KR101453554B1 - Method for detecting molecular interactions and kit therefor - Google Patents

Abstract

The present invention relates to a method for exploring the interaction of molecules such as physiologically active substances and a method for screening target molecules both in vivo and in vitro. The present invention provides a first search material and a second search material, wherein the first search material binds to a magnetic material that is displaced by a magnetic field and the second search material binds to a label. Thus, the complex of the first search material and the second search material can be reversibly searched by changing the intensity of the magnetic field, thereby effectively screening the target molecule.

Description

[0001] METHOD FOR DETECTING MOLECULAR INTERACTIONS AND KIT THEREFOR [0002]

The present invention relates to an in- vitro and in for example, a method for dynamically searching for the interaction of physiologically active substances in vivo . In addition, the present invention in vitro and in for example, the interaction of physiologically active substances for analyzing interactions in vivo , and a method for detecting a target substance of interest.

In general, a bioactive chemical compound refers to a compound that binds to a biological material including proteins, nucleic acids, sugars or lipids in vivo to control their function or biological activity. Such physiologically active compounds are obtained by extraction from an organism or 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 developed as a new drug after testing their pharmacological activity and clinical trials using animal models and human models. However, many of the natural bioactive compounds are not suitable for industrial mass production because of their complexity of structure, limitations of purification and separation techniques, and the like. Therefore, combinatorial chemicals and novel organic synthesis methods have been studied as a method for mass production of useful physiologically active compounds (Schreiber, S., 2000, Target-oriented and diversity-oriented organic synthesis in drug discovery, Science 287, 1964-1969).

Proteins in vivo exhibit their biological functions by binding to other proteins. Generally, two proteins of complementary structure interact, and the physiologically active compound specifically binds to a specific site of the protein tertiary structure. In general, the interaction between two proteins strongly suggests that these proteins are functionally related, and a physiologically active compound that specifically binds to a specific site of a protein can be obtained by controlling the activity of the protein, Suggests the possibility of development as a therapeutic agent capable of diagnosing, preventing, treating or alleviating a disease. Therefore, in the field of drug development, new target proteins can be detected by confirming the interaction between "bait", a substance whose function and characteristics have already been revealed, and "prey" Various methods for screening a physiologically active substance as a candidate substance have been studied. Up to now, about 500 target proteins have been identified in relation to disease and are expected to increase to over 5,000 in the next few years.

After the interpretation of the human genome, various studies on the role of physiologically active substances in various metabolism and signal transduction processes in cells have been conducted more rapidly. Research areas such as chemical genomics, chemical biology and chemical proteomics have emerged, including analysis design, functional genomics, proteomics, It is concerned with the development of various technologies such as automatic engineering and bioinformatics.

In to search for the interaction between the protein As an in vitro method there has traditionally been used 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, for example, 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 a protein production and isolation process that requires much effort, and in but it does not provide information consistent with intracellular interactions because it is an interaction carried out in vitro .

To overcome this disadvantage, several functional cloning such as yeast two-hybrid (Y2H), yeast tree-hybrid (Y3H; see Licitra, EJ, and Liu, A three-hybrid system for detecting small ligand-protein receptor interactions, Proc Natl Acad Sci USA 93, 12817-12821, drug-western (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 , 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; Reference: Moshinsky DJ, Ruslim L., Blake RA, Tang F., A widely applicable, high- (4): 447-52) and mRNA display cloning (Reference: McPherson, M., Yang, K. et al., Proc. Natl. 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, yeast two-hybrid systems are difficult to apply to humans because of the large size and complexity of the genome, resulting in poor reliability and difficult interpretation. In addition, the system should be selected according to the original intracellular distribution of the protein, the problem of securing a good library, the transcription activity depending on the cell metabolism and growth, and the hybrid protein may be toxic.

On the other hand, Y3H is characterized in that the search for the interaction between the physiologically active compound and the protein takes place in living cells. However, Y3H, like Y2H, has limitations to be performed in simple cells such as yeast cells, is not suitable for analysis of full-length membrane proteins, and is posttranslational it is difficult to apply to the interaction between a protein and a physiologically active compound requiring modification or an accessory protein. In particular, yeast cells have a lower genetically lower permeability to bioactive compounds than mammalian cells.

Other bioinformatic methods have been developed to predict interactions between proteins. This method is a method for predicting the interactions between proteins through systematic classification or multiple sequence alignment based on the database-based genome sequence information. Structural genomics, molecular fingerprint, and various cluster analysis methods have also been developed relatively recently and are a virtual screening method.

Thus, many methods have been proposed for exploring the interactions between proteins, but there is a continuing need for more effective and easier to manipulate methods. In addition, various techniques for exploring physiologically active compounds using interaction with target proteins have been developed. For example, as a biochemical method, Ding et al. (2002) screened a library of purine-like molecules using affinity chromatography to identify P19 stem cells (See 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 disadvantage in that it can be applied to a search only if the affinity is very high, and effective detection can be performed when a large amount of the target protein is present. In addition, most of the physiologically active compounds are hydrophobic and bind competitively with target proteins in an aqueous environment. Therefore, when an extract of a detection subject is passed through an agarose or other support on which a physiologically active compound is immobilized, Binds not only with specific binding partners but also with non-specific binding partners. Therefore, in this method, a washing step for eliminating binding molecules having low affinity is indispensably required.

Genetic methods have been proposed to overcome the disadvantages of such biochemical methods. Zheng et al. Used the yeast tree-hybrid system to explore heterozygotes and increased drug susceptibility in the host. The greatest advantage of this genetic approach is the strong association between phenotype and genotype. That is, one hit found in the search results using this system directly provides a clone of the gene for the target protein (see 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 protein complex. However, this system has a disadvantage that it should be used only in the east.

Chemo-genetics utilizes physiologically active compounds to indicate the effect of mutations on cells, and identifies the desired active compound from a group of compounds or a combinatorial library (Schreiber, SL 2003, 1999, chemical genetics: exploring and controlling cellular processes with chemical probes Trends Biochem. Sci., 24, 81, 51-61; Crews, CM, and Splittgerber, 317-320). This method is usually performed by arranging hundreds to thousands of compounds with genetically engineered cells in a well of a microtiter plate to search for the desired phenotype (cf. 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. ). For example, if the desired result is one that results in the expression of a particular gene or group of genes, the cell may be transfected with an associated promoter that results in the expression of green fluorescent protein (GFP) or other common marker Reporter constructs. However, this method also proved to have two major problems: First, the chemistry of genetics is not suitable for exploring physiologically active compounds acting in trace amounts of nM units. Second, this method can typically find only a few active substances through screening for hundreds of thousands of substances.

As described above, the conventional techniques for searching for the interactions between proteins or searching for the interaction between proteins and physiologically active compounds are mostly in can be carried out in vitro or can only be carried out in limited cells such as yeast or bacteria and must be subjected to a process of gene manipulation and expression so that it is limited to the size of the DNA and due to the false positive hit rate due to other parameters The reliability of the detected result is lowered.

The present inventors have overcome the problems of the prior art, in not only in vitro As a result of research into dynamic interaction of physiologically active substances in vivo , it has been found that a physiologically active substance can be bonded to a magnetic material or a particle containing a magnetic substance that can move by a magnetic field And then introducing another physiologically active substance having a labeling substance bound thereto into the cell, applying a magnetic field to the cell, measuring the position of the label moved under the magnetic field, and searching for the binding between these physiologically active substances Thereby completing the present invention.

In general, the invention is in vivo i.e. intracellular, intratracheal or in vivo and in vitro that is, for the material (molecules) to be analyzed the interaction examples outside the cell, to provide a method for searching for the interaction between the physiologically active substance in real time and detecting the target material for the purpose. More specifically, the invention is in vitro and in It is an object of the present invention to provide a method for directly searching for an interaction between a "first search substance" that is a specific physiologically active substance in vivo and a "second search substance" that is a physiologically active substance to be searched and detected.

In addition, the present invention in vitro and in The present invention relates to a method for directly searching for an interaction between a "first search substance" and a "second search substance"

In addition, the present invention in vitro and in it is an object of the present invention to provide a kit or a chip for directly searching for the interaction of the "first search substance" and the "second search substance" in vivo .

The present invention also contemplates searching for a target substance for blocking or inhibiting or activating or inducing the interaction of a "first search substance" and a "second search substance" Can be used as a method for detecting a target substance.

In addition, the present invention in vitro and in quot; first search substance "and" second search substance "by observing changes in cell morphology or function in vivo .

As described above, according to the present invention, the second component comprising the first component of the physiologically active substance and the other physiologically active substance having the label attached thereto for the search, the complex formed by the interaction can be directly searched in real time from the position-changed marker by reacting in the system or field. In addition, the change of the position of the coupling body is reversible because it can be dynamically measured according to the strength of the external 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 vivo . Therefore, it can be very useful for searching for and detecting target proteins and new drug candidates for new drug development, as well as for improving existing drugs or discovering new pharmacological uses.

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

In order to achieve the above object, the present inventors have designed two constructs. Specifically, there is provided a localizer in which the position is changed according to an externally applied driving force, and one or more "first search substance" Were directly or indirectly bonded using a linker or the like. The mobile reactant of the first construct and the search material to be analyzed may be provided as a single directly fused construct. In addition, the first construct may be provided with two or more components in which a probe capable of recognizing a search substance to be analyzed is fused and a mobile reaction material is indirectly combined with at least one search substance to be analyzed. The second construct comprises one or several second search substances, for example, a biologically active substance, to which one or more labels are bound, so that search is possible. The labeling substance of the second construct and the physiologically active substance to be analyzed may be provided as a single directly fused construct. In addition, the second construct may be provided with two or more constructs in which a marker capable of recognizing a physiologically active substance to be analyzed is fused and a one or more physiologically active substances to be analyzed are indirectly combined. Approaching a position where the first and second components can interact with each other in the same field or system and then applying an action force from the outside and changing the position or movement to a simple device By measuring, a complex formed by interaction can be dynamically searched 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" and when the second search material is "bait", the first search material is " " In the present invention, the position or movement of the marker may be detected by an optical method including a microscope, a scanner, a radioactive label detection means, a fluorescence polarization reader, a spectrophotometer, a magnetic resonance imaging (MRI) Fluorescence detector, luminescence detector, and the like.

Specifically, a method for searching for a substance interaction in the present invention comprises the steps of: i) locating a locator moving according to an externally applied driving force and a first construct combining a first search material ; ii) providing a second construct comprising a second search material coupled with a label for search; iii) approaching the first and second components in the same field or system to a location where they can interact; And iv) applying an external force and measuring a marker whose position or movement has changed.

In addition, the present invention provides a method for detecting a target substance, comprising the steps of: i) providing a first construct that combines a first reaction substance with a mobile reaction material that moves according to an external force; ii) providing a second construct comprising a second search material coupled with a label for search; iii) approaching the first construct and the second construct in a mutually operable position in the same field or system; iv) measuring an indicator of a change in position or motion by applying an external force; And v) isolating and identifying a substance contained in the first construct or the second construct. Detection of the target substance can be performed by known methods, for example, RT-PCR, PCR for genomic DNA, or mass spectroscopy.

In addition, the present invention can produce three constructs to search for the interaction of a substance and to detect the target substance. Specifically, the method includes: i) providing a first construct that combines a first search material with a mobile reaction material that moves according to an external force; ii) providing a second construct comprising a second search material to which a label is attached; iii) providing a third construct comprising a material for the search; iv) approaching said first, second and third components in a mutually operable position in the same field or system; And v) applying an external force and measuring a marker whose position or movement has changed; And vi) isolating and identifying a substance contained in the first, second, or third construct. Detection of the target substance can be performed by known methods, for example, RT-PCR, PCR for genomic DNA, or mass spectroscopy.

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

In one embodiment, the present invention utilizes a magnetic force as an externally applied force. As a mobile reaction material constituting the first constituent, a nano-particle containing a magnetic substance, preferably a substance having a size of 5 nm to 2,000 nm is provided, and a specific substance such as a physiological substance The active material is used by bonding or coating. The mobile reaction material may be provided to be bonded to or coated with two or more specific materials. The second construct makes use of other physiologically active substances with a label attached thereto for searching as a partner interacting with the physiologically active substance in the first construct. The target substance is searched in real time by introducing the first construct and the second construct into the same cell, applying a magnetic field from the outside, and measuring the label moved under the magnetic field. In addition, a more dynamic search can be performed by changing the intensity of the magnetic field, and a substance that does not change or change position while changing the intensity of the magnetic field can be comparatively searched using a positive or negative control.

In another embodiment, the present invention provides a method for preparing a magnetic particle, comprising the steps of i) coating a magnetic particle with a specific bioactive compound, ii) providing the coated magnetic particle of step i) with a target molecule Iii) applying a magnetic field to the cells of step ii) to move the intracellular magnetic particles, iv) measuring the position or movement of the label, and v) And detecting and identifying the target substance to which the label is attached in the visible cells.

The present invention in eukaryotic or prokaryotic, when performed in vivo ; In vivo, intratracheally, and intracellularly of mammals; Can be carried out in vivo, in a tissue and in a cell of a plant. In particular, the method of the present invention can be carried out intracellularly, intracisternally or in vivo of Zebra fish, C. elegans, Yeast, Fly or Frog. In the present invention, the first construct and the second construct may be prepared by using a transduction peptide or a fusogenic peptide, a lipid gene or a liposome, or a combination thereof, or using a first construct and a second construct using Opti-MEM Can be easily delivered into cells by generally known methods such as electroporation or magnetofection, or cultured with cells in a medium. In particular, when the method of the present invention is carried out intracellularly, it can be carried out in a culture plate / dish or a microarrayed reactor, for example, living cells.

In the present invention, the exertion force externally applied can be appropriately selected according to the characteristics including the physical, chemical, biological and electrostatic characteristics of the mobile reaction material contained in the first constituent.

The term "physiologically active substance" in the present invention means a nucleic acid, a nucleotide (mono- / oligo- / poly- nucleotide), a protein, a peptide (mono- / oligo- / poly- And includes all substances exhibiting physiological activity in vivo, including mono- / oligo- / poly-saccharides, lipids, vitamins, chemical compounds and the like.

"Bait " in the present invention means a physiologically active substance used for searching for interaction with other physiologically active substances.

In the present invention, "prey" means a physiologically active substance to be sought or detected as an interaction partner of the "bait ".

In the present invention, the term "target molecule" refers to activating, inducing or blocking the interaction of "prey" or "bait" and "prey" materials interacting with " Inhibiting " refers to all the substances to be identified.

In the present invention, the term "localizer " means a substance that moves in response to an externally applied driving force.

The "acting force" applied externally in the present invention refers to any kind of force that can result in a change in the position of the defined mobile reaction material in the first construct, including electrical, electromagnetic, magnetic and gravitational forces.

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 physiologically active substance and the transfer reaction substance or the binding between the physiologically active substance and the label substance includes physical, chemical, electrostatic and biological direct or indirect binding. The physiologically active substance may be a transferring substance or a labeling substance Can be used.

In the present invention, the probe constituting the first construct or the second construct and used in the search may physically, chemically, electrostatically, and biologically bind to the physiologically active substance to bind the search substance, the regulatory factor or physiologically active substance, And includes substances that can bind a substance directly or indirectly, for example, an antibody, a protein, a protein domain, a protein motif, a peptide, 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

Phosphor dyes FITC Coated with nanoparticles Intracellular  Confirm location change

(FITC-MNP) coated with superparamagnetic nanoparticles (MNPs) by reacting superparamagnetic nanoparticles conjugated with streptavidin (50 nm; streptavidin nanoparticles) with FITC-biotin and TATHA2- -TATHA2 complex. ≪ / RTI > The TATHA2 peptide was synthesized according to the prior art (Wadia, et al., Nat Med. 10: 310-315 (2004)) and the N-terminal was labeled with biotin. As a negative control, QD565-TATHA2 complex was obtained by reacting nanoparticle QD565 with TATHA2-biotin, which does not cause positional change by an externally applied magnetic field. Pre-cultured Hela cells (purchased from HeLa cell, ATCC) were washed with serum-free DMEM, and a solution of FITC-MNP-TATHA2 complex and QD565-TATHA2 complex in Opti-MEM (Invitrogen) Cells, and the cells were cultured in a CO ₂ incubator fixed at 37 캜 for 12 hours to introduce FITC-MNP-TATHA2 and QD565-TATHA2 complex into Hela cells. The cells were transferred onto a focusing plate of a confocal microscope and magnetic force was applied to the bottom surface with a commercially available permanent magnet or electromagnet. According to the known method, the focus plate of the confocal microscope was adjusted and the morphology of the nanoparticles transferred to the cell bottom surface was observed (cf. 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). It was confirmed that the FITC-MNP-TATHA2 complex shifted by the external magnetic field only in the cells into which FITC-MNP-TATHA2 was introduced. In the cells in which QD565-TATHA2 was used as a negative control, I did not see it.

Example  2

Among the various physiologically active substances Intracellular  Explore Interactions

1) Search for intracellular interactions using nanoparticles coated with fusion proteins and physiologically active low molecular compounds with fluorescent proteins :

Streptavidin nanoparticles were reacted with FK506-biotin, and superparamagnetic nanoparticles (MNPs) were coated with a physiologically active low molecular weight (FK506) (MNP-FK506). HeLa cells expressing EGFP protein or FKBP-RFP fusion protein were washed with serum-free DMEM and the cells were incubated with MNP-FK506 and Opti-MEM (Invitrogen) for 12 hours in a CO ₂ incubator fixed at 37 ° C. (Invitrogen), and MNP-FK506 was introduced into Hela cells. The cells were transferred onto a focusing plate of a confocal microscope and magnetic force was applied to the bottom surface with permanent magnets or electromagnets. The focusing plate of the confocal microscope was adjusted and the fluorescent protein migrated to the cell bottom surface was observed (FIG. 3). Only the cells expressing the FKBP12-RFP fusion protein (red) were displaced by the external magnetic field, and the cells (green) expressing EGFP used as a negative control did not show any positional change due to the magnetic field.

2) FK506 and its pharmacologically relevant binding partner, FKBP12 ( FK506 binding protein 12) in cells:

Streptavidin nanoparticles were reacted with FK506-biotin and TATHA2-biotin at 4 DEG C for 1 hour while mixing to obtain FK506-MNPs complex. The reaction solution was purified using electromagnet or permanent magnet to remove unbound ligand. Hela cells were transfected with RFP-fused FKBP12 (FKBP-RFP) or EGFP-expressing plasmids, respectively, and then these cells were detached and cultured together in an incubator. After the FK506-MNPs complex prepared above was introduced into these cells, a magnetic field (M.F., magnetic field) was externally applied using a permanent magnet or an electromagnet. Confocal images of living cells were taken using a confocal laser microscope (Carl Zeiss) under magnetic field and with no magnetic field applied. In the photograph of FIG. 4, only cells expressing FKBP12 fused with RFP (indicated by a red arrow) are moved by an external magnetic field, and in the case of cells expressing EGFP used as a negative control (indicated by a green arrow) And no change in position was observed.

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

The streptavidin nanoparticles were reacted with the caspase-3 inhibitor II (DEVD) -bototin conjugate (CALBIOCHEM) and TATHA2-biotin at 4 ° C for 1 hour while mixing with the DEVD-MNPs complex ≪ / RTI > The complex was purified using a permanent magnet to remove unbound ligand. After plasmids expressing mEFFP-3 (mRFP-Caspase3) or EGFP fused with mRFP were introduced into Hela cells, these cells were separated and cultured together in an incubator. Next, DEVD-MNPs prepared above were introduced into the cells, and then a magnetic field was externally applied by using a permanent magnet or an electromagnet. Confocal images of living cells were taken using a confocal laser microscope (Carl Zeiss) under magnetic field and with no magnetic field applied. In the photograph of FIG. 5, only cells expressing mRFP-Caspase3 (indicated by a red arrow) are moved by an external magnetic field and cells expressing EGFP (indicated by a green arrow) used as a negative control No change in position due to magnetic field was observed.

4) Verification of intracellular interactions of IκBα protein and its binding partner RelA protein :

Streptavidin nanoparticles were reacted with FK506-biotin and TATHA2-biotin at 4 DEG C for 1 hour while mixing to obtain FK506-MNPs complex. The complex was purified using a permanent magnet to remove unbound ligand. HeLa cells were co-transfected with ECFP and a plasmid expressing IκBα (IκBα-ECFP-FKBP12) fused with FKBP12 and RelA (EYFP-RelA) fused with EYFP, and then FK506-MNPs prepared above were introduced into these cells , The permanent magnet or the electromagnet was used to apply the magnetic field from the outside. Confocal images of living cells were taken using a confocal laser microscope (Carl Zeiss) under magnetic field and with no magnetic field applied. As shown in the photograph of FIG. 6, IκBα-ECFP-FKBP12 fusion protein and EYFP-RelA fusion protein were shifted 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 Verification of interaction:

Streptavidin nanoparticles were mixed and reacted with anti-phospho-IκBα-biotin and TATHA2-biotin at 4 ° C. for 1 hour to obtain an anti-phospho-IκBα-MNPs complex. The complex was purified using a permanent magnet to remove unbound ligand. After introducing plasmids expressing IκBα (IκBα-mRFP) fused with mRFP into HeLa cells, the anti-phospho-IκBα-MNPs prepared above were introduced into these cells, treated with TNF-α for 5 minutes, Or a magnetic field was externally applied using an electromagnet. Confocal images of living cells were taken using a confocal laser microscope (Carl Zeiss) under magnetic field and with no magnetic field applied (FIG. 7).

6) Verification of intracellular interaction of oligonucleotide ( UAS ) with binding site of GAL4 protein and GAL4 protein :

An oligonucleotide containing UAS (SEQ ID NO: 1), biotin-CCCAGTTTCTAGACGGAG (UAS-biotin), which is a DNA base sequence to which the GAL4 protein as a transcriptional activator of yeast can bind, was synthesized. Streptavidin nanoparticles were reacted with UAS-biotin and TATHA2-biotin at 4 DEG C for 1 hour while mixing to obtain a UAS-MNPs complex. The complex was purified using a permanent magnet to remove unbound ligand. After introducing a plasmid expressing GAL4 (GAL4-EGFP) fused with EGFP into Hela cells, the UAS-MNPs prepared above were introduced into these cells, and the magnetic field was externally applied using permanent or electromagnet. Confocal images of living cells were taken using a confocal laser microscope (Carl Zeiss) under magnetic field and with no magnetic field applied (FIG. 8).

7) miRNA Wow mRNA of Intracellular  Verification of interaction:

To verify the interaction of miRNA with its intracellular target mRNA, let-7b miRNA (SEQ ID NO: 2) was selected which binds to lin-28 mRNA (Fig. 9). The let-7b miRNA was synthesized by the structure of UGAGGUAGUAGGUUGUGUGGUU-biotin (let-7b-biotin) and FITC-CCCTATAGTGAGTCGTATTA (FITC-lin28p) was synthesized to mark lin-28 mRNA (SEQ ID NO: 3). Streptavidin nanoparticles were reacted with let-7b-biotin and TATHA2-biotin at 4 ° C for 1 hour while mixing to obtain a let7b-MNPs complex. The complex was purified using a permanent magnet to remove unbound ligand. After introducing plasmids expressing lin-28 mRNA into HeLa cells, let7b-MNPs and FITC-lin28p prepared above were introduced into these cells, and the magnetic field was externally applied by using a permanent magnet or electromagnet. Confocal images of living cells were taken using a confocal laser microscope (Carl Zeiss) under magnetic field and with no magnetic field applied (FIG. 10).

8) Beta- Cyclodextrin and MBP  Protein Intracellular  Verification of interaction:

Beta-cyclodextrin (Sigma) was biotinylated (βCD-biotin) using an EZ-Link NHS-PEO Solid Phase Biotinylation Kit (Pierce). Streptavidin nanoparticles,? CD-biotin and TATHA2-biotin were reacted while mixing at 4 占 폚 for 1 hour to obtain? CD-MNPs complex. The complex was purified using a permanent magnet to remove unbound ligand. A plasmid expressing MBP (MBP-EGFP) fused with EGFP was introduced into HeLa cells, and the thus prepared βCD-MNPs were introduced into these cells, and then the magnetic field was externally applied using a permanent magnet or an electromagnet. Confocal images of living cells were taken using a confocal laser microscope (Carl Zeiss) under magnetic field and with no magnetic field applied (FIG. 11).

Example  3

Microarrayed Within the cell  Verification of binding partner proteins

About 1,000 full-length ORFs (open reading frames) derived from humans were distributed from the Human Genome Function Research Project. About 1,000 genes were propagated 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. The microarrayed cells were prepared according to conventional techniques (Reference: Ziauddin and Sabatini, Nature 411: 107-110 (2001)) and verified by expression of the fused GFP fluorescent protein. The 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 the magnetic field was externally applied using a permanent magnet or electromagnet. Confocal images of living cells were taken using a confocal laser microscope under magnetic field and with no magnetic field applied. RT-PCR for positive clones and sequencing of mRNA confirmed that positive clones were FKBP12, the pharmacologically relevant target of FK506 (FIG. 12).

Example  4

Cell signaling (signal After the translation of the protein of the formula and the changes verified by transduction protein complexes)

The TNF-α / IκBα signal transduction system was selected to verify post-translational modification of proteins and protein complexes by intracellular signaling. IκBα complexes with the NF-κB proteins RelA / p65, p50 and c-Rel to regulate various signals. Treatment of TNF-α with cells increases the activity of IκB kinase (IKK), phosphorylating 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 was constructed by fusing the IκBα gene and the mRFP gene, so that the IκBα-mRFP fusion protein was expressed. 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. EYFP-RelA fusion protein was expressed in cells by preparing expression vector fused with EYFP gene and RelA gene. mRFP-βTrCP fusion protein was also prepared by using an expression vector obtained by fusing each gene. Anti-phospho-IκBα antibody (Cell Signaling Technology) was biotinylated using EZ-Link NHS-PEO Solid Phase Biotinylation Kit (Pierce) and validated by Western blot.

1) Verification of protein phosphorylation by cell signaling:

Streptavidin nanoparticles were mixed with biotin-SS-FITC, biotin-TATHA2 and biotin-antiphophot-IκBα (Ser32) antibodies at 4 ° C for 1 hour to obtain IκBα (Ser32) -MNPs complexes. The IκBα (Ser32) -MNPs complex was introduced into HeLa cells into which the IκBα-mRFP expression vector was introduced in the same manner as in Example 1, treated with 10 ng / ml of TNF-α for 5 minutes, and subjected to confocal laser microscopy Confocal images of living cells were photographed under magnetic field and with no magnetic field applied (FIGS. 14 and 15). The IKK inhibitor SC-514 was treated at a concentration of 1 mM. IκBα phosphorylated by TNF-α treatment was found to bind to the IκBα (Ser32) -MNPs complex and to be displaced by externally applied magnetic force. The IKK inhibitor, SC-514, did not phosphorylate IκBα, It was verified that no position movement occurred.

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

HeLa cells were transfected with IκBα-ECFP-FKBP12 fusion protein expression vector, EYFP-RelA fusion protein expression vector and mRFP-βTrCP fusion protein expression vector, and the FK506-MNPs complex was transformed into the same And introduced into cells. 10 ng / ml of TNF- [alpha] was treated for 5 minutes, and confocal images of living cells were taken under a magnetic field and a magnetic field using a confocal laser microscope (Figs. 16 and 17). The IκBα-ECFP-FKBP12 fusion protein and the EYFP-RelA fusion protein, which form a complex independent of signal transduction, cause localized migration by externally applied magnetic forces, whereas the mRFP-βTrCP fusion protein, which binds to IκBα phosphorylated by signaling TNF-α. The IKK inhibitor, SC-514, inhibited the binding of the IκBα-ECFP-FKBP12 fusion protein to the mRFP-βTrCP fusion protein without interfering with the binding of the IκBα-ECFP-FKBP12 fusion protein to the EYFP-RelA fusion protein.

Example  6

Pharmacological Investigation of Interaction Inhibitors and Accelerators drug screening )

1) Search for interaction promoters:

IκBα-mRFP fusion protein expression vector and IκBα (Ser32) -MNPs complex were introduced into HeLa cells in the same manner as in Example 4-1), the cells were treated with various compounds and growth promoting factors, And the positional shift of IκBα-mRFP fusion protein was observed with a confocal microscope. It was confirmed that only TNF-α, IL-2, LPS, Fas, and the like among the physiologically active substances treated in the cells caused localization of IκBα-mRFP fusion protein by external magnetic force (FIGS. 15 and 17).

2) FK506 Inhibition of Interaction by:

The FK506-MNPs complex and the FKBP12-mRFP expression vector were introduced into cells in the same manner as in Example 3, and various compounds were treated to observe the movement of the FKBP12-mRFP fusion protein. Of the treated compounds, only FK506 competitively inhibited the translocation of the FKBP12-mRFP fusion protein (Fig. 19).

Claims (26)

(i) providing a first construct comprising a first bioactive molecule coupled with a magnetic material that moves according to a magnetic field;
ii) providing a second construct comprising a second physiologically active substance conjugated with a label for search;
iii) approaching the first construct and the second construct in a location where they can interact with each other within the cell; And
iv) measuring a marker having a change in location or motion by applying a magnetic field to the cell, and searching for an interaction between the first physiologically active substance and the second physiologically active substance,
Wherein the magnetic material has a particle size of from 5 nm to 2,000 nm. delete The method of claim 1, wherein the first and second physiologically active substances are at least one selected from the group consisting of a nucleic acid, a nucleotide, a protein, a peptide, an amino acid, a sugar, a lipid, a chemical compound, ≪ / RTI > delete The method of claim 1, wherein the magnetic field is an electromagnetic field or a permanent magnetic field. The method according to claim 1, wherein the magnetic field intensity is varied while searching. delete delete The method according to claim 1 or 3, wherein the labeling substance is a radioactive substance, a fluorescent substance or a luminescent substance. The method according to claim 9, characterized in that the fluorescent or luminescent material itself exhibits fluorescence or luminescence, or fluorescence or luminescence is exhibited by the binding of the first constituent to a specific substance or to another substance. 10. The method of claim 9, wherein the fluorescent material is a fluorescent dye, or a tetracysteine motif, or a fluorescent protein comprising GFP, YFP, CFP and RFP, or a fluorescent nanoparticle. 4. The method of claim 1 or 3, wherein the magnetic material in the first construct is directly or indirectly bonded or coated with two or more specific materials. The method of claim 1, wherein the binding of the magnetic material and the first physiologically active substance in the first construct is an electrostatic, physical, chemical or biological coupling. 4. The method according to any one of claims 1 to 3, wherein in the second composition, the label is directly or indirectly bonded or coated with the second physiologically active substance. 15. The method of claim 14, wherein the binding of the labeling substance and the second physiologically active substance in the second construct is an electrostatic, physical, chemical or biological coupling. 4. The method according to claim 1 or 3, wherein the binding of the magnetic substance and the first physiologically active substance in the first composition or the binding of the label substance and the second physiologically active substance in the second composition to the binding of the antibody, A protein domain, a protein motif, and a peptide. 4. The method according to claim 1 or 3, wherein the cell is a eukaryotic cell or a prokaryotic cell. 2. The method of claim 1, wherein the intracellular delivery of the first and second constructs comprises a transduction peptide or a fusogenic peptide, a lipid or liposome or a combination thereof, electroporation ), Or magnetofection. ≪ Desc / Clms Page number 13 > 2. The method of claim 1, wherein the interaction of the first construct with the second construct is performed in a cell in a culture container, or in a microarrayed cell. The method according to claim 6, wherein the substance which does not change or change position when changing the intensity of the magnetic field is compared and compared as a positive or negative control. A method for detecting a target substance, further comprising the step of searching for the interaction of the substance according to the method of claim 1 or 3, and then isolating and identifying the physiologically active substance contained in the first construct or the second construct. i) providing a first constituent comprising a magnetic material and a first physiologically active substance, the magnetic material being displaceable according to a magnetic field;
ii) providing a second construct comprising a second physiologically active substance to which a label is bound;
iii) providing a third construct comprising a material for the search;
iv) accessing the first construct, the second construct, and the third construct in a cell where they can interact; And
v) applying a magnetic field to the cell and measuring the label of a change in location or motion to search for the interaction of the substance,
Wherein the magnetic material has a particle size of from 5 nm to 2,000 nm. cell;
1. A magnetic resonance imaging apparatus comprising: a first component comprising a magnetic material whose position moves according to a magnetic field; and a first physiologically active substance which binds to the magnetic substance; And
A second construct comprising a labeling substance and a second physiologically active substance which binds to the labeling substance,
Introducing the first construct and the second construct into the cell such that the first construct and the second construct interact with each other, and when the magnetic field is applied, the labeling substance is in contact with the first physiologically active substance and the second physiologically active substance The position or movement of which is changed according to the interaction of the user,
Wherein the magnetic material has a particle size of from 5 nm to 2,000 nm. cell;
1. A magnetic resonance imaging apparatus comprising: a first component comprising a magnetic material whose position moves according to a magnetic field; and a first physiologically active substance which binds to the magnetic substance;
A second construct comprising a labeling substance and a second physiologically active substance binding to the labeling substance; And
A third construct comprising a substance for the search,
Introducing the first construct, the second construct, and the third construct into the cell such that the first construct, the second construct, and the third construct interact with each other, and when the magnetic field is applied, And the position or motion thereof is changed according to the action,
Wherein the magnetic material has a particle size of from 5 nm to 2,000 nm. The kit according to claim 23 or 24, further comprising means for searching for changes in the shape or function of the cells depending on the interaction of the physiologically active substances. 2. The method of claim 1, further comprising the step of searching for a change in cell morphology or function associated with said interaction.

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