WO2002074950A1 - Procede d'analyse d'interaction intermoleculaire - Google Patents

Procede d'analyse d'interaction intermoleculaire Download PDF

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Publication number
WO2002074950A1
WO2002074950A1 PCT/JP2002/001719 JP0201719W WO02074950A1 WO 2002074950 A1 WO2002074950 A1 WO 2002074950A1 JP 0201719 W JP0201719 W JP 0201719W WO 02074950 A1 WO02074950 A1 WO 02074950A1
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protein
molecule
substance
group
nucleic acid
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PCT/JP2002/001719
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English (en)
Japanese (ja)
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Naoto Nemoto
Takashi Funatsu
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Mitsubishi Chemical Corporation
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Publication of WO2002074950A1 publication Critical patent/WO2002074950A1/fr

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules

Definitions

  • the present invention relates to a method for detecting a protein molecular interaction. More specifically, the present invention relates to a method for quantitatively detecting a protein molecular interaction by using a single molecule labeling method and a single molecule imaging method.
  • Nemoto and colleagues discovered that adding a molecule of puromycin and a fluorescent molecule to a cell-free translation system at a certain concentration adds a fluorescent molecule specifically to the C-terminus of the synthesized protein. (Nemoto, N., et al (1999) FEBS Lett. 462, 43-46).
  • the one-molecule imaging method and the one-molecule labeling method as described above can be combined, it becomes possible to construct a system with unprecedented speed and quantification. Furthermore, it is technically very significant if a protein chip such as a DNA chip that enables large-scale parallel processing is successfully made. However, unlike DNA, proteins have extremely difficult issues in terms of stability and purification methods. For example, those already reported as protein chips are limited to extremely stable proteins such as antibodies. At present, a method for immobilizing a protein encoded by cDNA or the like on a substrate while maintaining its function has not yet been established. Disclosure of the invention
  • An object of the present invention is to provide a method for rapidly and quantitatively measuring the intermolecular interaction between such a protein, nucleic acid, sugar chain or bioactive molecule and a target molecule.
  • the present inventors have conducted intensive studies to solve these problems and found that puromycin conjugated with a fluorescent label and biotin for immobilization can be converted to the C-terminal of the protein to be immobilized by using a cell-free translation system. It has been found that the protein can be immobilized on a support or the like via a streptavidin on a glass membrane or the like without losing its function by specifically introducing the protein into the protein.
  • the present inventors have shown that when the protein immobilized as described above is measured using the single-molecule imaging method, the number of immobilized molecules can be identified very accurately. I found it.
  • the present invention has been completed based on these findings.
  • Protein labeling in which one molecule of a labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group bind to one molecule of a substance capable of binding to the C-terminus of a protein A C-terminal labeled protein using a reagent.
  • a labeling substance and an affinity substance or a covalent bond-forming reactive group are respectively bound to a substance having an ability to bind to the C-terminus of a protein via a spacer.
  • the protein according to (1) In a protein labeling reagent, a labeling substance and an affinity substance or a covalent bond-forming reactive group are respectively bound to a substance having an ability to bind to the C-terminus of a protein via a spacer.
  • the affinity substance is a substance selected from the group consisting of biotin, maltose, guanine nucleotide, metal ion, daltathione, protein binding DNA, antigen molecule, calmodulin binding peptide, ATP, and estradiol.
  • the bond-forming reactive group is a ketone group, a diol group, an azide group or a psoralen;
  • the protein according to any one of (1) to (3).
  • a protein labeling reagent consisting of one molecule of a substance capable of binding to the C-terminus of a protein and one molecule of a labeling substance bound to one molecule of an affinity substance or a covalent bond-forming reactive group Using nucleic acids encoding proteins in the presence of The protein according to any one of (1) to (5), which is obtained by performing protein synthesis in a transcription / translation system.
  • the combination of the specific polypeptide bound to the solid phase / the affinity substance in the molecule or the covalent bond-forming reactive group is biotin binding protein / biotin, maltose binding protein Z maltose, G protein / guar Nin nucleotides, polyhistidine peptide, metal ions, daltathione-S-transferase gnolection, DNA binding protein ZDNA, antibody / antigen molecule, calmodulin / calmodulin binding peptide, ATP binding protein ZATP, and estradiol receptor protein Z estradiol, ketone Group / hydrazide group, diol group Z hydrazide group, azide group / alkyl group, psoralen z nucleobase (nucleic acid base such as pyrimidine ring or purine ring or analog thereof), (1) or Protein according to any one of (8).
  • a protein chip comprising a protein aggregate bound to a solid phase according to any one of (7) to (9).
  • the affinity substance is a substance selected from the group consisting of biotin, maltose, guanine nucleotide, metal ion, daltathione, protein-binding DNA, antigen molecule, calmodulin-binding peptide, ATP, and estradiol.
  • a covalent bond-forming reactive group which is a ketone group, a diol group, an azide group or a psoralen,
  • Specific polypeptide bound to solid phase ⁇ Affinity substance in molecule or combination of covalent bond forming reactive groups is biotin binding protein ⁇ Piotin, maltose binding protein ⁇ Maltose, G protein ⁇ Guanin Nucleotide, polyhistidine peptide / metal ion, glutathione s_transferase ⁇ gunoletathione, DNA binding protein ZDNA, antibody Z antigen molecule, calmodulin / calmodulin binding peptide, ATP binding protein / ATP, and estradiol receptor protein / estradiol , A ketone group Z hydrazide group, a diol group / hydrazide group, an azide group / alkyl group, and a psoralen Z nucleic acid base. The substance described in Crab.
  • a chip comprising an aggregate of substances bound to a solid phase according to any one of (15) to (17).
  • (21) A method for analyzing the interaction between a protein, nucleic acid, sugar chain or bioactive molecule and a target molecule, comprising the following steps:
  • (24)-A s a protein having one molecule of the first labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group, one molecule having the ability to bind to the C-terminus of the protein
  • a protein labeled at the C-terminus with a protein labeling reagent consisting of one molecule of a labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group from (21) (23) The method according to any one of the above.
  • a labeling substance and an affinity substance or a covalent bond-forming reactive group are respectively bound to a substance having an ability to bind to the C-terminus of a protein via a spacer. The method described in (24).
  • the covalent bond-forming reactive group is a ketone group, a diol group, an azide group or a psoralen;
  • (29) A substance having the ability to bind to the C-terminus of a protein as a protein having one molecule of the first labeling substance and one molecule of an affinity substance or 3 ⁇ 4a bond-forming reactive group.
  • Transcription / translation system using a nucleic acid that encodes a protein in the presence of a protein labeling reagent consisting of one molecule of a labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group bound to a molecule The method according to any one of (21) to (28), wherein a protein obtained by performing protein synthesis in is used.
  • (30) As a nucleic acid having one molecule of a first labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group, one of the 5 ′ end and the 3 ′ end is one molecule.
  • (31) A protein or nucleic acid having one molecule of a first labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group in a molecule is converted into an affinity substance or a covalent bond-forming reaction in the molecule.
  • the combination of an affinity substance or a covalent bond-forming reactive group in a specific polypeptide molecule bound to a solid phase is a combination of biotin-binding protein / biotin, maltose-binding protein Z-maltose, G-protein Z guanine.
  • step (a) Prior to the above step (a), the amount of the protein, nucleic acid, sugar chain or bioactive molecule immobilized on the solid phase is determined using the signal of the first labeled substance using evanescent light.
  • step (34) Using the signal intensity of the second labeling substance detected in step (b) and the amount of the protein, nucleic acid, sugar chain, or bioactive molecule immobilized on the solid phase, which has been previously quantified, (34) The method according to (34), further comprising a step of measuring the signal intensity per unit amount of the immobilized substance by using an Epane light to quantitatively determine the intensity of the interaction between the molecules.
  • FIG. 1 shows a schematic diagram of detection of a binding protein by single-molecule imaging.
  • fluorescent molecules that move randomly are not detected as a background
  • light is emitted according to the residence time.
  • C uses the C-terminal labeling method.
  • FIG. 2 shows a schematic diagram of a technique for identifying an unknown gene that interacts with a target molecule.
  • (D) adds a functional group or the like to the C-terminus of the target molecule in a cell-free translation system
  • (E) performs label synthesis at the same time as protein synthesis in a cell-free translation system
  • (F) shows each target molecule.
  • the amount of protein immobilized on the gel is quantified, and in (G), the magnitude of the interaction is measured in a few seconds in proportion to the fluorescence intensity.
  • Figure 3 shows the results of analyzing the interaction between kinesin molecules and microtubule molecules using single molecule labeling and single molecule imaging.
  • Figure 4 shows the structural formula of a puromycin derivative (Cy5-biotin-puro) containing the fluorescent dye Cy5 and biotin.
  • FIG. 5 shows the results of labeling a protein with a pure mouth mycin derivative (Cy5-biotin-puro) having a fluorescent dye Cy5. And biotin.
  • FIG. 6 shows the results of an experiment in which Cy5-biotin-puro was immobilized on a support (streptavidin membrane or streptavidin slide glass).
  • FIG. 7 shows the results of quantitative determination of a biotin-bound fluorescent molecule (DNA labeled with biotin and a fluorescent substance) using evanescent light.
  • a protein comprising a molecule having the ability to bind to the C-terminus of a protein and one molecule of a labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group bonded thereto
  • a C-terminal labeled protein (hereinafter, also referred to as C-terminal labeled-protein) is provided using a labeled ⁇ drug.
  • the C-terminal labeled protein of the present invention comprises a protein part and a label part in which the C-terminal of the protein is labeled (labeled) with a labeling reagent.
  • 'Material part' refers to an interaction subject whose function is known or unknown. And the presence or absence of an interaction between this protein part and a target molecule described later.
  • This protein part may be any of a natural protein or a mutant thereof, and an artificial protein or a mutant thereof.
  • the natural protein also includes a diverse library of proteins transcribed and translated from cDNA libraries derived from various organs, tissues or cells of organisms.
  • the artificial protein includes a sequence obtained by combining all or a partial sequence of a natural protein, or a sequence containing a random amino acid sequence.
  • labeling reagent is a reagent consisting of one molecule of a substance capable of binding to the C-terminus of a protein and one molecule of a labeling substance bound to one molecule of an affinity substance or a covalent bond-forming reactive group. It is. That is, one molecule of the label reagent has one molecule of a labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group, and has a substance capable of binding to the C-terminal of a protein.
  • Each of the labeling substance and the affinity substance or the covalent bond-forming reactive group may be directly chemically bonded to a substance capable of binding to the C-terminus of the protein, or may be chemically bonded via a spacer. They may be combined.
  • the labeling substance is usually selected from non-radioactive labeling substances such as fluorescent substances.
  • the fluorescent substance has a free functional group (for example, a hydroxyl group that can be converted to an active ester, a hydroxyl group that can be converted to a phosphoramidide, or an amino group), and has the above-mentioned nucleic acid derivative such as Rescueomycin or puromycin-like compound.
  • Various fluorescent dyes that can be linked, for example, any of a fluorescein series, a rhodamine series, an eosin series, and an NBD series.
  • the fluorescent substance examples include Cy5 (Amersham), Cy3 (Amersham), IC5 (Dohjin Chemical), IC3 (Dohjin Chemical), fluorescein, tetramethylrhodamine, Texas Red, Atalizine Orange, etc. Is mentioned.
  • a chemiluminescent substance for example, luminol, atarizinimu I, etc. may be used as the labeling substance.
  • the substance has affinity for a specific substance
  • the types such as proteins, peptides, saccharides, lipids, and low molecular weight compounds are not particularly limited.
  • Specific examples of the affinity substance include biotin, maltose, guanine nucleotide, metal ion, glutathione, protein-binding DNA, antigen molecule, calmodulin-binding peptide, ATP, and estradiol.
  • Examples of the covalent bond-forming reactive group include a ketone group, a diol group, an azide group, and a psoralen. .
  • nucleic acid derivative As the "substance having the ability to bind to the C-terminus of a protein" constituting the labeling reagent, a nucleic acid derivative is usually used. This nucleic acid derivative is limited as long as it is a compound capable of binding to the C-terminus of the synthesized protein when protein synthesis (translation) is performed in a cell-free protein synthesis system or a living cell. However, it is possible to select one whose 3 ′ end has a similar chemical structure skeleton to aminoacyl tRA.
  • Representative compounds include Puromycin having an amide bond, 3, -N-aminoacylpuromycin aminonucleoside (3'-N-Aminoacylpuromycin aminonucleoside, PANS-amino acid), and 7-amino acid.
  • PANS-Gly with the glycine part PANS-Val with the amino acid part of palin
  • PANS-Ala with the amino acid part of alanine and other PANS monoamino acid compounds in which the amino acid part corresponds to all amino acids .
  • AANS-amino acid 3 -N-aminoacyladenosine aminonucleoside (AANS-amino acid) is linked by an amide bond formed by the dehydration condensation of the amino group of 3'-aminoadenosine and the amino group of amino acid.
  • the amino acid part corresponds to MNS-Gly of glycine
  • the amino acid part corresponds to AANS-Val of palin
  • the amino acid part corresponds to AANS-Ala of alanine
  • the amino acid part corresponds to each amino acid of all amino acids.
  • AANS-amino acid compounds can be used.
  • nucleosides or nucleosides and ester bonds of amino acids can also be used.
  • all compounds chemically linked to a nucleic acid or a substance having a chemical structure skeleton and a base similar to a nucleic acid and a substance having a chemical structure skeleton similar to an amino acid are included in the nucleic acid derivative used in the present method. It is. 'As a substance having the ability to bind to the c-terminus of a protein, puromycin, a compound in which a PANS-amino acid or AANS-amino acid is bound to a nucleoside via a phosphate group is more preferred.
  • puromycin ribocytidine ⁇ puromycin (rCpPur), deoxydilpuromycin (dCpPur), and deoxyperidylpuromycin (dUpPur)
  • rCpPur ribocytidine ⁇ puromycin
  • dCpPur deoxydilpuromycin
  • dUpPur deoxyperidylpuromycin
  • Specific examples of the substance constituting the protein labeling reagent of the present invention include a general formula: X— (L 1 ) m —A— (L 2 ) n -Y
  • X represents a residue of a labeling substance
  • ⁇ represents a residue of an affinity substance or a covalent bond-forming reactive group
  • L 1 and L 2 each independently represent a divalent spacer group.
  • M and n each independently represent an integer of 0 or 1
  • A represents a residue of a substance capable of binding to the C-terminus of the protein
  • Residue of labeling substance represented by X, affinity substance or residue of covalent bond forming group represented by Y, residue of substance capable of binding to C-terminal of protein represented by A Specific examples include the labeling substance and the affinity substance described above, or the residue of a covalent bond-forming reactive group.
  • divalent spacer group L 1 and L 2 examples are shown, fat aliphatic hydrocarbon group of 1 about 0-1 carbon atoms (e.g., an alkylene group, an alkenylene group) or a polyethylene emissions, polyethylene da recall And the like.
  • n and n are preferably 1.
  • the labeling reagent is a chemical bond known per se between the above-mentioned labeling substance and the above-mentioned affinity substance or a covalent bond-forming reactive group and a substance having the ability to bind to the C-terminus of the protein via a spacer, if desired. It can be manufactured by bonding by a method. Specifically, for example, a “substance capable of binding to the C-terminus of a protein” protected with an appropriate protecting group is bound on a solid-phase carrier, and is used as a spacer using a nucleic acid synthesizer.
  • Spacer phosphoramidite, fluorescent substance or affinity substance or It can be prepared by sequentially linking a phosphoramidite having a covalent bond-forming reactive group, followed by deprotection.
  • the components may be combined by a liquid phase synthesis method, or both may be used in combination.
  • the binding of a metal ion such as nickel as an affinity substance can be performed using a chelating reagent such as uritrilotriacetic acid or iminodiacetic acid to which the metal ion can be coordinated.
  • an aliphatic hydrocarbon group having about 1 to 10 carbon atoms may be used. (Eg, an alkylene group, an alkylene group, etc.) and a polymer derived from a polymer such as polyethylene and polyethylene glycol.
  • the method for preparing the C-terminal labeled protein of the present invention is not particularly limited. For example, in the presence of the labeling reagent, the coding region encoding the protein is
  • ⁇ It is ligated under the control of a promoter region derived from a virus or cell such as 7 and transcribes it to synthesize mRNA.
  • a promoter region derived from a virus or cell such as 7
  • transcribes it to synthesize mRNA As the coding region D N ⁇ ⁇ , a sequence from which a stop codon has been deleted is preferably used in order to improve the efficiency of labeling by several tens of times.
  • mRA obtained from a living body by a method known per se can be used. These mRNAs can be prepared by expressing them in a translation system and performing protein synthesis. .,
  • Examples of the translation system used include a cell-free translation system and living cells.
  • the cell-free translation system or living cell is not limited as long as protein synthesis is performed by adding or introducing a nucleic acid encoding a protein into the cell-free translation system or living cell.
  • a cell-free translation system composed of an extract of a prokaryotic or eukaryotic organism, for example, an Escherichia coli, a heron reticulocyte, a wheat germ extract, or the like can be used.
  • Prokaryotic or eukaryotic organisms such as E. coli cells, can be used as a living cell translation system.
  • RNA transcribed and synthesized by a method using a known RNA polymerase or the like is introduced as type III.
  • the C-terminus of the protein can be labeled with the labeling reagent by allowing the labeling reagent to be present at an appropriate concentration.
  • concentration of the labeling reagent to be present varies depending on the labeling reagent, nucleic acid or translation system actually used, but generally the final concentration is preferably in the range of 0.1 to 200M.
  • the concentration of the nucleic acid derivative to which the labeling substance, the affinity substance, or the covalent bond-forming reactive group used in the present invention is bound varies depending on the actually used RNA, labeling substance, nucleic acid derivative, translation system, etc. Those skilled in the art can appropriately determine the concentration by the method described above. That is, in the above-described system for preparing a C-terminal labeled protein, a nucleic acid derivative to which a label substance is bound is added at a different concentration, and the resulting translation product is separated using SDS polyacrylamide electrophoresis or the like. Then, measure the signal intensity emitted from the labeling substance bound to the C-terminus, and select the concentration showing the highest signal intensity.
  • the thus synthesized C-terminal labeled protein is lysed by a method known per se, and then gel filtration or the like (for example, Bio-Spin 6; BIO-Rad)
  • the unreacted labeling reagent can be removed and obtained.
  • unreacted labeling reagent may be removed by gel filtration.
  • the C-terminal labeled protein may be bound to a solid phase, but it is preferable to bind to the solid phase via an affinity substance or a reactive group capable of forming a covalent bond.
  • the affinity substance or covalent bond-forming reactive group used in the present invention is a molecule that specifically binds to a specific polypeptide, and binds the specific polypeptide that binds to the molecule to the solid phase surface.
  • the term “specific polypeptide” used herein includes a binding protein, a receptor protein constituting a receptor, an antibody, and the like.
  • Specific polypeptides / affinity substances bound to the solid phase or combinations of covalent bond forming reactive groups include, for example, a biotin-binding protein such as avidin and streptavidin, zubiotin, a maltose-binding protein / maltose, and a G-protein.
  • biotin-binding proteins such as avidin and streptavidin, maltose-binding protein / maltose, metal ions such as polyhistidine peptide nonnickel or covanolate, glutathione s-transferase / daltathione, antibody Z antigen molecule (Epitope) and the like, and a combination of streptavidin / pyotin is particularly preferred.
  • biotin-binding proteins such as avidin and streptavidin, maltose-binding protein / maltose, metal ions such as polyhistidine peptide nonnickel or covanolate, glutathione s-transferase / daltathione, antibody Z antigen molecule (Epitope) and the like, and a combination of streptavidin / pyotin is particularly preferred.
  • These binding proteins are known per se, and the DNA encoding the protein has already been cloned.
  • the binding of the above-mentioned specific polypeptide to the solid phase surface can be carried out by a method known per se. Specifically, for example, tannic acid, formalin, glutaraldehyde, pyrvic aldehyde, bis-one Benzizone diazotate, toluene-2,4-diisocyanate, an amino group, a carboxyl group, or a method utilizing a hydroxyl group or an amino group can be used.
  • a protein chip containing an aggregate of proteins is constructed by binding a large number of the C-terminally labeled proteins of the present invention to a solid phase. be able to.
  • Such a protein chip is useful for analyzing a molecular interaction between a protein and a target molecule.
  • nucleic acid having one molecule of a labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group in one molecule (hereinafter, also referred to as a labeled nucleic acid).
  • the labeling substance, affinity substance, or covalent bond-forming reactive group here is as described above in (1).
  • the nucleic acid of the present invention has a single-stranded nucleic acid in which either the 5 ′ end or the 3 ′ end is labeled with one molecule of a labeling substance, and a base sequence complementary to the base sequence of the single-stranded nucleic acid, It can be prepared by annealing one end of the 5 or 3 end with a single-stranded nucleic acid labeled with one molecule of an affinity substance or a reactive group capable of forming a covalent bond.
  • the type of the nucleic acid is not particularly limited, and may be DNA or RNA, and may be a naturally occurring nucleic acid, a nucleic acid produced by a genetic recombination technique, or a chemically synthesized nucleic acid.
  • nucleic acid of the present invention may be used by binding to a solid phase, and specific embodiments thereof are as described above in (1).
  • nucleic acids described above not only the nucleic acids described above, but also sugar chains or bioactive molecules may be used.
  • a means for holding and holding a support having a solid phase a means for introducing the C-terminal labeled protein or labeled nucleic acid of the present invention into the support, and a means for washing the support.
  • An apparatus for producing a chip such as a protein chip or a nucleic acid chip is provided.
  • a known suitable method is used. It is also possible to construct an apparatus by combining various means. Each means in the apparatus is itself known, and the operations of these means, such as holding the base, adding a C-terminal labeled protein or a label nucleic acid solution, and washing, are performed by methods known per se. ' Combining these operations, fully automatic or semi-automatic,
  • An apparatus for immobilizing a C-terminal labeled protein or a labeled nucleic acid can be constructed.
  • a method for analyzing an interaction between a protein, a nucleic acid, a sugar chain, or a bioactive molecule and a target molecule comprising the following steps.
  • the protein having one molecule of the first labeling substance and one molecule of a hydrophilic substance or a covalent bond-forming reactive group in one molecule has an ability to bind to the C-terminus of the protein.
  • Labeling of a protein in which one molecule of a labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group bind to one molecule of a substance that has a C-terminal labeled with a reagent Can be.
  • Specific examples of these C-terminally labeled proteins and nucleic acids having one molecule of a first labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group in one molecule are described in the present specification. It is as described above in the specification.
  • the target molecule in the analysis method of the present invention means a molecule that interacts with a C-terminal labeled protein or a labeled nucleic acid, and specifically includes a protein, a nucleic acid, a sugar chain, a low molecular compound, and the like.
  • the protein is not particularly limited as long as it has an ability to interact with a C-terminal labeled protein or a labeled nucleic acid, and may be a full-length protein or a partial peptide containing a binding activity site.
  • the protein may be a protein whose amino acid sequence and function are known, or may be an unknown protein.
  • the synthesized peptide chain may be a glycoprotein having a sugar chain bonded thereto.
  • the nucleic acid is not particularly limited as long as it has an ability to interact with a C-terminal labeled protein or a label nucleic acid, and DNA or RNA can also be used. Further, the nucleic acid may have a known nucleotide sequence or function, or may have an unknown nucleic acid. Preferably, those having a known function as a nucleic acid capable of binding to a protein and a base sequence, or those which have been cut and isolated from a genomic library or the like using a restriction enzyme or the like can be used.
  • the sugar chain is not particularly limited as long as it has an ability to interact with the C-terminal labeled protein or the labeled nucleic acid, and may be a sugar chain whose sugar sequence or function is known or unknown.
  • a sugar chain which has already been separated and analyzed and whose sugar sequence or function is known is used.
  • the low molecular weight compound is not particularly limited as long as it has the ability to interact with a C-terminal labeled protein or labeled nucleic acid. Either those with known functions or those with known ability to bind to proteins can be used.
  • the term “interaction” performed by these target molecules with a C-terminally labeled protein or labeled nucleic acid usually means a covalent bond, a hydrophobic bond, a hydrogen bond, a van der Waals bond, and a covalent bond between the protein or nucleic acid and the target molecule. It refers to the effect of forces acting between molecules resulting from at least one of the electrostatic couplings, but this term should be interpreted in the broadest sense. And should not be construed as limiting in any way.
  • the covalent bond includes a coordination bond and a dipole bond.
  • the coupling by electrostatic force includes not only electrostatic coupling but also electric repulsion.
  • the interaction also includes a binding reaction, a synthesis reaction, and a decomposition reaction resulting from the above-described actions.
  • interaction examples include binding and dissociation between an antigen and an antibody, binding and dissociation between a protein receptor and a ligand, binding and dissociation between an adhesion molecule and a partner molecule, and binding and dissociation between an enzyme and a substrate. Binding and dissociation between nucleic acids and their associated proteins, binding between proteins in the signal transduction system; association and dissociation, binding and dissociation between glycoproteins and proteins, or between sugar chains and proteins Bonding and dissociation between them.
  • the target molecule used in the present invention is used after being labeled with a labeling substance.
  • the labeling substance used for labeling the target molecule should be different from the labeling substance used for labeling the C-terminal labeled protein or labeled nucleic acid.
  • the labeling substance is usually selected from non-radioactive labeling substances such as fluorescent substances.
  • the fluorescent substance include various fluorescent dyes having a free functional group (for example, a carboxyl group, a hydroxyl group, an amino group, etc.) and capable of being linked to the above-mentioned target substances such as proteins and nucleic acids, for example, fluorescein series, rhodamine series, Any type such as eosin series and NBD series may be used.
  • the compound can be labeled, such as a dye, the type and size of the compound are not limited.
  • the fluorescent substance examples include Cy5 (Amersham), Cy3 (Amersham), IC5 (Dojindo), IC3 (Dojindo), fluorescein, tetramethylrhodamine, Texas red, and acrylic. Gin orange and the like.
  • a chemiluminescent substance for example, luminol, Ataridi-Pmumu I, etc. may be used as the labeling substance.
  • the binding of the labeling substance to the target molecule can be carried out using an appropriate method known per se. Specifically, for example, when the target molecule is a protein, the method of labeling the C-terminus described in (1) above can be used. When the target molecule is a nucleic acid, an oligo DNA primer to which a label Labeling can be carried out easily by a method of performing PCR using PCR.
  • the amount of the protein, nucleic acid, sugar chain, or biologically active molecule immobilized on the solid phase is determined using the first label with Kesen 10 light. It is preferable to include a step of quantifying using the signal of the substance as an index. Further, in the method of the present invention, the signal intensity of the second label detected in the step (b) is compared with the protein, nucleic acid, -sugar chain or physiological protein immobilized on a solid phase previously quantified. Measuring the signal intensity per unit amount of the immobilized protein or nucleic acid using the amount of the active molecule using evanescent light, thereby quantifying the intensity of the interaction between the molecules. preferable. In such a preferred embodiment of the present invention, it becomes possible to relatively easily quantify the intermolecular interaction between a protein, a nucleic acid, a sugar chain or a bioactive molecule and a target molecule.
  • the method for detecting the second labeling substance labeled on the target molecule is not particularly limited.
  • it can be detected using a fluorescent imaging analyzer.
  • a labeled molecule is brought into contact with a solid-phased molecule, and the interaction between the two molecules causes the fluorescent light emitted from the labeled dig molecule remaining on the solid-phased molecule to be converted into fluorescent light.
  • This is a method of measuring or analyzing using a commercially available fluorescent imaging analyzer.
  • a C-terminal labeled protein or a labeled nucleic acid is immobilized, and a target molecule labeled with a labeling substance is brought into contact with it.
  • C-terminal labeling The base for immobilizing proteins or labeled nucleic acids is ditrosenololose membrane or Nymouth membrane, which is usually used for immobilizing proteins and nucleic acids. A plastic microplate or the like can also be used.
  • the method for bringing the labeled target molecule into contact with the solid-phased molecule in the present method may be any method as long as the two molecules come into contact with each other to an extent sufficient for interaction.
  • Molecules into commonly used biochemical buffers It is preferable to prepare a solution in which the solution is dissolved at an appropriate concentration, and to contact the solution with the surface of the solid phase. After the two molecules are brought into contact with each other, a step of washing the excess of the labeled target molecule with the same buffer or the like is preferably performed, and a signal (fluorescent signal) emitted from the target molecule labeled substance remaining on the solid phase is obtained. Can be measured or analyzed using a commercially available imaging analyzer to identify molecules that interact with the immobilized molecule.
  • a method for performing a large number of analyzes at the same time includes, for example, a method of addressing a plurality of C-terminal labeled proteins or labeled nucleic acids on the solid surface and immobilizing the same, A method in which a plurality of types of labeled target molecules are brought into contact with an end-labeled protein or a labeled nucleic acid or the like is used.
  • the primary structure of the target molecule that has been found to interact with the C-terminal labeled protein or labeled nucleic acid by the above method is unknown, its primary structure is analyzed by an appropriate method known per se. can do. Specifically, when the target molecule recognized as interacting is a protein, the primary structure can be identified by analyzing the amino acid sequence using an amino acid analyzer or the like. In addition, when the target molecule is a nucleic acid, a base sequence can be determined by a base sequence determination method using an automatic DNA sequencer or the like. Thereby, the target molecule can be identified.
  • a device can also be constructed by combining known appropriate means.
  • Each means in the apparatus is itself known, and the operations of these means, such as holding of the substrate, addition of the target molecule, washing, and signal detection, may be performed by methods known per se. .
  • an apparatus for fully or semi-automatically measuring the interaction between a protein or nucleic acid and a target molecule can be constructed. All of the contents disclosed in the specification of Japanese Patent Application No. 2001-5202798, which is the application on which the priority of the present application is based, are disclosed herein by reference.
  • Example 1 Interaction analysis between kinesin molecule and microtubule molecule using single molecule labeling method and single molecule imaging method
  • Pure mouth mycin dihydrochloride (Wako Pure Chemical Industries) 250 mg in 30 ml of 0.3 M sodium carbonate-0.2 M sodium bicarbonate-2.0 M aqueous sodium salt solution (aqueous solution A) and 20 ml was dissolved in methylene chloride and stirred, and the organic layer was separated. Methylene chloride (20 ml) was added to the aqueous layer, liquid separation was performed, the organic layers were combined, concentrated by an evaporator, and then a small amount of pyridine was added and concentrated twice. It was dissolved in cetonitrile (5 ml) and pyridine (2.5 ml).
  • Trifluoroacetic anhydride (1.0 g) was added, the mixture was stirred at room temperature for 30 minutes, cooled with ice, water (5 ml) was added, and further 20 ml of aqueous solution A was gradually added. The operation of extracting twice with methylene chloride (20 ml), concentrating the obtained organic layer, adding pyridine and concentrating the mixture was repeated twice to obtain crude N0; -trif ⁇ uoroacetyl-puromycin (250 mg). .
  • the filtrate was added to a DMF suspension containing NovaSyn TG amino resin (novabiochem) and a catalytic amount of DMAP, followed by stirring at room temperature for 16 hours.
  • the resin was collected with a glass filter, washed with DMF, methanol, and ethyl acetate, and dried with a vacuum pump. Then, the amino groups of the unreacted resin were acetylated by the normal cabbing method in DNA synthesis. Puromycin was released from one part of the obtained puromycin resin by the usual deprotection method and quantified, and it was calculated as 67 ⁇ mol / gram resin, which was 7 times.
  • the extracted solution was concentrated, diluted with water, neutralized by gradually adding 30% acetic acid in an ice bath, freeze-dried, and the resulting solid was purified by reversed-phase high-performance liquid chromatography (HPLC). .
  • the column was C0SM0SIL 0DS AR-300 (20 vulgar 250 vulgar) (Nacalai) and eluted with a linear gradient of 0.1 M aqueous triethylamine solution and 80% acetonitrile-0.02 M aqueous triethylamine solution. About 6 micromol of the desired product, aminolink-dC-puromycin, was obtained.
  • Cy5 Monofunctional Dye (Amersham Pharmacia Biotech) Add 50 DMF to 1 tube, and immediately add 80 nmol of Aminolink-dC-Puromycin to 0.1 ⁇ M 0.15 M sodium carbonate buffer (pH 9.0) 100 ⁇ l. The dissolved solution was added and left for 1 hour at room temperature with occasional stirring. Shim-Pack CLC-0DS (4.6 mm x 250 mm) (Shimadzu Cy5-Puro was purified by reverse phase HPLC using the above solvent system. It was considered that labeling proceeded preferentially at the 5'-terminal amino group, and no product with a labeled amino group was detected.
  • a Kinesin fragment having Nco I and Sac I restriction enzyme sites at both ends is a pUC8 vector (phskinZ) that encodes kinesin lacking the C-terminal side, is a type I primer, and has the nucleotide sequence shown in SEQ ID NO: 1. And a primer having the nucleotide sequence described in SEQ ID NO: 2 was used for PCR. After purifying the obtained PCR product, the restriction enzymes Nco I and Sac I restriction enzyme sites at both ends is a pUC8 vector (phskinZ) that encodes kinesin lacking the C-terminal side, is a type I primer, and has the nucleotide sequence shown in SEQ ID NO: 1. And a primer having the nucleotide sequence described in SEQ ID NO: 2 was used for PCR. After purifying the obtained PCR product, the restriction enzymes Nco I and Sac I restriction enzyme sites at both ends is a pUC8 vector (phskinZ) that encodes kinesin lacking the C-
  • the linearized Kinesin DNAplasmid was applied to Transription Mix (Invitrogene) to a final concentration of 10 nM, and reacted at 30 ° C for 15 minutes.
  • Translation Mix (Invitrogene) 30 1 and Cy5-puro (final concentration 30 ⁇ ) are added and reacted for 1 hour, and translated at the same time. Cy5-purokinesin) is obtained. 7.5 ⁇ ⁇ ⁇ ⁇ ° / translated sample. Separation was performed by SDS-PAGE on acrylamide gel, and bands were visualized without staining using a 530DF30 band-pass filter of Fluor Imager (BioRad Co).
  • Streptavidin is immobilized on quartz glass via Biotinylated BSA, and a partially biotinylated Microtubule (extracted from pig brain and stained with TMR-SE) is bound thereto.
  • a partially biotinylated Microtubule (extracted from pig brain and stained with TMR-SE) is bound thereto.
  • Cy5-puro-kinesin coat the glass surface with lmg / ml Casein (Sigma), and gel-filter the Ki esin sample with NAP5 using AssayBuffer (80 mM PIPES, 2 mM MgC12, ImM EGTA).
  • FIG. 4 shows the structural formula of the obtained puromycin derivative (Cy5-biotin-puro) having the fluorescent dye Cy5 and biotin.
  • GFPuv4 As GFP, we use GFPuv4 '(Ito, Y. et al., Biocnemical and Biophysical Research Communication 264, 556-560, (1999)) discovered by Ito et al., And easily excite even with 488 nm laser light. It can be confirmed with an image analyzer.
  • a DNA construct having a T'7 promoter region and Kozak sequence upstream of this GFP was prepared as follows.
  • the DNA (SEQ ID NO: 3) having the T'7 promoter region and Kozak sequence is referred to as type I DNA.
  • a primer containing the 5′-side sequence of this type III (SEQ ID NO: 4) and a primer containing a part of the 3′-side complementary strand and the 5′-side complementary sequence of GFP (SEQ ID NO: 5) were used.
  • PCR was performed. The condition of PCR was 25 cycles of 20 seconds at 95 ° C, 20 seconds at 68 ° C and 20 seconds at 72 ° C, and was performed using EX Taq polymerase (Takara).
  • PCR was performed using a plasmid having the nucleotide sequence of SEQ ID NO: 6 and a primer having the nucleotide sequence of SEQ ID NO: 7, using the plasmid encoding GFPuv4 as type I.
  • PCR conditions were 95 ° C for 20 seconds, 68 ° C for 20 seconds and 72 ° C. Performed using EX Taq polymerase (Takara) with 30 seconds as 30 cycles o
  • PCR products were purified by phenol extraction and ethanol precipitation using a primer remover (Edge Biosystem). These DNA templates were added in equal amounts, and PCR was performed using a primer having the nucleotide sequence of SEQ ID NO: 4 and a primer having the nucleotide sequence of SEQ ID NO: 7. PCR conditions are 95 ° C for 20 seconds, 68. 20 seconds and 72 in C. C was performed using EX Taq polymerase (Takara) with a cycle of 40 seconds at 30 cycles. These PCR products were purified by phenol extraction and ethanol precipitation using a primer remover (Edge Biosystem). The purified DNA was transcribed using RiboMAX (Promega) to obtain mRNA for translation. '
  • the GFP mRNA having the T7 promoter and Cy5-biotin-puro synthesized by the above method were added to the wheat germ cell-free translation system so that the final concentrations would be 20 ⁇ ⁇ and 40 ⁇ , respectively.
  • the reaction was carried out at ° C for 1 hour.
  • experiments were also performed using Cy5-dC-Puro instead of Cy5-biotin-puro.
  • These reaction products were separated by 15% SDS-atarylamide electrophoresis and confirmed by a fluorescence image analyzer, Molecular Imager (Bio-Rad).
  • the results are shown in FIG. 5 (in FIG. 5, the results obtained using Cy5-dC-Puro are referred to as Cy5-Puro).
  • FIG. 5 the results obtained using Cy5-dC-Puro are referred to as Cy5-Puro).
  • Cy5-biotin-puro synthesized in Example 2, or without biotin for comparison Each of Cy5-puro was dissolved in 2 O mM (pH 8.0) Tris buffer, and the concentrations were adjusted to 5001, 100, and 10 ⁇ m. 50 of each solution was spotted on a streptavidin membrane (Promega) and allowed to stand for 20 minutes. Then, the plate was washed about 10 times with a 2 O mM (H8.0) tris buffer, and then confirmed with a fluorescence image analyzer Molecular Imager (Bio-Rad). Fig. 6 shows the results.
  • Cy5-biotin-puro can bind more specifically to streptavidin membrane or streptavidin slide glass than to Cy5-puro.
  • the immobilization ratio of Fluorpuro and Fluor-biotin-puro to streptin-conjugated vidin-methylene in Figure 6 was 15% for Fluorpuro and 88% for Fluor-biotin-puro after two washes. .
  • the amount of Fluor-biotin-puro immobilized on the slide glass was about 3.5 times that of Fluorpuro. That is, it can be seen that the immobilization of Fluor-biotin-puro on the slide glass is a sufficiently specific binding even in consideration of non-specific adsorption.
  • Example 4 Quantification of biotin-binding fluorescent molecules (DNA labeled with biotin and a fluorescent substance) by evanescent light
  • the number of biotin- and fluorescent-labeled DNAs adsorbed on the glass surface increases according to the concentration of the flowed biotin- and fluorescent-labeled DNA. 5
  • the number of bright spots is also proportional to about 30, about 60, and about 180 do it It was confirmed that it increased.
  • streptavidin was not immobilized on the glass surface as a control, no such increase was observed.
  • a C-terminal is used by using a substance having one molecule of a labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group in one molecule.
  • the amount of nucleic acid can be easily measured quantitatively.

Abstract

L'invention concerne un procédé de mesure, rapide et quantitative, d'interaction entre une protéine, un acide nucléique, une chaîne de polysaccharides ou une molécule à activité physiologique et une molécule cible. Elle concerne ainsi une protéine marquée à son C terminal à l'aide d'un réactif de marquage de protéine dans lequel une molécule de marqueur et une molécule d'une substance d'affinité, ou un groupe réactif formant une liaison covalente, sont liés à une molécule d'une substance pouvant se lier au C terminal d'une protéine.
PCT/JP2002/001719 2001-02-27 2002-02-26 Procede d'analyse d'interaction intermoleculaire WO2002074950A1 (fr)

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EP1617204A4 (fr) * 2003-04-23 2008-07-30 Olympus Corp Procede pour analyser la fluorescence moleculaire par eclairage evanescent
US7657133B2 (en) * 2004-08-18 2010-02-02 University Of Basel Single analyte molecule detection by fibre fluorescence probe
WO2006062200A1 (fr) * 2004-12-09 2006-06-15 Universal Bio Research Co., Ltd. Bibliothèque d’acides nucléiques et bibliothèque de protéines
JP5219144B2 (ja) * 2007-12-28 2013-06-26 公益財団法人新産業創造研究機構 新規アフィニティーラベル化方法及びラベル化方法を用いたスクリーニング方法
CN105418764A (zh) * 2010-08-27 2016-03-23 国立大学法人东京大学 蛋白质或胜肽的印刷方法、及蛋白质或胜肽阵列、功能性蛋白质或功能性胜肽的鉴定方法
JP2012202742A (ja) * 2011-03-24 2012-10-22 Fujifilm Corp 検出方法および検出装置
KR101167649B1 (ko) 2011-04-20 2012-07-20 한국과학기술원 세포 환경 내에서의 단일 분자 수준의 단백질-단백질 상호작용 분석 장치
JP5930286B2 (ja) * 2012-02-10 2016-06-08 国立大学法人金沢大学 薬物と高分子化合物相互作用確認用試薬及び相互作用の検査方法
KR20180117529A (ko) 2017-04-19 2018-10-29 주식회사 프로티나 단백질-단백질 상호작용 분석에 의한 약물 반응성 예측 방법

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11322781A (ja) * 1998-05-15 1999-11-24 Mitsubishi Chemical Corp タンパク質のラベル化化合物およびその化合物を用いたタンパク質のラベル化方法
WO2000068690A1 (fr) * 1999-05-07 2000-11-16 Riken Methode de detection de proteines en interaction mutuelle
WO2001004265A2 (fr) * 1999-07-12 2001-01-18 Phylos, Inc. Marquage de proteines c-terminales
WO2001016600A1 (fr) * 1999-08-31 2001-03-08 Mitsubishi Chemical Corporation Methode d'analyse d'une interaction mutuelle entre une proteine et une molecule
JP2001128690A (ja) * 1999-08-26 2001-05-15 Mitsubishi Chemicals Corp タンパク質−ｄｎａ連結分子及びその利用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11322781A (ja) * 1998-05-15 1999-11-24 Mitsubishi Chemical Corp タンパク質のラベル化化合物およびその化合物を用いたタンパク質のラベル化方法
WO2000068690A1 (fr) * 1999-05-07 2000-11-16 Riken Methode de detection de proteines en interaction mutuelle
WO2001004265A2 (fr) * 1999-07-12 2001-01-18 Phylos, Inc. Marquage de proteines c-terminales
JP2001128690A (ja) * 1999-08-26 2001-05-15 Mitsubishi Chemicals Corp タンパク質−ｄｎａ連結分子及びその利用
WO2001016600A1 (fr) * 1999-08-31 2001-03-08 Mitsubishi Chemical Corporation Methode d'analyse d'une interaction mutuelle entre une proteine et une molecule

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NAOTO NEMOTO ET AL.: "Fluorescence labeling of the C-terminus of proteins with a puromycin analogue in cell-free translation systems", FEBS LETTERS, vol. 462, 1999, pages 43 - 46, XP004260584 *

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