KR20130001739A - Method for detecting molecular interactions and kit therefor - Google Patents

Abstract

PURPOSE: A method for detecting the interaction of molecules such as physiologically active substances is provided to directly detect the interaction in real time. CONSTITUTION: A method for detecting molecular interaction comprises: a step of providing a first component containing a first detection material and a localizer of which position moves according to an externally applied driving force; a second component containing a second detection material which is conjugated with a label; a step of placing the first and second components to a position enabling interaction in a cell; and a step of applying the externally applied driving force and measuring the changed label; a step of detecting the interaction between the first and second detection materials. The first and second detection materials are a physiologically active substance. The externally applied driving force is a magnetic field. [Reference numerals] (*) Marker material; (A) Detection material A; (AA) Analysis criterion of interaction; (B) Detection material B; (B1) Position change of the marker material; (B2) Movement change of the marker material; (B3) Function or shape change of cells; (C) Detection material C; (CC) First component; (DD) Third component; (EE) Second component; (FF) Interaction; (M) Magnetic material; (P1,P2) Probe or intermediate(s)

Description

METHOD FOR DETECTING MOLECULAR INTERACTIONS AND KIT THEREFOR}

The present invention in vitro and in The present invention relates to a method for dynamically searching for the interaction of bioactive materials, for example, for analyzing the interaction in vivo . In addition, the present invention is in vitro and in The present invention relates to a method for detecting interaction of a substance for analyzing an interaction in vivo , for example, a bioactive substance, and detecting a desired target substance.

In general, a bioactive chemical compound refers to a compound that binds to biomaterials including proteins, nucleic acids, sugars or lipids and modulates their function or biological activity in vivo. Such bioactive compounds are extracted from organisms or obtained by chemical synthesis. For example, many antibiotics, including "cyclosporin A" (Novartis AG) and "FK506" (Fujisawa), which are used to alleviate the immune rejection that occurs during organ transplantation, Separated. Such natural or synthetic physiologically active compounds are tested as their pharmacological activity and developed into a new drug through clinical trials using animal and human models. However, many of the natural bioactive compounds are not suitable for industrial mass production due to the complexity of their structure, limitations in purification and separation techniques, and the like. Therefore, combinatorial chemicals and new organic synthesis methods have been studied as methods for the production of useful bioactive compounds in large quantities (Ref. Schreiber, S., 2000, Target-oriented and diversity-oriented organic synthesis in drug discovery, Science 287, 1964-1969).

Proteins in vivo exhibit their biological function by binding to other proteins. In general, two proteins of complementary structure interact with each other, and the bioactive compound specifically binds to a specific portion of the protein tertiary structure. In general, the interaction between two proteins strongly suggests that these proteins are functionally related, and bioactive compounds that specifically bind to specific regions of the protein can be regulated by, for example, disease-related proteins. In the case of the present invention suggests the development as a therapeutic agent that can diagnose, prevent, treat or alleviate the disease. Therefore, in the field of drug development, new target proteins can be detected or new drugs can be identified by confirming the interaction between "bait", a substance whose function and characteristics have already been identified, and "prey," an interaction partner to be searched and detected. Various methods for screening bioactive substances as candidates have been studied. To date, about 500 target proteins have been identified in relation to disease and are expected to increase to more than 5,000 in the next few years.

Since the interpretation of the human genome, various studies on the role of bioactive substances in various metabolic and signaling processes in cells have been carried out more rapidly. Research areas such as chemical genomics, chemical biology and chemical proteomics have emerged. These include analysis design, functional genomics, proteomics, He is involved in the development of various technologies, including automatic engineering and bioinformatics.

In to search for the interaction between the protein As in vitro methods, traditionally biochemical methods such as cross-linking, radiolabeled ligand binding, X-ray crystallography and affinity chromatography, affinity blotting ( affinity blotting, immunoprecipitation and mass spectrometry have been developed and used (see EM Phizicky and S. Fields, Microbiol. Rev., 59: 94-123, 1995; AR Mendelsohn and R. Brent, Science, 284: 1948-1950, 1999). However, these methods require protein production and isolation that require a lot of effort, and in Because of the interactions performed in vitro , it does not provide information consistent with intracellular interactions.

In order to overcome this disadvantage, some functional cloning such as yeast two-hybrid (Y2H), yeast tree-hybrid (Y3H; references: Licitra, EJ, and Liu, JO, 1996, A three-hybrid system for detecting small ligand-protein receptor interactions.Proc. Natl. Acad. Sci. USA 93, 12817-12821), drug-western; references: Tanaka, H., Ohima, N. , and Hidaka, H. 1999, Isolation of cDNAs encoding cellular drug-binding proteins using a novel expression cloning procedure: drug-western.Mol.Pharmacol. 55, 356-363), phage-display cloning (Ref. Sche, PP , McKenzie, KM, White, JD, and Austin, DJ 1999, Display cloning: functional identification of natural product receptors using cDNA-phage display.Chem. Biol. 6, 707-716), fluorescence resonance energy transfer , FRET; References: Moshinsky DJ, Ruslim L., Blake RA, Tang F., A widely applicable, high- throughput TR-FRET assay for the measurement of kinase autophosphorylation: VEGFR-2 as a prototype.J Biomol Screen. 2003 Aug; 8 (4): 447-52) and mRNA display cloning (Ref. McPherson, M., Yang, Y., Hammond, PW, and Kreider, BL, 2002, Drug receptor identification from multiple tissues using cellular-derived RNA display libraries. Chem. Biol. 9, 691-698).

However, among these, the yeast two-hybrid system is difficult to apply to humans when the size and complexity of the genome is low, the reliability of the results is difficult and difficult to interpret. In addition, the system should be selected according to the protein's original cell distribution position, and the transcriptional activity may be affected by the problem of securing a high quality library and the metabolism and growth, and the hybrid protein may be toxic.

Y3H, on the other hand, is characterized by the discovery of interactions between bioactive compounds and proteins in living cells. However, Y3H, like Y2H, also has limitations to be performed in simple cells such as yeast cells, and is not suitable for analysis of full-length membrane proteins, and posttranslational expression of endogenous proteins in yeast. It is difficult to apply to interactions between bioactive compounds and proteins that require modifications or accessory proteins. In particular, there is a problem that yeast cells have a genetically lower permeability to bioactive compounds compared to mammalian cells.

In addition, bioinformatics methods have been developed that use bioinformatics to predict the interactions between proteins. This method predicts interactions between proteins through phylogenetic or multiple sequence alignment based on database genomic sequence information. In addition, structural genomics, molecular fingerprints, and various cluster analysis methods have been relatively recently developed and are virtual screening methods.

As such, many methods have been proposed to explore the interactions between proteins, but there is a continuing need for more effective and easier to manipulate methods. In addition, various techniques have been developed for screening bioactive compounds using interactions with target proteins. For example, as a biochemical method, Ding et al. (Ding, S. et al.) Screened a library of purine-like molecules using affinity chromatography to neuron P19 stem cells. Compounds that induce differentiation into cells were detected (reference: Ding, S., TYH, Brinker, A., Peters, EC, Hur, W., Gray, NS, and Schultz, PG 2003, Synthetic small molecules that control stem) cell fate.Proc. Natl. Acad. Sci. USA 100, 7632-7637). However, this method has a drawback that it can be applied to the search only when the affinity is very high, and the effective detection is possible only when a large amount of the target protein is present. In addition, most of the bioactive compounds are hydrophobic and competitively bind to the target protein of interest in an aqueous environment. Therefore, when the extract to be passed through the agarose or other support to which the bioactive compound is immobilized, the protein is May form a bond not only with a specific binding partner but also with a nonspecific binding partner. Therefore, in this method, a washing step to remove binding molecules having low affinity is essential.

Genetic methods have been proposed to overcome the shortcomings of such biochemical methods. Zheng et al. Used an yeast tri-hybrid system to explore increased drug sensitivity in heterozygotes and hosts. The greatest advantage of this genetic method is the strong association between phenotype and genotype. In other words, one hit found in the search using this system provides a direct clone of the gene for the target protein (Ref. Jaeger, S., Eriani, G., and Martin, F. 2004, Results and prospects). of the yeast three-hybrid system.FEBS Lett. 566, 7-12). In this system new target protein candidates can be fused and used as part of a multivalent protein complex. However, this system has a disadvantage in that it should be limited to yeast.

Chemogenetics uses bioactive compounds to indicate the effects of gene mutations on cells, identifying the active compound of interest from a group of compounds or from a combination library (Ref .: Schreiber, SL 2003, The small molecule approach to biology.Chem. & Eng. 1199 News 81, 51-61; Crews, CM, and Splittgerber, U. 1999, chemical genetics: exploring and controlling cellular processes with chemical probes.Trends Biochem. Sci. 24, 317-320). This method is typically performed by arranging hundreds to thousands of compounds in a well of a microtiter plate with genetically engineered cells to search for the desired phenotype (Ref .: Clemons, PA, Koehler, AN, Wager, BK, Springs, TG, Spring, DR, King, RW, Schreiber, SL, and Foley, MA 2001, A one-bead, one-stock solution approach to chemical genetics: part 2. Chem. Biol. 6, 71-83 ). For example, if the desired result is to induce the expression of a particular gene or group of genes, then the cell may be inserted with an associated promoter that induces the expression of green fluorescence protein (GFP) or other commercial markers. And reporter constructs. However, this method has also been found to have two major problems: First, chemical genetics is not suitable for screening bioactive compounds that function in trace nM units. Second, this method typically finds only a few active substances by screening hundreds of thousands of substances.

As described above, the search for interactions between proteins, or the prior art to explore the interaction between proteins and physiologically active compounds, in most It can be performed in vitro or only in restricted cells such as yeast or bacteria, and must be genetically modified and expressed, thereby limiting the size of the DNA and due to the high false positive hit rate due to other parameters. The reliability of the detected result is low.

The present inventors overcome the problems of the prior art, in as well as in vitro In order to explore the dynamic interaction of bioactive materials in vivo in real time, studies have shown that bioactive materials can be incorporated into magnetic-materials or particles containing magnetic materials that can be moved by magnetic fields. In addition, other bioactive substances bound to the labeling substance for detection were introduced into the cell, and then the magnetic field was applied to the cells, and the position of the label moved under the magnetic field was measured to detect and verify the binding between the bioactive substances. By this, the present invention was completed.

Generally, the present invention is in in vivo, ie intracellular, tissue or in vivo and in It is an object of the present invention to provide a method for detecting target substances in real time and searching for interactions between molecules, for example, bioactive substances, which are intended to analyze interactions in vitro or extracellularly. Specifically, the present invention is in vitro and in and in vivo for the purpose of providing a specific biomolecule is "first search substance" and, the physiologically active substance and the target of a search method that directly explore the interaction between the "second search substance".

In addition, the present invention in vitro and in The present invention relates to a method for directly detecting the interaction between a "first searcher" and a "second searcher" in vivo and detecting a target material of interest.

In addition, the present invention is in vitro and in An object of the present invention is to provide a kit or chip for directly detecting the interaction between the "first searcher" and the "second searcher" in vivo .

In addition, the present invention is to search and target a target material for blocking or inhibiting, activating or inducing the interaction of the "first search material" and the "second search material" It can be used as a method for detecting a target material.

In addition, the present invention is in vitro and in The present invention relates to a method of indirectly exploring the interaction between a "first searcher" and a "second searcher" by observing a change in the form or function of a cell in vivo .

As described above, according to the present invention, the same reaction system includes a first reactant which is changed in position according to an action force applied from the outside, and a first construct of the bioactive material and another bioactive material having a label for the search. By reacting in a system or field, the complex formed by interaction can be directly searched in real time from the repositioned label. In addition, the position change of the conjugate is reversible because it can be measured dynamically according to the strength of the external action force. Accordingly, the invention is in without destroying the cell, without limitation to the size of the target material to be searched, and detecting an interaction between the biomolecule vitro and in Since it can be effectively performed in vivo , it can be very useful for not only searching and detecting target proteins and drug candidates for drug development but also for improving existing drugs or discovering new pharmaceutical uses.

1 shows a schematic of the method of the invention.
2 is a confocal microscope photograph of the change in the position of the FITC-coated nanoparticles by applying a magnetic field from the outside after introducing the nanoparticles coated with the FITC green fluorescent dye into the cell.
Figure 3 is a confocal micrograph showing the shift of fluorescence in cells expressing FKBP12-RFP fusion protein (red) by an externally applied magnetic field compared to cells expressing EGFP (green).
4 is a confocal microscope showing the positional shift of a conjugate under reversible fluctuations of an externally applied magnetic field to cells (indicated by red arrows) as a result of the interaction between the FKBP12-RFP fusion protein and its pharmaceutical binding partner, FK506-MNPs. It is a photograph.
FIG. 5 is a confocal micrograph showing the positional shift under reversible fluctuations of an externally applied magnetic field of mRFP-Caspase3 expressing cells (indicated by red arrows).
FIG. 6 is a confocal micrograph showing the positional shift of the IκBα protein and RelA protein complex under reversible fluctuations of an externally applied magnetic field as a result of the interaction between the IκBα protein and RelA protein.
FIG. 7 is a confocal micrograph showing the position shift of anti-phospho-IκBα antibody-IκBα protein complex under reversible fluctuation of externally applied magnetic field as a result of detecting phosphorylated IκBα protein using anti-phospho-IκBα antibody. .
8 is a confocal micrograph showing the positional shift of the UAS oligonucleotide and GAL4 protein complex under reversible fluctuations of an externally applied magnetic field.
Figure 9 is a conceptual diagram showing the formation of the complex and its position shift according to the interaction of let-7b miRNA and lin-28 mRNA binding thereto.
10 is a confocal micrograph showing the positional shift of miRNA and mRNA complexes under reversible fluctuations of an externally applied magnetic field.
FIG. 11 is a confocal micrograph showing the position shift of beta-CD and MBP protein complexes under reversible fluctuations of an externally applied magnetic field.
12 is a conceptual diagram of an experiment verifying binding partner proteins in microarrayed cells.
13 is a schematic diagram of IκBα-mRFP, IκBα-ECFP-FKBP12, EYFP-RelA, and mRFP-βTrCP fusion proteins.
14 is a schematic diagram showing that phosphorylated IκBα-mRFP binds to nanoparticles by anti-phospho-IκBα (Ser32) antibody after TNF-α treatment.
FIG. 15 is a confocal microscope photograph of the change in position of a nano-particle complex formed after TNF-α treatment by an externally applied magnetic field as shown in the schematic diagram of FIG. 14.
FIG. 16 is a schematic diagram showing that the fusion proteins described in FIG. 13 form nanoparticle complexes after TNF-α treatment.
17 is a confocal micrograph of observing that the nanoparticle composite formed as shown in the schematic diagram of FIG. 16 is changed in position by an externally applied magnetic field.
18 shows the results of confirming the inhibition of HDAC protein activity by CGK1026.
19 is a confocal micrograph showing that the binding of Biotin-FK506 and FKBP12 is competitively inhibited by the treatment of FK506.

In order to achieve this object, the inventors have designed two constructs. Specifically, one or more "first search material" to provide a localizer that is changed in position according to an externally applied driving force and to be analyzed, for example, a bioactive material Was prepared directly or indirectly bonded using a linker or the like. The mobile reactant of the first component and the searcher to be analyzed may be provided as one directly fused component. In addition, the first component may be provided as two or more components indirectly combined with a mobile reactant fused with a probe capable of recognizing a probe to be analyzed and one or more probes to be analyzed. The second construct may comprise one or several second searchers, eg, bioactive substances, to which one or more labels may be bound, to enable search. The labeling material of the second component and the bioactive material to be analyzed may be provided as one component directly fused. In addition, the second component may be provided as two or more components indirectly combined with a label fused with a probe capable of recognizing a bioactive substance to be analyzed and one or more bioactive substances to be analyzed. After approaching the first component and the second component to a position where they can interact in the same field or system, applying a force from the outside and changing the position or movement of the marker to a simple device By measuring, the complex formed by the interaction can be dynamically explored in real time. In addition, the interaction can be indirectly searched by measuring changes in the morphology or function of the cells after application of the external force. In the present invention, when the first search material is "bait", the second search material is "prey", when the second search material is "bait", the first search material is "prey" Can be used as ". In the present invention, the position or movement of the label may be changed by an optical method including a microscope, a scanner, a radioactive label detecting means, a FP (fluorescence polarization reader), a spectrophotometer, a magnetic resonance imaging (MRI), a SQUID, It can be measured using generally known methods such as a fluorescence detector and a luminescence detector.

Specifically, in the present invention, the method for searching for interaction of materials includes: i) a first construct combining the first search material with a localizer which is moved in accordance with an externally applied driving force. Providing; ii) providing a second construct comprising a second search material bound to a label for search; iii) approaching the first component and the second component to an interactive position in the same field or system; And iv) applying an external action force and measuring the label whose position or movement has changed.

In addition, the detection method of the target material in the present invention, i) providing a first component combined with a mobile reaction material and the first search material to be moved in accordance with the external action force; ii) providing a second construct comprising a second search material bound to a label for search; iii) approaching the first component and the second component to a position where they can interact in the same field or system; iv) applying an external force and measuring the marker whose position or movement has changed; And v) separating and identifying a substance contained in the first component or the second component. Detection of the target material can be carried out by known methods such as RT-PCR, PCR for genomic DNA or Mass Spectroscopy.

In addition, the present invention can prepare three constructs to explore the interaction of substances and detect target substances. Specifically, i) providing a first structure in which the mobile reaction material and the first search material coupled to the position moving according to the external action force; ii) providing a second construct comprising a second search substance bound to a label; iii) providing a third component comprising a substance for screening; iv) approaching the first component, the second component and the third component to a position where they can interact in the same field or system; And v) applying an external action force and measuring a label whose position or movement has changed; And vi) separating and identifying a material contained in the first, second, or third constructs. Detection of the target material can be carried out by known methods such as RT-PCR, PCR for genomic DNA or Mass Spectroscopy.

In the present invention, the target material to be searched constituting the first component, the second component or the third component may be provided in the form of a library.

In one embodiment, the present invention uses a magnetic force as an action force applied from the outside. As a mobile reactant constituting the first component, a nano-particle comprising a magnetic material, preferably a material having a size of 5 nm to 2,000 nm, is provided to the mobile reactant and a specific material, for example, physiological It is used by combining or coating the active material. The transfer reaction material may be provided in combination with or coated with two or more specific materials. The second construct is a partner that interacts with the bioactive material in the first construct to make and use another bioactive material with a label for search. The target substance is searched for in real time by introducing the first and second constructs into the same cell, applying a magnetic field from the outside, and measuring a label moved under the magnetic field. In addition, a more dynamic search can be performed by changing the strength of the magnetic field, and a comparative search can be performed by using a material that does not change position or changes while changing the strength of the magnetic field as a positive or negative control.

In another embodiment, the present invention comprises the steps of: i) coating a specific bioactive compound on a magnetic particle, and ii) providing the coated magnetic particle of step i) with a target molecule bound to a labeling substance. Delivering into the cell, iii) applying a magnetic field to the cells of step ii) to move the magnetic particles in the cell, iv) measuring the position or movement of the label, and v) the label is capable of changing the position or movement. Detecting and identifying a labeled target substance in the cells shown.

Invention in when performed in vivo , in eukaryotic or prokaryotic cells; Mammalian in vivo, tissue and intracellular; It can be performed in vivo, in tissues and in cells of plants. In particular, the methods of the present invention can be carried out in the cells, tissues or in vivo of Zebra fish, C. elegans, yeast, Fly or Frog. In the present invention, the first and second constructs may be cell-transfer peptides (transducible peptides or fusogenic peptides), lipid gene carriers (lipids or liposomes), or combinations thereof, or the first and second constructs may be used in Opti-MEM. The cells may be cultured with the cells in the medium, or easily transferred into the cells by a generally known method such as electroporation or magnettofection. In particular, when the method of the present invention is carried out in cells, it can be carried out in a culture plate / dish or microarrayed reactor, for example in living cells.

In the present invention, the action force applied from the outside can be appropriately selected according to the characteristics including physical, chemical, biological and electrostatic properties of the mobile reactant included in the first component.

In the present invention, the "physiologically active substance" is a nucleic acid (nucleic acid), nucleotides (mono- / oligo- / poly-nucleotide), protein (protein), peptide (mono- / oligo- / poly-peptide), amino acid (amino acid) ), Including sugars (mono- / oligo- / poly-saccharides), lipids (lipids), vitamins, chemical compounds (chemical compounds, etc.) and constituents thereof, including all substances exhibiting physiological activity in vivo.

In the present invention, "bait" refers to a bioactive material used to explore the interaction with other bioactive materials.

In the present invention, "prey" refers to a biologically active substance to be searched or detected as an interaction partner of the "bait".

In the present invention, a "target molecule" means a "prey" that interacts with a "bait" or "activating" or "inducing" or "blocking" the interaction of a "bait" and a "prey" material. Means all substances to be inhibited.

In the present invention, the term "localizer" refers to a material which is moved in response to an externally applied driving force.

In the present invention, the "action force" applied from the outside refers to any kind of force that can bring about the change of position of the defined mobile reactant in the first component, including electric force, electromagnetic force, magnetic force and gravity.

In the present invention, the labeling substance constituting the second constituent and used in the search may be a fluorescent material which exhibits fluorescence by itself or exhibits fluorescence by interaction with the first construct or another substance, for example, FITC, rhodamine, fluorescent proteins such as GFP, YFP, CFP and RFP, and tetracysteine motifs or fluorescent nanoparticles, or they can emit light on their own, interact with the first construct or with other substances Luminescent material such as luciferase and the like and radioactive labels such as ³² P, ³⁵ S, ³ H and ¹⁴ C and the like which are generally used Include all possible markings.

In the present invention, the binding between the bioactive material and the transfer reaction material or the binding between the bioactive material and the labeling material includes physical, chemical, electrostatic and biological direct or indirect bonds, and the bioactive material is a moving reaction material or labeling material. It may be used to be coated on the surface of.

In the present invention, the probe (probe) constituting the first component or the second component and used for searching is combined with the bioactive material physically, chemically, electrostatically and biologically so that the probe material, the position regulator or the bioactive material and the label Substances capable of directly or indirectly binding a substance include, for example, antibodies, proteins, protein domains, protein motifs, peptides, and the like.

Hereinafter, the present invention will be described in more detail based on examples. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The references cited in the present invention are incorporated herein by reference.

Example

Example  One

Fluorescent dyes FITC Using nanoparticles coated with Intracellular  Check position change

FITC-MNP coated superparamagnetic nanoparticles (MNPs) with FITC by reacting streptavidin conjugated superparamagnetic nanoparticles (50 nm; streptavidin nanoparticles) with FITC-biotin and TATHA2-biotin -TATHA2 complex was obtained. TATHA2 peptides were synthesized according to the prior art (Wadia, et al., Nat Med. 10: 310-315 (2004)) and the N-terminus was labeled with biotin. As a negative control, QD565, a nanoparticle which does not cause position change by an externally applied magnetic field, was reacted with TATHA2-biotin to obtain a QD565-TATHA2 complex. After pre-cultured HeLa cells (purchased from ATCC) were washed with DMEM containing no serum, a solution of FITC-MNP-TATHA2 complex and QD565-TATHA2 complex, respectively, was mixed with Opti-MEM (Invitrogen) medium. The cells were treated and the cells were incubated for 12 hours in a CO ₂ incubator fixed at 37 ° C. to introduce the FITC-MNP-TATHA2 and QD565-TATHA2 complexes into HeLa cells. The cells were transferred onto the focal plate of the confocal microscope and magnetically applied to commercially available permanent or electromagnets at the bottom. According to known methods, the focal plate of the confocal microscope was adjusted and the morphology of the nanoparticles migrated to the cell bottom was observed (Molecular Cloning: A Laboratory Manual, CSHL Press; Current Protocols in Cell Biology or Molecular Biology, John Wiley and Sons Inc .; Cells: A Laboratory Manual, CSHL Press; Cell Biology: A Laboratory Handbook, Academic Press) (FIG. 2). It was confirmed that the FITC-MNP-TATHA2 complex was moved by an external magnetic field only in the cells into which FITC-MNP-TATHA2 was introduced. I didn't see it.

Example  2

Between various bioactive substances Intracellular  Interactive navigation

1) Screening of intracellular interactions using nanoparticles coated with fusion proteins and bioactive low molecular weight compounds with fluorescent proteins :

Streptavidin nanoparticles were reacted with FK506-biotin to superparamagnetic nanoparticles (MNPs) coated with bioactive small molecules (FK506) (MNP-FK506). HeLa cells expressing EGFP protein or FKBP-RFP fusion protein were washed with serum-free DMEM and then cells were washed with MNP-FK506 and Opti-MEM for 12 hours in a CO ₂ incubator fixed at 37 ° C. (Invitrogen) was cultured in a mixed medium to introduce MNP-FK506 into HeLa cells. The cells were transferred onto the focal plate of the confocal microscope and magnetically applied to the bottom with permanent or electromagnets. The focal plate of the confocal microscope was adjusted and the fluorescent protein moved to the cell bottom surface (Fig. 3). Only cells expressing FKBP12-RFP fusion protein (red) were moved by an external magnetic field, and cells expressing EGFP (green) used as a negative control (green) showed no change in position by magnetic field.

2) FK506 and its pharmaceutically related binding partner, FKBP12 ( FK506 binding Validation of intracellular interactions of protein 12):

Streptavidin nanoparticles were reacted with FK506-biotin and TATHA2-biotin for 1 hour at 4 ° C. while mixing to obtain FK506-MNPs complex. The reaction solution was purified using electromagnets or permanent magnets to remove unbound ligands. The plasmids expressing FKBP12 (FKBP-RFP) or EGFP fused to HeLa cells were introduced into HeLa cells, and the cells were separated, mixed, and cultured together in an incubator. Next, after the FK506-MNPs complex prepared above was introduced into these cells, a magnetic field (M. F., Magnetic Field) was applied from outside using a permanent magnet or an electromagnet. Confocal laser microscopy (Carl Zeiss) was used to take confocal images of living cells under and without magnetic fields. Only cells expressing FKBP12 fused with RFP (indicated by a red arrow) in the photograph of FIG. 4 are displaced by an external magnetic field, and in the case of cells expressing EGFP (indicated by a green arrow) used as negative controls, There was no change in position.

3) Case-phase inhibitor -3 (-3 caspase inhibitor) Verification of in interaction between the peptide and its binding part neoin Case -3 phase cells:

Streptavidin nanoparticles were reacted with caspase-3 inhibitor II (DEVD) -biotin conjugate (CALBIOCHEM) and TATHA2-biotin for 1 hour at 4 ° C. for 1 hour to mix the DEVD-MNPs complex. Obtained. The complex was purified using permanent magnets to remove unbound ligands. The plasmids expressing mEFP-mspFP-3 (mRFP-Caspase3) or EGFP fused to HeLa cells were introduced, and the cells were separated, mixed, and incubated together in an incubator. Next, after introducing the prepared DEVD-MNPs into these cells, a magnetic field was applied from outside using a permanent magnet or an electromagnet. Confocal laser microscopy (Carl Zeiss) was used to take confocal images of living cells under and without magnetic fields. Only cells expressing mRFP-Caspase3 (indicated by red arrows) in the photograph of FIG. 5 are displaced by an external magnetic field, and in the case of cells expressing EGFP (indicated by green arrows) used as negative controls. There was no change in position due to the magnetic field.

4) Validation of intracellular interactions of IκBα protein with its binding partner RelA protein :

Streptavidin nanoparticles were reacted with FK506-biotin and TATHA2-biotin for 1 hour at 4 ° C. while mixing to obtain FK506-MNPs complex. The complex was purified using permanent magnets to remove unbound ligands. After introducing plasmids expressing IκBα (IκBα-ECFP-FKBP12) fused with ECFP and FKBP12 and RelA (EYFP-RelA) fused with EYFP, HeLa cells were introduced with the FK506-MNPs prepared above. The magnetic field was applied from outside using a permanent magnet or an electromagnet. Confocal laser microscopy (Carl Zeiss) was used to take confocal images of living cells under and without magnetic fields. As shown in the photograph of FIG. 6, IκBα-ECFP-FKBP12 fusion protein and EYFP-RelA fusion protein were moved by an externally applied magnetic field by an externally applied magnetic field.

5) IκBα protein and antibody thereto for anti-phospho within -IκBα antibodies of the cell Validation of the interaction:

Streptavidin nanoparticles were reacted with anti-phospho-IκBα-biotin and TATHA2-biotin for 1 hour at 4 ° C. while mixing to obtain an anti-phospho-IκBα-MNPs complex. The complex was purified using permanent magnets to remove unbound ligands. After introducing a plasmid expressing IκBα (IκBα-mRFP) fused with mRFP to HeLa cells, the anti-phospho-IκBα-MNPs prepared above were introduced into these cells, followed by treatment with TNF-α for 5 minutes, followed by permanent magnetic material. Alternatively, a magnetic field was applied from the outside using an electromagnet. Confocal laser microscopy (Carl Zeiss) was used to take confocal images of living cells under magnetic fields and without magnetic fields (FIG. 7).

6) Validation of intracellular interaction of oligonucleotides ( UAS ) with binding sites of GAL4 protein and GAL4 protein :

An oligonucleotide, biotin-CCCAGTTTCTAGACGGAG (UAS-Biotin), comprising a DNA sequence UAS (SEQ ID NO: 1) capable of binding a yeast transcriptional activator GAL4 protein was synthesized. Streptavidin nanoparticles were reacted with UAS-biotin and TATHA2-biotin at 4 ° C. for 1 hour to obtain a UAS-MNPs complex. The complex was purified using permanent magnets to remove unbound ligands. After introducing a plasmid expressing GAL4 (GAL4-EGFP) fused with EGFP to HeLa cells, UAS-MNPs prepared above were introduced into these cells, and then a magnetic field was applied from outside using a permanent magnet or an electromagnet. Confocal laser microscopy (Carl Zeiss) was used to take confocal images of living cells under magnetic fields and without magnetic fields (FIG. 8).

7) miRNA Wow mRNA of Intracellular  Validation of the interaction:

To verify the interaction of miRNAs with their target mRNAs, let-7b miRNA (SEQ ID NO: 2) was selected that binds to lin-28 mRNA (FIG. 9). let-7b miRNA was synthesized with the structure of UGAGGUAGUAGGUUGUGUGGUU-biotin (let-7b-biotin), and FITC-CCCTATAGTGAGTCGTATTA (FITC-lin28p) was synthesized to label lin-28 mRNA (SEQ ID NO: 3). Streptavidin nanoparticles were reacted with let-7b-biotin and TATHA2-biotin for 1 hour at 4 ° C. while mixing to obtain a let7b-MNPs complex. The complex was purified using permanent magnets to remove unbound ligands. After introducing plasmids expressing lin-28 mRNA into HeLa cells, let7b-MNPs and FITC-lin28p prepared above were introduced into these cells, and then a magnetic field was applied from outside using a permanent magnet or an electromagnet. Confocal laser microscopy (Carl Zeiss) was used to take confocal images of living cells under magnetic fields and without magnetic fields (FIG. 10).

8) beta- With cyclodextrin MBP  Protein Intracellular  Validation of the interaction:

Beta-cyclodextrin (Sigma) was biotinylated (βCD-biotin) using the EZ-Link NHS-PEO Solid Phase Biotinylation kit (Pierce). Streptavidin nanoparticles, βCD-biotin and TATHA2-biotin were reacted with mixing at 4 ° C. for 1 hour to obtain βCD-MNPs complex. The complex was purified using permanent magnets to remove unbound ligands. After introducing plasmids expressing MBP (MBP-EGFP) fused with EGFP to HeLa cells, βCD-MNPs prepared above were introduced into these cells, and then a magnetic field was applied from outside using a permanent magnet or an electromagnet. Confocal laser microscopy (Carl Zeiss) was used to take confocal images of living cells under magnetic fields and without magnetic fields (FIG. 11).

Example  3

Microarrayed Intracellularly  Validation of Binding Partner Proteins

More than 1,000 human-derived full-length open reading frames (ORFs) genes were distributed by the Human Genome Functional Research Project. After over 1,000 genes were grown in E. coli cells, plasmid DNA was extracted and cloned into pcDNA-GFP-DEST vector (Invitrogen) for gene expression in animal cells. The gene inserted into the pcDNA-GFP-DEST vector produces a fusion protein in which the GFP protein is fused to the C-terminus of the target protein. Microarrayed cells were prepared according to conventional techniques (Ziauddin and Sabatini, Nature 411: 107-110 (2001)) and verified by expression of fused GFP fluorescent proteins. FK506-MNPs obtained in the same manner as in Example 2 were introduced into the microarrayed cells in the same manner as in Example 1, and then a magnetic field was applied from the outside using a permanent magnet or an electromagnet. Confocal laser microscopy was used to take confocal images of living cells under and without magnetic fields. RT-PCR and mRNA sequencing of the positive clones confirmed that the positive clones were FKBP12, a pharmaceutically relevant target of FK506 (FIG. 12).

Example  4

Cell signaling (signal post-translational modification of proteins and changes in protein complexes by transduction )

The TNF-α / IκBα signal transduction system was chosen to verify the intracellular translation and protein complex changes in cellular signaling. IκBα is complexed with NF-κB proteins RelA / p65, p50, c-Rel and the like to regulate various signals. Treatment of TNF-α with cells increased the activity of IκB kinase (IKK), resulting in phosphorylation of the 32nd serine and 36th serine of IκBα, resulting in the binding of IκBα and βTrCP proteins. Brown, et al., Science 267: 1485, (1995). In order to verify the phosphorylation of IκBα and the formation of IκBα-βTrCP complex by TNF-α treatment, the following expression constructs were prepared (FIG. 13). An expression vector in which the IκBα gene and the mRFP gene were fused was prepared to express the IκBα-mRFP fusion protein. In addition, an expression vector obtained by fusing the IκBα gene, the ECFP gene, and the FKBP12 gene was prepared to express the IκBα-ECFP-FKBP12 fusion protein. The EYFP-RelA fusion protein was expressed in cells by constructing an expression vector in which the EYFP gene and the RelA gene were fused. mRFP-βTrCP fusion protein was also used to construct an expression vector fusion of each gene. Anti-phospho-IκBα antibody (Cell Signaling Technology) was biotinylated using EZ-Link NHS-PEO Solid Phase Biotinylation kit (Pierce) and verified by Western blot.

1) Validation of Protein Phosphorylation by Cell Signaling:

Streptavidin nanoparticles and biotin-SS-FITC, biotin-TATHA2 and biotin-anti-phopho-IκBα (Ser32) antibodies were mixed at 4 ° C. for 1 hour to obtain IκBα (Ser32) -MNPs complex. After introducing the IκBα (Ser32) -MNPs complex into the HeLa cells into which the IκBα-mRFP expression vector was introduced in the same manner as in Example 1, 10 ng / ml of TNF-α was treated for 5 minutes and a confocal laser microscope was used. Confocal images of living cells were taken under magnetic fields and without magnetic fields (FIGS. 14 and 15). SC-514, an IKK inhibitor, was treated at 1 mM concentration. IκBα phosphorylated by TNF-α treatment was confirmed to be relocated by an externally applied magnetic force in combination with IκBα (Ser32) -MNPs complex, and the treatment of IKK inhibitor SC-514 did not cause phosphorylation of IκBα. It was verified that no position shift occurred.

2) The following experiment was performed to confirm the formation of protein complex by signal transduction .

The HeLa cells were transformed by introducing an IκBα-ECFP-FKBP12 fusion protein expression vector, an EYFP-RelA fusion protein expression vector, and an mRFP-βTrCP fusion protein expression vector into HeLa cells, and the FK506-MNPs complex was identical to that of Example 2. Obtained by the method and introduced into the cells. 10 ng / ml of TNF-α was treated for 5 minutes and a confocal laser microscope was used to take confocal images of living cells under a magnetic field and without a magnetic field (FIGS. 16 and 17). Irrespective of signal transduction, IκBα-ECFP-FKBP12 fusion protein and EYFP-RelA fusion protein caused site shift by externally applied magnetic force, whereas mRFP-βTrCP fusion protein which binds to phosphorylated IκBα by signal transduction Only when the TNF-α was treated, the position shifted. SC-514, an IKK inhibitor, did not interfere with the binding of IκBα-ECFP-FKBP12 fusion protein and EYFP-RelA fusion protein, but only inhibited the binding of IκBα-ECFP-FKBP12 fusion protein and mRFP-βTrCP fusion protein.

Example  6

Pharmaceutical exploration of interaction inhibitors and promoters drug screening )

1) Exploration of interaction promoters:

After introducing the IκBα-mRFP fusion protein expression vector and the IκBα (Ser32) -MNPs complex into HeLa cells in the same manner as in Example 4), the cells were treated with various compounds and growth promoters and applied with an external magnetic force. Position shift of IκBα-mRFP fusion protein was observed by confocal microscopy. It was confirmed that only TNF-α, IL-2, LPS, Fas, etc. among the physiologically active substances treated to the cells cause the position shift of IκBα-mRFP fusion protein due to external magnetic force (FIGS. 15 and 17).

2) FK506 Interaction suppression by:

After introducing the FK506-MNPs complex and the FKBP12-mRFP expression vector into cells in the same manner as in Example 3, various compounds were treated to observe the position shift of the FKBP12-mRFP fusion protein. Only FK506 in the treated compounds was found to competitively inhibit the position shift of the FKBP12-mRFP fusion protein (FIG. 19).

Claims (26)

i) providing a first component comprising a localizer whose position moves in accordance with an externally applied driving force and a first search material;
ii) providing a second construct comprising a second search material bound to a label for search;
iii) accessing the first construct and the second construct to a location in which the cells can interact; And
iv) exploring the interaction of the first searcher with the second searcher by applying an external force and measuring a label whose location or motion has changed. . The method of claim 1, wherein the first searcher or the second searcher is a bioactive molecule. 3. The method of claim 2, wherein the bioactive material is at least one material selected from the group consisting of nucleic acids, nucleotides, proteins, peptides, amino acids, sugars, lipids, chemical compounds, and constituents thereof. . 4. A method according to any one of claims 1 to 3, wherein the external force is a magnetic field. The method of claim 4 wherein the magnetic field is an electromagnetic or permanent magnetic field. 5. The method of claim 4, wherein the search is performed while varying the strength of the magnetic field. The method of any one of claims 1 to 3, wherein the transfer reactant comprises a magnetic material. 8. The method of claim 7, wherein the particle size of the mobile reactant with magnetic material is between 5 nm and 2,000 nm. The method of any one of claims 1 to 3, wherein the labeling substance is a radioactive substance, a fluorescent substance or a luminescent substance. 10. The method of claim 9, wherein the fluorescent or luminescent material exhibits fluorescence or luminescence by itself, or exhibits fluorescence or luminescence by combining with a particular substance or other substance of the first component. The method of claim 10, wherein the fluorescent material is a fluorescent dye, or a tetracysteine fluorescent motif, or a fluorescent protein comprising GFP, YFP, CFP, and RFP, or a fluorescent nanoparticle. The method of claim 1, wherein the mobile reactant in the first component is bonded or coated directly or indirectly with at least two specific materials. 13. The method of claim 12 wherein the combination of the mobile reactant and the first search material in the first construct is an electrostatic, physical, chemical or biological bond. The method of claim 1, wherein the label in the second component is bonded or coated directly or indirectly with the second search material. 15. The method of claim 14, wherein the binding of the labeling agent to the second searcher in the second construct is an electrostatic, physical, chemical or biological bond. 4. The antibody of any one of claims 1 to 3, wherein the antibody binds to the transfer reactant and the first searcher in a first construct or to the bond of the label and the second searcher in a second construct. And at least one probe selected from the group consisting of proteins, protein domains, protein motifs and peptides. The method of any one of claims 1 to 3, wherein said cell is a eukaryotic cell or prokaryotic cell. The method of claim 1, wherein the intracellular delivery of the first and second constructs is transducible peptide or fusogenic peptide, lipid gene carrier (lipid or liposome) or combinations thereof, electroporation. ), Or magnetofection. The method of claim 1, wherein the interaction of the first and second constructs is performed in cells in a culture vessel, or in microarrayed cells. 7. A method according to claim 6, characterized in that the material is searched comparatively as a positive or negative control for a substance which does not change position or change when the intensity of the magnetic field is changed. After detecting the interaction of the substances according to the method of any one of claims 1 to 3, further comprising the step of separating and identifying the target substance contained in the first component or the second component to detect the target substance How to. i) providing a first component comprising a localizer and a first search material which are moved in accordance with an externally applied driving force;
ii) providing a second construct comprising a second search substance bound to a label;
iii) providing a third component comprising a substance for screening;
iv) accessing the first construct, the second construct, and the third construct to a location in which the cells can interact; And
v) exploring the interaction of the substance by applying an external action force and measuring a label with a changed location or motion. cell;
A first component comprising a mobile reactant that is moved in position according to an external force, and a first search material that binds to the mobile reactant; And
A second substance comprising a labeling substance and a second search substance bound to the labeling substance,
The first component and the second component are introduced into the cell so that the first component and the second component interact with each other, and when the external force is applied, the labeling substance is formed of the first and second searching substances. A kit for exploring interactions of a substance, characterized in that the position or movement of the interaction changes. cell;
A first component comprising a mobile reactant that is moved in position according to an external force, and a first search material that binds to the mobile reactant;
A second component comprising a labeling substance and a second search substance which binds to the labeling substance; And
A third component comprising a substance for screening,
When the first component, the second component and the third component are introduced into the cell so that the first component, the second component, and the third component interact with each other, and the external force is applied, the labeling substance is formed by the interaction of the search substances. A kit for exploring the interaction of matter, characterized in that its position or movement changes with action. 25. The kit of claim 23 or 24, further comprising means for detecting changes in morphology or function of cells upon interaction of said search agents. The method of claim 1, further comprising exploring changes in morphology or function of the cell according to the interaction.

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