a signal absorbing atomic group Q and an atomic group Y, wherein the atomic group X is the atom.
  • a specific binding partner of group Y wherein when atomic group X and atomic group Y are linked by a non-covalent bond and the first compound and the second compound are associated, atomic group Q is said atomic group
  • the target biopolymer absorbs a signal emitted by P, competitively inhibits the connection between the atomic group X and the atomic group Y, and the target biopolymer is connected to the first compound via the atomic group X.
  • the atomic group Q cannot absorb the signal emitted by the atomic group P.
  • the atomic group X and the atomic group Y are each a receptor atomic group capable of forming a complex with a metal coordinating oligopeptide via a metal ion, the metal coordinating oligopeptide, A lectin and a sugar chain that specifically binds to the lectin, avidin and biotin, a protein or fragment thereof, and a protein or a protein that specifically binds to the protein or fragment thereof, or An antigenic determinant to which the antibody specifically binds to an antibody or an antigen-binding fragment thereof, or a fragment thereof, a single-stranded oligonucleotide and a single-stranded oligonucleotide complementary to the oligonucleotide A single-stranded or double-stranded oligonucleotide and a protein that specifically binds to the oligonucleotide or There are cases there between one and the other of the fragment.
  • the atomic group Y is R 1 or R 2
  • R 1 is -CONH- (Aaa 1 ) h- (Aaa 2 ) i- (Aaa 3 ) j- (Aaa 4 ) k -COOH
  • R 2 is NH 2- (Aaa 5 ) l- (Aaa 6 ) m- (Aaa 7 ) n- (Aaa 8 ) o -CONH- Aaa 1
  • Aaa 3 , Aaa 5 and Aaa 7 are each independently any one of 18 kinds of natural amino acids other than L-histidine and L-aspartic acid
  • Aaa 2 , Aaa 4 , Aaa 6 and Aaa 8 are each independently an amino acid residue of L-histidine or L-aspartic acid
  • h, j, l and n are each independently 0, 1 or 2
  • i, k, m and o may each independently be an integer of
  • each of the signal transmitting atomic group P and the signal absorbing atomic group Q is a fluorescent dye and a quencher specific to the fluorescent dye, or a nuclide that can be detected as an MRI contrast agent Or an MRI quencher for the element.
  • the signal transmitting atomic group P is pyrene, 7-methoxycoumarin, Cascade Blue, Alexa Fluor (registered trademark) 350, 7 Aminiocoumarin-X, Pacific Blue, dimethylaminocoumarin, BODIPY 493/503, BODIPY- FI-X, DTAF, 6-FAM, dansyl-X, Oregon Green 500, Alexa Fluor (registered trademark) 488, dT-FAM, Oregon Green 488, Rhodol Green, Oregon Green-N, 514, Rhodamine-X, RhodamineX TET, Alexa Fluor® 430, 2 ′, 4 ′, 5 ′, 7′-tetrabromosulfone fluorescein, BODIPY-FI Br2, 6-JOE, BODI Selected from the group consisting of PY 530/550, Alexa Fluor® 532 and HEX, wherein the signal absorbing group Q is BHQ1 or DABCY
  • the signal transmitting atomic group P may be an atomic group including a 19 F nuclide
  • the signal absorbing atomic group Q may be an atomic group including a Gd 3+ complex.
  • the labeling agent of the present invention may contain or consist of a complex of the first compound and the second compound.
  • the labeling agent of the present invention may contain a metal ion that specifically forms a complex with the first compound and the second compound.
  • the first compound is HisZiFiT
  • the second compound is BHQ2-His 6 or DABCYL-His 6
  • the first compound is TMR-triNTA and the second compound is DABCYL-His 6
  • the metal ion contained in the labeling agent of the present invention may be Zn 2+ when the first compound is HisZiFiT and the second compound is BHQ2-His 6 or DABCYL-His 6 , and the first compound is TMR-triNTA.
  • the second compound when the second compound is DABCYL-His 6 , it may be Ni 2+ .
  • the present invention provides a biopolymer labeling method.
  • the labeling method of the present invention comprises the step of preparing any labeling agent of the present invention and a biopolymer containing an atomic group that competitively inhibits the connection between the atomic group X and the atomic group Y of the labeling agent; And contacting the labeling agent with the biopolymer.
  • the labeling agent associates without removing the labeling agent not associated with the biopolymer.
  • the method may include a step of detecting a signal transmitted from the biopolymer.
  • the biopolymer labeling method of the present invention provides the first compound and the second compound of the labeling agent, and a biopolymer containing an atomic group that competitively inhibits the connection between the atomic group X and the atomic group Y. And the step of bringing the first compound and the second compound into contact with the biopolymer at the same time, or bringing either the first compound or the second compound into contact with the biopolymer first. And then contacting the other with the biopolymer.
  • the step of bringing the first compound and the second compound into contact with the biopolymer at the same time, or either the first compound or the second compound is performed first, After contacting the molecule, after the step of contacting the other with the biopolymer, the biopolymer with which the first compound is associated is removed without removing the first compound that is not associated with the biopolymer.
  • the method may include detecting a signal to be transmitted.
  • the present invention provides a method for labeling a protein.
  • the protein labeling method of the present invention comprises the steps of preparing a first compound, a second compound, and a fusion protein comprising an amino acid sequence comprising an amino acid sequence having 4 to 10 consecutive histidine residues, and a metal divalent ion Associating the first compound with the second compound in the presence of M 2+ , purifying the complex of the first compound and the second compound, and bringing the complex into contact with the fusion protein
  • the first compound is HisZiFiT and the second compound is BHQ2-His 6 or DABCYL-His 6
  • the first compound is TMR-triNTA and the second compound is DABCYL-His 6 .
  • the complex that is associated with the complex is not removed without removing the complex that is not associated with the fusion protein.
  • the method may include detecting a signal to be transmitted.
  • preparing the fusion protein may include introducing a gene construct that expresses the fusion protein into a cell.
  • the step of bringing the complex into contact with the fusion protein may include injecting the complex into the cell.
  • the first compound may be HisZiFiT
  • the second compound may be BHQ2-His 6 or DABCYL-His 6
  • the metal divalent ion M 2+ may be Zn 2+ .
  • the first compound may be TMR-triNTA
  • the second compound may be DABCYL-His 6
  • the metal divalent ion M 2+ may be Ni 2+ .
  • a certain atomic group is a fluorescent dye, biotin or other compound, lectin, antibody or other protein or fragment thereof, or oligonucleotide
  • the atomic group is the compound, A derivative group capable of covalent bonding with any atom constituting the molecule of the protein or fragment thereof and the oligonucleotide, and substantially functions as the compound, the protein or fragment thereof, or the oligonucleotide. It means that it does not interfere with.
  • a certain atomic group includes a fluorescent dye, biotin and other compounds, a lectin, an antibody and other proteins or fragments thereof, and an oligonucleotide” refers to the atomic group described above.
  • a derivative group capable of covalent bonding with any atom constituting the compound, the protein or fragment thereof and the molecule of the oligonucleotide, and the function of the compound, the protein or fragment thereof, or the oligonucleotide Means that it does not substantially inhibit.
  • the target biopolymer competitively inhibits the connection between the atomic group X and the atomic group Y because the target biopolymer is the atomic group Y or an atom that is not identical to the atomic group Y.
  • the group Z includes the group Z, and the group Z is connected to the group X to competitively inhibit the connection between the group Y and the group X.
  • the atomic group Z may have the same structure as the atomic group Y, a structure including the atomic group Y, or a structure listed below.
  • Group Z consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted or added to the amino acid sequence of the oligopeptide of group Y, and is a complex of group X and group Y It may contain a metal-coordinating oligopeptide that forms a complex with the atomic group X via the same metal ion that participates in the formation.
  • the atomic group Z includes an atom having a structure of a derivative or an analog of the atomic group Y.
  • the atomic group X and the atomic group Y are avidin and biotin, respectively, and the atomic group Z includes a biotin derivative or analog structure that binds to avidin at the same level or stronger than biotin. There is.
  • the atomic group Z is a protein of the atomic group Y or A protein of atomic group X having an amino acid sequence in which one or several amino acid residues are substituted, deleted, or added to the amino acid sequence of the fragment, and at the same level or stronger than the protein of atomic group Y or a fragment thereof Or it may be a protein or fragment thereof that binds to the fragment.
  • the atomic group Z is a single-stranded oligonucleotide of the atomic group Y.
  • the atomic group Z is Whether the amino acid sequence of the antibody or antigen-binding fragment thereof has an amino acid sequence in which one or several amino acids are substituted, deleted or added, or a derivative of an atomic group of the antigenic determinant or an atomic group of an analog In some cases, the atomic group is the same as or stronger than the atomic group Y.
  • Group Z has a nucleotide sequence in which one or several nucleotides are substituted, deleted or added to the nucleotide sequence of the single-stranded or double-stranded oligonucleotide, or 1 in the amino acid sequence of the protein or fragment thereof. It may be an atomic group that has an amino acid sequence in which one or several amino acid residues are substituted, deleted, or added and binds to the same degree or stronger than the atomic group Y.
  • the amino acid sequence of the fusion protein of the present invention is the amino acid sequence of 4 to 10 consecutive histidine residues at any position from the amino terminus to the carboxyl terminus, provided that the function of the fusion protein is not substantially inhibited. It may be included.
  • the amino acid sequence having 4 to 10 consecutive histidine residues is preferably arranged at a position where no steric hindrance occurs when the labeling agent of the present invention is accessed in the three-dimensional structure of the fusion protein. Alternatively, it is preferably arranged at the carboxyl terminus.
  • the fusion protein of the present invention When preparing the fusion protein of the present invention, it may be chemically synthesized by a solid-phase method or other well-known artificial synthesis methods, or may be produced cell-free or in a host cell by recombinant DNA technology. May be produced. When preparing the fusion protein of the present invention, it may be isolated and purified, but isolation and purification are not necessarily required. The preparation of the fusion protein of the present invention may be to prepare a cell, tissue, organ or individual that is derived from an organism into which DNA encoding the fusion protein has been introduced and expresses the fusion protein.
  • Fluorescence micrograph after washing of cells stained with TMR-triNTA quencher complex The graph of the fluorescence intensity before and behind washing
  • Fluorescence micrograph of a fluorescent background 1 ⁇ 10 2 seconds after DABCYL-His 6 was added to cells stained with TMR-triNTA.
  • a fluorescence micrograph of a fluorescent background 4 ⁇ 10 2 seconds after DABCYL-His 6 was added to cells stained with TMR-triNTA.
  • Quenching Agent BHQ2-His 6 Using a peptide synthesizer according to a conventional method, a quenching group BHQ2 group (chemical name: 4 ′-(4-nitro-phenyldiazo) -2′-) was added to the amino terminus of the hexamer peptide of histidine.
  • Quenching Agent DABCYL-His 6 Using a peptide synthesizer in accordance with a conventional method, quenching group DABCYL group (chemical name: 4-([4- (dimethylamino) phenyl] azo) benzoic acid) at the amino terminus of hexamer peptide of histidine acid succinimidyl ester (09278, sigma Aldo ridges) were reacted to, DABCYL histidine hexamer peptide linked to the amino terminus (hereinafter, "DABCYL-His 6" that.) of chemical formula .DABCYL-His 6 were synthesized Is as follows.
  • reaction products were purified using HPLC (Shimadzu Prominence, Shimadzu Corporation).
  • the elution conditions were 0.01M TFA (trifluoroacetic acid) aqueous solution as solution A, 0.01M TFA acetonitrile solution as solution B, and a flow rate of 1.0 mL per minute.
  • TFA trifluoroacetic acid
  • concentration gradient of solution B was 0-20% in 5 minutes, 20-50% in 15 minutes, 50-80% in 5 minutes.
  • DABCYL-His 6 was used, the concentration gradient of solution B was 0-10% for 2 minutes, 10-40% for 10 minutes, and 40-70% for 2 minutes.
  • the molecular weight of the purified sample was measured using a mass spectrometer, and it was confirmed that BHQ2-His 6 or DABCYL-His 6 was obtained.
  • HisZiFiT which forms a zinc ion-specific complex with an oligopeptide of histidine in vivo
  • HisZiFiT which forms a zinc ion-specific complex with an oligopeptide of histidine in vivo
  • HisZiFiT intermediate 1 is represented by the following chemical formula.
  • HisZiFiT intermediate 2 is represented by the following chemical formula.
  • HisZiFiT Intermediate 1 (100 mg, 0.222 mol) was dissolved in 52 mL of 1,2-dichloroethane-ethanol mixed solvent (5: 2), and 370 mg of palladium-carbon was added under a hydrogen atmosphere. After stirring for 2 hours and removing palladium-carbon by Celite filtration, 77 mg of a concentrated sample was obtained.
  • FIG. 1 shows emission spectrum of the fluorescent dye solution
  • the solid line in FIG. 1 is an absorption spectrum of the fluorescent dye solution.
  • the peak of the absorption spectrum of the fluorescent dye solution to which Zn 2+ ions were added was 526 nm
  • the peak of the emission spectrum was 553 nm. Therefore, a change in the emission spectrum at an excitation wavelength of 500 nm was observed.
  • FIG. 2 shows emission spectra before and after the addition of BHQ2-His 6 to the fluorescent dye solution to which Zn 2+ ions were added.
  • FIG. 2 is the emission spectrum of the fluorescent dye solution before the addition of BHQ2-His 6
  • the solid line in FIG. 2 is the emission spectrum of the fluorescent dye solution after the addition of BHQ2-His.
  • FIG. 2 when BHQ2-His 6 was added, a remarkable quenching phenomenon was observed in which the emission intensity at the emission peak wavelength of 553 nm decreased to 1/680 (excitation: slit width 3 nm, emission : Slit width 3 nm).
  • Results The emission spectra before and after the addition of DABCYL-His 6 to the fluorescent dye solution to which Zn 2+ ions were added are shown in FIG.
  • the solid line in FIG. 3 is the emission spectrum of the fluorescent dye solution before DABCYL-His 6 is added, and the broken line in FIG. 3 is the emission spectrum of the fluorescent dye solution after DABCYL-His 6 is added.
  • FIG. 3 when DABCYL-His 6 was added, a remarkable quenching phenomenon was observed in which the emission intensity at the emission peak wavelength of 553 nm decreased to 1%.
  • the emission spectra before and after the addition of His 6 to the fluorescent dye solution are shown in FIG.
  • the solid line in FIG. 4 is the emission spectrum of the fluorescent dye solution before the addition of His 6
  • the broken line in FIG. 4 is the emission spectrum of the fluorescent dye solution after the addition of His 6 .
  • FIG. 4 when His 6 was added, fluorescence recovery was observed in which the emission intensity at the emission peak wavelength (553 nm) decreased by addition of BHQ2-His 6 increased 5.4 times (excitation: slit width 5 nm). , Emission: slit width 5 nm).
  • Aqueous DABCYL-His 6 fluorescence recovery His 6 by His 6 of HisZiFiT which is quenched by the contained 5 mM 2 [mu] L (1 equivalents ratio relative HisZiFiT) is, after the Zn 2+ ion and DABCYL-His 6 were mixed Added to the fluorescent dye solution, an emission spectrum with an excitation wavelength of 470 nm was measured. Results The emission spectra before and after the addition of His 6 to the fluorescent dye solution are shown in FIG.
  • the solid line in FIG. 5 is the emission spectrum of the fluorescent dye solution before the addition of His 6
  • the broken line in FIG. 5 is the emission spectrum of the fluorescent dye solution after the addition of His 6 .
  • TMR-TriNTA The chemical formula of TMR-triNTA is as follows.
  • TMR-triNTA Quenching of TMR-triNTA by DABCYL-His 6
  • a DMSO solution containing 5 mM TMR-triNTA and a 10 mM NiCl 2 aqueous solution are prepared, and TMR-triNTA is dissolved in 20 mM Tris-HCl buffer (pH 7.4).
  • 0.5 mL of a fluorescent dye solution in which 0.3 mM and NiCl 2 were diluted to 3 mM (equivalent ratio 10 times with respect to TMR-triNTA) was prepared.
  • the fluorescent dye solution is purified using ion exchange chromatography HiTrap Q HP (17-1153-01, GE Healthcare) and contains 0.16 mM TMR-triNTA-3Ni (20 mM Tris-HCl buffer).
  • ion exchange chromatography HiTrap Q HP 17.1153-01, GE Healthcare
  • Results The emission spectra before and after the addition of DABCYL-His 6 to the fluorescent dye solution are shown in FIG.
  • the solid line in FIG. 6 is the emission spectrum of the fluorescent dye solution before the addition of DABCYL-His 6
  • the broken line in FIG. 6 is the emission spectrum of the fluorescent dye solution after the addition of DABCYL-His 6 .
  • FIG. 6 when DABCYL-His 6 was added, a remarkable quenching phenomenon was observed in which the emission intensity at the emission peak wavelength of 576 nm decreased to 8%.
  • the emission spectra before and after the addition of His 6 to the fluorescent dye solution are shown in FIG.
  • the solid line in FIG. 7 is the emission spectrum of the fluorescent dye solution before the addition of His 6
  • the broken line in FIG. 7 is the emission spectrum of the fluorescent dye solution after the addition of His 6 .
  • FIG. 7 when His 6 was added, fluorescence recovery was observed in which the emission intensity at the emission peak wavelength (576 nm) decreased by the addition of DABCYL-His 6 increased 5.4 times. From the above results, it was shown that TMR-triNTA-3Ni can react reversibly with DABCYL-His 6 and / or His 6 by association and dissociation.
  • a DMSO solution containing 5 mM TMR-triNTA and a 10 mM NiCl 2 aqueous solution are prepared, and TMR-triNTA is 0.3 mM and NiCl 2 is 3 mM in 20 mM Tris-HCl buffer (pH 7.4).
  • 0.5 mL of a fluorescent dye solution diluted to an equivalent ratio of 10 times with respect to TMR-triNTA was prepared.
  • the fluorescent dye solution is purified using ion exchange chromatography HiTrap Q HP (17-1153-01, GE Healthcare) and contains 0.16 mM TMR-triNTA-3Ni (20 mM Tris-HCl buffer).
  • 0.4 mL of a fluorescent dye solution in which TMR-triNTA was diluted to 5 ⁇ M in 20 mM Tris-HCl buffer (pH 7.4) was prepared.
  • TMR-triNTA 1 ⁇ L of an aqueous solution containing 0.5 mM of His 6 is added in a total amount of 4 ⁇ L, 2 ⁇ L of an aqueous solution containing 1 mM is added in a total of 6 ⁇ L, and an aqueous solution containing 5 mM is 2.4 ⁇ L (equal ratio of 0.25 to 10). Times) was added.
  • the fluorescence intensity of HisZiFiT was measured with an excitation wavelength of 500 nm and an emission wavelength of 553 nm, and TMR-triNTA with an excitation wavelength of 500 nm and an emission wavelength of 576 nm. The results were plotted in a graph in which the relative value of the fluorescence intensity with the fluorescence intensity of the solution sample not containing His 6 being 1 was plotted on the vertical axis and the concentration of His 6 on the horizontal axis.
  • Results A graph showing the relationship between the concentration of His 6 added to the complex of HisZiFiT or TMR-triNTA and the quencher and the fluorescence intensity is shown in FIG.
  • Black plot of rhombus ( ⁇ ) of FIG. 8 shows that the fluorescence of TMR-triNTA-3Ni complex is quenched by complexation with Dabcyl-His 6 was recovered by competition His 6.
  • Black triangular plot ( ⁇ ) of FIG. 8 shows that the fluorescence of HisZiFiT-2Zn complex is quenched by complexation with Dabcyl-His 6 has not recovered by competition His 6.
  • TMR-triNTA significantly recovers fluorescence in the presence of the histidine hexamer peptide to be labeled. On the other hand, it was revealed that almost no fluorescence recovery was observed with HisZiFiT.
  • COS7 cells are seeded in a culture dish having a diameter of 35 mm so as to form 7 ⁇ 10 4 cells, 10% fetal bovine serum, 100 ⁇ g / mL penicillin and 100 ⁇ g / mL in a 37 ° C., 5% CO 2 and saturated water vapor atmosphere. Cultured using Dulbecco's modified Eagle medium (D-MEM) supplemented with streptomycin.
  • D-MEM Dulbecco's modified Eagle medium
  • His-tagged protein Transgenic His 6 minus EGF-TD construct, as His 6 and EGF domain and PDGF transmembrane domain and is fused proteins (hereinafter, referred to as "His-tagged protein”.) Is expressed in mammalian cells, His 6 And a DNA fragment encoding the EGF domain was cloned into a pDisplay TM (Invitrogen) vector. 0.5 ⁇ g of the construct was transfected into COS7 cells using the effecten reagent (Qiagen).
  • COS7 cells transfected with a fluorescent staining His 6 -EGF-TD construct were cultured for 1 to 2 days, then washed twice with Hank's balanced buffer solution (hereinafter referred to as “HBSS”), and 1 ⁇ M HisZiFiT ( Stained at room temperature with 10 ⁇ M ZnCl 2 ), 1 ⁇ M TMR-triNTA (3 ⁇ M NiCl 2 ) or 5 ⁇ g / mL anti-His 6 antibody (11922416001, Roche). HisZiFiT and TMR-triNTA were diluted in 200 mM Tris-HCl buffer (pH 7.4).
  • the anti-His 6 antibody was diluted in D-MEM containing 10% fetal calf serum and used as a positive control. After the antibody was added as a primary antibody, the cells were washed twice with HBSS, and 6.67 ⁇ g / mL Alexa Fluor-633 antibody (A21050, molecular probe) was added as a secondary antibody. Thereafter, the cells were washed twice with HBSS and observed with a confocal microscope (FV1000, Olympus).
  • FIGS. 9A, 9B and 9C Fluorescence micrographs of COS7 cells that transiently express His-tagged proteins stained with anti-His 6 antibodies, TMR-triNTA and HisZiFiT are shown in FIGS. 9A, 9B and 9C, respectively. It was shown by immunostaining that a protein having His 6 was expressed in the transfected cells and localized on the cell surface. The fluorescence emission intensity on the cell surface reached a plateau within 1 minute after the addition of HisZiFiT and TMR-triNTA. It was shown that the fluorescence intensity of TMR-triNTA in cell staining was stronger compared to the fluorescence intensity of HisZiFiT. From the above results, it was shown that HisZiFiT and TMR-triNTA can selectively detect target molecules in vivo.
  • TMR-triNTA quencher complex DABCYL-His 6 and complexes detected DABCYL-His 6 and TMR-triNTA of His-tagged protein with the complex of TMR-triNTA (hereinafter, referred to as "TMR-triNTA quencher complex.") Is the target molecule in vivo It was evaluated whether it could be detected selectively.
  • TMR-triNTA-3Ni 1 ⁇ M TMR-triNTA-3Ni alone or TMR-triNTA-3Ni quencher complex (TMR-triNTA-3Ni (final concentration 1 ⁇ M) and DABCYL-His 6 (final)
  • TMR-triNTA-3Ni final concentration 1 ⁇ M
  • DABCYL-His 6 final
  • the mixture was stained at room temperature with a mixture of 1 ⁇ M).
  • the TMR-triNTA-3Ni alone and the TMR-triNTA-3Ni quencher complex were prepared in 200 mM Tris-HCl buffer (pH 7.4). The confocal microscope was used for observation.
  • FIGS. 10A and 10B Results Fluorescence micrographs before and after washing of cells stained with TMR-triNTA-3Ni alone are shown in FIGS. 10A and 10B, respectively.
  • FIGS. 10C and 10D show fluorescence micrographs before and after washing of cells stained with TMR-triNTA-3Ni quencher complex, respectively.
  • the TMR-triNTA-3Ni quencher complex caused the cell surface to specifically emit light without generating background fluorescence.
  • FIG. 11 shows a graph of fluorescence intensity (arbitrary unit) before and after washing on the surface and inside of the cells stained with fluorescence.
  • the error bar for each experimental condition indicates the standard deviation of the measured value of the experimental result repeated 6 to 9 times under the same condition.
  • An asterisk (*) indicates that the p-value is less than 1% in a two-sided unpaired t-test.
  • the fluorescence intensity of TMR-triNTA-3Ni alone is 3500 on the cell surface and 400 inside the cell, and the fluorescence intensity of TMR-triNTA-3Ni quencher complex is 3300 on the cell surface and 200 inside the cell. Met.
  • the fluorescence intensity of TMR-triNTA-3Ni alone was 3600 on the cell surface and 500 inside the cell, and the fluorescence intensity of the TMR-triNTA quencher complex was 3500 on the cell surface and 200 inside the cell. It was.
  • the fluorescence intensity of TMR-triNTA-3Ni separated from the TMR-triNTA-3Ni quencher complex is similar to the fluorescence intensity of TMR-triNTA-3Ni alone, and the TMR-triNTA-3Ni quencher complex
  • the body was shown to statistically significantly reduce the background fluorescence inside the cells, with or without cell washing, compared to TMR-triNTA-3Ni alone.
  • another selective labeling agent of the present invention can also easily and selectively detect a target molecule in vivo without a washing step, and the throughput of fluorescence detection for disease analysis, drug discovery, etc. is improved.
  • various fluorescent agents can be used for the selective labeling agent of the present invention, it was suggested that a plurality of target molecules can be selectively multiplexed.
  • a luminescent agent that emits near-infrared rays having high biological permeability it was suggested that individual level monitoring can be performed in real time.
  • DABCYL-His According to the 6 TMR-triNTA reduction DABCYL-His 6 single background fluorescence is to reduce the background fluorescence of TMR-triNTA alone was evaluated.
  • the combination of BHQ2-His 6 or DABCYL-His 6 with HisZiFiT can react reversibly with His 6 and therefore the combination is also non-specific in vivo. It was suggested that the target molecule can be selectively detected without generating background fluorescence. In addition, the detection system using this feature allows the target to be injected by simultaneously or differentially injecting the luminescent agent and the quencher, even when the selective target agent cannot penetrate the sample containing the target molecule. It was suggested that molecules can be selectively detected in vivo.

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Abstract

L'invention porte sur un marqueur fluorescent sélectif pour un biopolymère, lequel marqueur est obtenu par développement d'un composé de colorant fluorescent sur la base d'un principe qui est applicable universellement aux colorants fluorescents existants ayant une diversité de propriétés fluorescentes, le composé de colorant fluorescent étant un composé qui forme un complexe avec un biopolymère par l'intermédiaire d'un ion métallique n'ayant pas de réactivité d'oxydoréduction, et est capable d'assurer une luminescence sélective seulement lorsqu'il est associé au biopolymère et ne génère aucune fluorescence d'arrière-plan non spécifique même sans nettoyage conducteur. Le marqueur comprend un premier composé comprenant un groupe d'atomes P qui envoie un signal et un groupe d'atomes X, et un second composé comprenant un groupe d'atomes Q qui absorbe le signal et un groupe d'atomes Y, le groupe d'atomes X étant un partenaire de liaison spécifique du groupe d'atomes Y et, lorsque le groupe d'atomes X est lié au groupe d'atomes Y par une liaison non covalente pour associer le premier composé au second composé, alors le groupe d'atomes Q absorbe le signal envoyé par le groupe d'atomes P. L'invention porte également sur un marqueur sélectif pour une protéine, le marqueur comprenant HisZiFiT comme premier composé et BHQ2-His6 ou DABCYL-His6 comme second composé ou comprenant TMR-triNTA comme premier composé et DABCYL-His6 comme second composé.
PCT/JP2010/063754 2009-08-20 2010-08-13 Marqueur sélectif pour biopolymère cible WO2011021581A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3288932A4 (fr) * 2015-04-29 2018-12-05 Sanford-Burnham Medical Research Institute Nouveaux inhibiteurs d'epha4 ciblant son domaine de liaison du ligand

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2007125822A1 (fr) * 2006-04-28 2007-11-08 Tokyo Institute Of Technology Complexe utilisable pour le criblage d'une substance

Patent Citations (1)

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WO2007125822A1 (fr) * 2006-04-28 2007-11-08 Tokyo Institute Of Technology Complexe utilisable pour le criblage d'une substance

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HAUSER CT ET AL.: "A hexahistidine-Zn2+-dye label reveals STIM1 surface exposure.", PROC NATL ACAD SCI U S A., vol. 104, no. 10, 2007, pages 3693 - 3697 *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3288932A4 (fr) * 2015-04-29 2018-12-05 Sanford-Burnham Medical Research Institute Nouveaux inhibiteurs d'epha4 ciblant son domaine de liaison du ligand

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