JP2005207823A - Spectrofluorometric analyzing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a spectrofluorometric analysis method of a single-molecule, capable of precisely measuring an interaction of molecules. <P>SOLUTION: The single-molecule fluorometric analyzing technique is to use fluorometric resonance energy transfer (FRET). That is, the spectrofluorometric analyzing method is adopted, in order to detect the association or interaction of a sample A and a sample B and includes a process for bringing the sample labeled with fluorescence into contact with the sample B labeled with a fluorescent substance, which converts the fluorescence energy of the fluorescent substance for use in labeling the sample A to exciting energy; a process of emitting light, having the wavelength for exciting the fluorescent substance for use in labeling the fluorescent substance to irradiate the fluorescent substance; a process for detecting the fluorescence emitted from the fluorescent substance, used for labeling the samples A and B; and a process for performing a fluorescence correlation spectral method or fluorescence intensity distribution analysis on the basis of the detected fluorescences to detect the change in the reaction of the samples A and B. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for measuring intermolecular interactions using fluorescence fluctuation analysis such as fluorescence correlation spectroscopy and fluorescence intensity distribution analysis.

  Conventionally, protein functional analysis research has mainly focused on analysis of gene sequences encoding proteins. However, due to the rapid development of human genome analysis in recent years, the focus of protein functional analysis research is shifting to the point of analyzing how proteins synthesized from genes existing in cells move and function. is there.

  For example, in vivo, various molecules with specific functions (functional proteins including enzymes and receptors; proteins that retain cell structures, lipids, glycoproteins, Biologically active molecules such as ions) are constantly changing and maintaining their life mechanisms. In order to observe and analyze such biological functions, it is necessary to observe the dynamics of a protein having a three-dimensional function at the molecular level.

  Many physiologically active substances such as hormones and neurotransmitters exert their actions by binding to receptor molecules present on the cell surface and regulate various life phenomena. Searching for substances that compensate, enhance, or inhibit the action of these physiologically active substances has become one of the main means for research and development of new medicines, and in particular, is the point of action in the process. It is very important to understand the nature of the receptor molecule.

  Recent developments in molecular biological techniques have made it possible to analyze many physiologically active receptors at the molecular level. Once the target receptor is determined, it will evolve into research on ligands for this and molecules that inhibit these interactions. By selecting various candidate substances as ligand-like substances such as ligands and inhibitors and investigating the interaction between these candidate substances and receptors, new receptors that are regulated by these receptors and ligands can be examined. It is expected that the physiological function or disease state will be clarified.

  One technique for observing molecular interactions is single-molecule fluorescence spectroscopy using FCS (Fluorescence Correlation Spectroscopy) as an analysis technique (Non-patent Document 1). This is a technique for measuring the minute movement of each target molecule accurately by measuring the fluctuation movement of the fluorescently labeled target molecule in the medium and using an auto-correlation function. By this method, physical quantities such as the number and size of molecules can be calculated.

  However, the analysis method using FCS limits the size of molecules that can be analyzed accurately. For example, there is a limit to the reaction of large molecules such as membrane fractions and molecules that are not uniform in size.

  Patent Document 1 discloses a method of analyzing a fluorescence intensity distribution for a measured fluorescence fluctuation signal as an analysis method for complementing this limit. Fluorescence intensity distribution analysis (FIDA) is similar to FCS in that it detects fluorescence fluctuations, but the intensity and measurement of fluorescence per molecule is measured by analyzing the statistical distribution of the obtained fluorescence signal. This is an analysis method that can calculate the number of molecules present in a certain system. By using this FIDA, the number of fluorescently labeled ligands bound to the receptor can be predicted by measuring the fluorescence intensity per molecule of the fluorescently labeled ligand and the number of fluorescent molecules present in the confocal region. For example, if there are multiple receptors in the membrane fraction and the ligand is present at a concentration sufficient to bind to the receptor, multiple labeled ligands will bind to the membrane fraction. At this time, the membrane fraction is measured as one molecule. Therefore, the number of molecules measured is smaller than the number of molecules of the fluorescently labeled ligand before binding to the receptor. In addition, when a plurality of fluorescently labeled ligands are bound to the membrane fraction, the detected fluorescence intensity per molecule is high. Furthermore, the concentration of the fluorescently labeled ligand is kept constant, the reaction is carried out by changing the concentration of the substance that inhibits the reaction between the fluorescently labeled ligand and the receptor, and the fluorescently labeled ligand reacting with the receptor at each concentration exists in the monomer state. It is also possible to calculate the binding constant of the receptor / ligand reaction by calculating the ratio of the fluorescently labeled ligand molecules. As described above, it is possible to know the reaction between the receptor and the ligand by performing FCS or FIDA analysis.

  On the other hand, when a protein on the cell membrane transmits an external stimulus, the protein often undergoes a structural change, or the protein associates and transmits the stimulus into the cell. For example, there are various things such as two proteins that associate to form a dimer, or a protein that functions as a tetramer.

  In the analysis using the FIDA for the reaction between the membrane fraction and the ligand described above, it was possible to know the reaction between the membrane fraction and the ligand by observing the change in the brightness of the fluorescently labeled ligand. However, it has been difficult to detect whether ligands bound to the membrane fraction are scattered in the membrane fraction or interact to form multimers. For example, it has been clarified that epidermal growth factor receptor (EGFR) forms a dimer when it binds to its ligand, epidermal growth factor (EGF). When the membrane fraction of cells expressing a large amount of EGFR is adjusted, the fluorescent labeled EGF is reacted, and the FIDA analysis is performed with a single molecule fluorescence analyzer, the fluorescence intensity per molecule increases. It can be seen that the membrane fractions interact. However, the formation of a dimer could not be confirmed.

  For example, when the receptor and the ligand react on the organelle membrane in the cell, depending on the type of the receptor, a multimer may be formed when the ligand binds to the receptor as in EGFR described above. In addition, proteins present on the membrane may be released or associated. In other words, from the viewpoint of fluorescence intensity, is there a one-to-one reaction between the receptor and the ligand, or a structural change of the receptor or an association between the receptors after the reaction between the receptor and the ligand? It was difficult to judge.

  Therefore, in the analysis of cell functions, there has been a demand for a method for detecting an interaction between membrane proteins and a structural change with high accuracy after detecting an interaction between a membrane protein and a ligand on the membrane fraction.

In addition, as a method for detecting association and interaction between molecules, there is fluorescence resonance energy transfer (FRET) (Non-patent Document 2). This is a technique in which molecules are labeled with a plurality of fluorescent substances having different spectral characteristics, and the movement of fluorescence energy between fluorescent molecules that occurs when the distance between the fluorescent molecules becomes very close to each other is observed. FRET uses two types of fluorescent materials with different spectral characteristics. A fluorescent material (donor) that gives fluorescence energy and a fluorescent material (acceptor) that receives the energy as excitation energy are used. A fluorescence resonance energy transition is observed when two fluorescent molecules mix and interact with each other and one absorption wavelength overlaps with the other emission wavelength. The efficiency of energy transition is proportional to r- ⁶ . Here, r is an average interval between two fluorescent molecules. Therefore, when the interaction between two molecules and the mutual affinity increase, the energy transition phenomenon is enhanced, and as a result, the fluorescence emission from the acceptor increases. Conventional detection of FRET has been observed with a fluorescence spectrophotometer or a fluorescence microscope, in which interacting molecules are labeled with respective fluorescent substances and used at a concentration of the mM order. However, since these methods detect a large number of molecules, the detected signal is average. For example, when a very small number of molecular reactions occur in many molecules, the energy transition is not necessary. It is difficult to detect a slight reaction because the fluorescent signal is buried in the fluorescent signals of a large number of molecules that have not reacted. Therefore, it was not possible to make a quantitative assessment of how the individual molecules are moving, or how many molecules interact with each other in the observed region.
Special Table 2002-5434142 Oliver Meissner and Hanns Haberlein, “Biochemistry”, 2003, 42, p.1667-1672 Theodorus WJ Gadella, Jr. and Thomas M. Jovin, “The Journal of Cell Biology”, 1995, 129, p.1543-1558

  In view of the above circumstances, an object of the present invention is to provide a single-molecule fluorescence analysis method capable of precisely measuring molecular interactions.

  In order to solve the above problems, a technique for analyzing single molecule fluorescence using fluorescence resonance energy transfer (FRET) was devised.

  That is, the present invention is a fluorescence spectroscopic analysis method for detecting the association or interaction between sample A and sample B, wherein the fluorescence energy of the fluorescently labeled sample A and the fluorescent substance labeled on the sample A is determined. Contacting the sample B that is fluorescently labeled with a fluorescent material such as excitation energy, irradiating the sample A and the sample B with light having a wavelength that excites the fluorescent material labeled on the sample A, and A step of detecting fluorescence generated from a labeled fluorescent substance, and a step of detecting a process of reaction change between the sample A and the sample B by performing fluorescence correlation spectroscopy or fluorescence intensity distribution analysis based on the detected fluorescence And a method characterized by comprising:

  The present invention also provides a fluorescence spectroscopic analysis method for detecting the association or interaction of sample A, sample B, and sample C, wherein the fluorescence-labeled sample A and the sample A The step of bringing the sample B fluorescently labeled with a fluorescent substance that uses the fluorescent energy of the labeled fluorescent substance as excitation energy, and the sample C, and the light having a wavelength for exciting the fluorescent substance labeled on the sample A are irradiated. A step of detecting fluorescence generated from the fluorescent substances labeled on the sample A and the sample B, and performing fluorescence correlation spectroscopy or fluorescence intensity distribution analysis based on the detected fluorescence, the sample A, the sample And B, and detecting a process of reaction change of the sample C.

  Furthermore, the present invention is the above method, wherein the fluorescently labeled sample A, and the fluorescent material labeled with the fluorescent material such that the fluorescent energy of the fluorescent material labeled with the sample A is used as excitation energy, Further, the step of contacting the sample C fluorescently labeled with a fluorescent substance that uses the fluorescent energy of the fluorescent substance labeled on the sample B as an excitation energy, and light having a wavelength for exciting the fluorescent substance labeled on the sample A Irradiating, detecting the fluorescence generated from the fluorescent substances labeled on the sample A, the sample B, and the sample C, and performing fluorescence correlation spectroscopy or fluorescence intensity distribution analysis based on the detected fluorescence, Detecting a process of reaction change of the sample A, the sample B, and the sample C.

  Furthermore, the present invention provides a fluorescence spectroscopic analysis method for detecting a structural change in a sample or an association state of intramolecular domains, wherein the first fluorescent material and the fluorescence energy of the first fluorescent material are excited energy. A step of irradiating a sample fluorescently labeled with both of the second fluorescent material and a light having a wavelength that excites the first fluorescent material, the first fluorescent material, and the second fluorescent material. A step of detecting fluorescence generated from the fluorescent substance, and a step of performing fluorescence correlation spectroscopy or fluorescence intensity distribution analysis based on the detected fluorescence to detect a structural change of the sample or an association state of intramolecular domains. A method characterized by comprising.

  According to the present invention, presence / absence of multimer formation, molecular binding and dissociation, molecular structural change, and association state of intramolecular domains can be easily detected.

  Here, fluorescent dyes are used for multiple types of fluorescent substances with different spectral characteristics, and single-molecule fluorescence analysis such as FCS and FIDA is used to detect the interaction between different molecules labeled with each fluorescent dye. Is used.

  The problem with analyzing molecular interactions is the order in which multiple molecules interact, the location of the molecular structure involved in the interaction, or how the molecular structure changes. . In the present invention, in order to measure such a fine interaction between molecules, measurement using FRET of a fluorescent dye is performed using an apparatus that performs single-molecule fluorescence spectrometry. Single-molecule fluorescence analysis such as FCS and FIDA can analyze fluorescence information from fluorescently labeled molecules at the single-molecule level, so it can detect microregional reactions, and the fluorescence signals of many unreacted molecules Will not be detected.

  Hereinafter, one embodiment of the present invention will be specifically described as an example.

  One aspect of the present invention is a fluorescence spectroscopic analysis method for detecting the association or interaction between sample A and sample B, wherein the first step comprises fluorescence-labeled sample A and sample A This is a step of contacting the sample B fluorescently labeled with a fluorescent substance that uses the fluorescent energy of the labeled fluorescent substance as excitation energy.

  Sample A and sample B are a sample pair for which interaction is to be detected. In this specification, the term “sample” refers to a sample prepared from a biological sample such as cells, tissues, organs, blood, serum, lymph, tissue, hair and earwax collected from an individual, as desired, or artificially. Including samples that are to be subjected to tests containing substances synthesized or produced, especially isolated molecules and recombinant proteins. In addition, the “sample” may be a sample obtained by subjecting a biological sample to any necessary pretreatment such as homogenate and extraction, if necessary, and is incorporated into an unisolated membrane fraction. Or a sample such as a protein immobilized on a cell membrane. Such a sample could be easily obtained by those skilled in the art depending on the sample of interest. The term “individual” as used herein includes any mammal, including humans, dogs, cats, cows, goats, pigs, sheep, and monkeys, and non-mammalian organisms such as plants and insects.

  In order to detect FRET in the sample, samples A and B are excited with the second fluorescent substance B by light having the emission wavelength of the first fluorescent substance, and the emission wavelength of the first fluorescent substance is It is labeled with a fluorescent substance pair that causes fluorescence resonance energy transfer in which light of another wavelength is emitted.

  The fluorescent substance that can be used as a label in the present invention may be any fluorescent substance known to those skilled in the art, but fluorescent glass particles and fluorescent proteins containing various fluorescent dyes, rare earth elements, and the like can be used. In addition, the method for fluorescently labeling the sample to be detected can be performed using any means known to those skilled in the art. Fluorescent proteins such as GFP, CFP, and YFP are fused to the sample to be detected. It is also included in the label in the present invention to introduce a gene so that it is expressed as a protein. For example, when detecting the reaction between samples A and B, sample A may be labeled with FITC, sample B with tetramethylrhodamine, or sample A with CFP, and sample B with GFP.

  Next, sample A and sample B are brought into contact. For example, a solution containing both samples may be mixed and contacted by reacting under appropriate conditions. Such conditions can be easily selected by those skilled in the art as shown in the following examples.

  Next, the sample A is irradiated with light having a wavelength that excites the labeled fluorescent substance. For example, when sample A is labeled with FITC, FITC is excited by irradiating the sample with light having a wavelength of 488 nm. In addition, when CFP and GFP are used as the fluorescent substance pair, the CFP is excited by irradiation with light having a wavelength of 450 nm.

  Next, the fluorescence generated from the fluorescent substances labeled on Sample A and Sample B is detected (see the right side of FIG. 1). The fluorescence signal from the excited labeled fluorescent substance is preferably detected by separating the fluorescence of the quantity label using a dichroic mirror. For example, FITC fluorescence and tetramethylrhodamine fluorescence are detected separately. Here, if the sample A and the sample B exist independently in the sample without interaction, only the fluorescence of FITC is detected. If samples A and B react and the distance between the molecules is close, the fluorescence signal of tetramethylrhodamine is detected by FRET in which the fluorescence energy of FITC moves to tetramethylrhodamine.

  The optical system that can be used for detection is, for example, a detector for detecting fluorescence, a dichroic mirror for separately inputting a fluorescence signal to the detector, or transmitting light for each wavelength width having specific spectral characteristics. This is an optical system to which a spectroscopic element capable of being added is added. The light source for exciting the fluorescent material may be a laser or the like, and the wavelength may be any wavelength from ultraviolet to visible and infrared. The excitation light is narrowed down through the objective lens and irradiated onto the sample. Fluorescence from the fluorescent material is collected by a condenser lens, and noise is removed by a pinhole. Fluorescence is first divided into two wavelength regions by a dichroic mirror. The divided fluorescence passes through the optical filter, and only the fluorescence having a specific wavelength is taken out. This fluorescence is detected by a detector and signal analysis is performed. The other light is further divided into two wavelength ranges by a dichroic mirror placed next. The fluorescence divided into two passes through a filter for extracting each wavelength and is detected by a detector.

  Next, fluorescence correlation spectroscopy or fluorescence intensity distribution analysis is performed based on the detected fluorescence, and the process of reaction change between sample A and sample B is detected.

  The detected fluorescence signal can be obtained as information on the brightness of fluorescence per molecule by performing distribution analysis on the brightness of fluorescence. In addition, the autocorrelation function analysis is performed on the fluorescence signal, and the time (translation diffusion time) for the fluorescent molecule to stay in the confocal region is calculated. In addition, the cross-correlation analysis of fluorescence is performed by simultaneously exciting multiple fluorescent substances at different wavelengths using lasers with different wavelengths and simultaneously separating and obtaining the fluorescence signals of each fluorescent substance. It is possible to know the ratio of interacting molecules among the fluorescent molecules. For example, as a result of the above analysis, when sample A and sample B are associated, the translational diffusion time by fluorescence correlation spectroscopy becomes longer than when sample A or sample B exists alone.

  Thus, by simultaneously measuring the diffusion constant, fluorescence cross-correlation analysis and FRET, it becomes possible to detect the reaction state with higher accuracy.

  By temporally and spatially controlling changes in the sample reaction, it is possible to obtain not only the binding strength of the interacting sample, but also information such as the speed of binding and the speed of dissociation. . In cell measurement, it is also possible to fluorescently label only the target location and examine the state of diffusion and movement of the target sample.

  Excitation of the fluorescent substance used in the above method, detection of fluorescence, analysis by FCS / FIDA, and the like can be performed using a commercially available single molecule fluorescence analysis system, for example, MS20 / 10S apparatus (Olympus Corporation).

  In another embodiment of the present invention, there is provided a fluorescence spectroscopic analysis method for detecting the association or interaction of sample A, sample B, and sample C, wherein the sample is labeled with fluorescence in the above method, Sample B, which is fluorescently labeled with a fluorescent substance that uses the fluorescent energy of the fluorescent substance labeled on A as excitation energy, and sample C are contacted, and light with a wavelength that excites the fluorescent substance labeled on sample A is irradiated. A step of detecting fluorescence generated from fluorescent substances labeled on sample A and sample B, and performing fluorescence correlation spectroscopy or fluorescence intensity distribution analysis on the basis of the detected fluorescence, sample A, sample B, and sample A method for detecting the process of C reaction change is provided. This method may detect the association or interaction of sample A, sample B, and sample C, but may also detect the influence of sample C on the association or interaction of sample A and sample B, for example, both Sample inhibition and enhancement are included.

  For example, when sample C is a substance that inhibits the interaction between sample A and sample B, the presence of sample C inhibits the interaction between sample A and sample B. When sample C does not exist, sample A and sample B are considered to interact. Therefore, when the fluorescent substance of sample A is excited, FRET from the fluorescent substance of sample A to the fluorescent substance of sample B is observed. As a result, the fluorescence from sample A is hardly observed, but the fluorescence from sample B is observed. It is thought that it is done. On the other hand, when sample C exists, it is considered that the interaction between sample A and sample B is inhibited depending on the concentration of sample C. As a result, it is considered that FRET is not observed even when the fluorescent substance of sample A is excited, and fluorescence of sample A alone is observed.

  In this way, the influence of sample C on the association or interaction between sample A and sample B can be detected.

  In another embodiment of the present invention, fluorescently labeled sample A, sample B fluorescently labeled with a fluorescent substance that uses the fluorescent energy of the fluorescent substance labeled on sample A as excitation energy, and further sample B Contacting the sample C that is fluorescently labeled with a fluorescent material that uses the fluorescent energy of the labeled fluorescent material as excitation energy, irradiating the sample A with light of a wavelength that excites the fluorescent material labeled, and the sample A step of detecting fluorescence generated from the fluorescent substances labeled on A, Sample B, and Sample C, and performing fluorescence correlation spectroscopy or fluorescence intensity distribution analysis based on the detected fluorescence, and Sample A, Sample B, and Sample C And detecting the process of the reaction change.

  For example, sample A is fluorescently labeled with CFP, sample B with GFP, and sample C with YFP. Here, YFP and GFP are fluorescent substances that use the fluorescence energy of CFP as excitation energy, and if each sample is at a distance that causes FRET between CFP and YFP or GFP, When excited, YFP or GFP emits light.

  Next, the CFP is excited with light of a wavelength of 450 nm, and the fluorescence generated from each fluorescent substance is detected in the same manner as described above, and fluorescence correlation spectroscopy or fluorescence intensity distribution analysis is performed to detect the reaction change process between the samples. To do. As a result, when CFP interacts with YFP and / or GFP, almost no fluorescence of CFP is observed, only fluorescence of YFP and / or GFP is observed, and when there is no interaction, CFP It is thought that only fluorescence is observed.

  In this way, by combining various fluorescent substances and labeling a plurality of samples with various fluorescent substances, association or interaction of the plurality of samples can be detected. Also, for example, sample A, sample B fluorescently labeled with a fluorescent material that uses the fluorescent energy of the fluorescent material labeled on sample A as excitation energy, and fluorescence energy of the fluorescent material labeled on sample B as excitation energy If the same method is used with sample C that is fluorescently labeled with a fluorescent substance, the fluorescence from sample C is detected only when samples A, B, and C are all in close proximity. be able to.

  In addition, according to the above method, not only the case where the sample A, the sample B, and the sample C associate simultaneously, but also the associated state of each can be detected over time. For example, in Sample A, Sample B, and Sample C, assume that Sample A and Sample B associate only in the presence of calcium, and Sample C further associates only when both molecules associate. First, sample A, sample B, and sample C are mixed in the reaction system. Since these molecules do not associate in the absence of calcium, when the fluorescent substance labeled on the sample A is excited at this time, fluorescence derived only from the fluorescent substance is detected. Next, when calcium is added to the reaction system, the sample A and the sample B are associated. Here, when the fluorescent substance labeled with sample A is excited, FRET from the fluorescent substance of sample A to the fluorescent substance of sample B is observed. As a result, almost no fluorescence from sample A is observed, but fluorescence from sample B is observed. Is observed. It is also observed that the translational diffusion time calculated from the fluorescence of sample B is longer than the translational diffusion time calculated from the fluorescence of sample A by FCS analysis. In sample C, reaction occurs only when sample A and sample B are associated, and FRET from the fluorescent material of sample B to the fluorescent material of sample C is gradually observed, resulting in a decrease in fluorescence from sample B, Fluorescence from C is observed. It is also observed that the translational diffusion time calculated from the fluorescence of sample C is longer than the translational diffusion time calculated from the fluorescence of sample B by FCS analysis. On the other hand, when the sample B is not present in the sample, none of the samples are associated with each other, so that only the fluorescence derived from the fluorescent material of the sample A is detected even when the fluorescent material labeled with the sample A is excited.

  Further, the number of labeled samples is not limited to the above, and can be further increased according to the type of sample if necessary.

  Thus, according to the present invention, it is possible to detect the reaction state of a plurality of molecules with higher accuracy by simultaneously measuring the diffusion constant, fluorescence cross-correlation analysis and FRET for the plurality of molecules.

  In the above method, the association between sample A and sample B and the association or interaction of two or more molecules such as binding and dissociation were detected. In another embodiment of the present invention, the structural change in the molecule or the intramolecular domain A fluorescence spectroscopic analysis method for detecting an association state, wherein the first fluorescent material and a second fluorescent material that uses the fluorescence energy of the first fluorescent material as excitation energy are fluorescently labeled. Irradiating the sample with light having a wavelength that excites the first fluorescent material, detecting the fluorescence generated from the first fluorescent material and the second fluorescent material, and detecting the detected And performing a fluorescence correlation spectroscopy or fluorescence intensity distribution analysis based on the fluorescence, and detecting a structural change of the sample or an association state of intramolecular domains.

  For example, in a first molecule and a second region that exist close together under certain conditions but exist in different positions under different conditions in a single molecule, Each is labeled with a first fluorescent material and a second fluorescent material. Under such conditions that the first region and the second region of the molecule are close to each other, the fluorescent substances in these regions are considered to be close. Therefore, when the sample is irradiated with light having a wavelength that excites the first fluorescent substance under such conditions, FRET from the first fluorescent substance to the second fluorescent substance is observed, and as a result, the first fluorescent substance is observed. Although the fluorescence from the substance is hardly observed, it is considered that the fluorescence from the second fluorescent substance is observed.

  Thus, structural changes of single molecules or association states of intramolecular domains can be detected by fluorescence spectroscopy.

  As an example of the detection of the reaction between the membrane fraction and the ligand, consider epidermal growth factor receptor (EGFR). EGFR forms a dimer when bound to the ligand EGF. When EGFR becomes a dimer, stimulation is transmitted into the cell and a reaction occurs. For example, in an experiment, human cultured cell A431 expressing a large amount of EGFR is used, and after this cell is treated with a hypotonic solution, a membrane fraction expressing EGFR is prepared with a homogenizer. Next, EGF (epidermal growth factor, fluorescein conjugate, Molecular Probes E-3481) labeled with FITC and EGF (epidermal growth factor, tetramethylrhodamine conjugate, Molecular Probes E-3478) labeled with tetramethylrhodamine were simultaneously added. Then, the reaction with EGFR is observed using FIDA-FRET analysis. In this case, the sample is excited with light having a wavelength of 488 nm that excites FITC. Next, as described above, fluorescence is detected by separating the fluorescence of FITC and tetramethylrhodamine. It is considered that the ratio of the detected fluorescence intensity of FITC and the fluorescence intensity of tetramethylrhodamine decreases due to the presence of the membrane fraction, and FRET is observed. At this time, it is considered that the reaction shown in FIG. That is, EGF fluorescently labeled with FITC and EGF labeled with tetramethylrhodamine are different only in fluorescent labeling, and thus cause a competitive reaction with EGFR. Therefore, even if a multimer is formed, a FITC-labeled EGF-only dimer and a tetramethylrhodamine-labeled EGF-only dimer are also produced, but with a certain probability (probably around 50%). It is thought that a dimer of EGF and tetramethylrhodamine labeled EGF is produced. In this case, the fluorescence emission from tetramethylrhodamine is increased by the excited FITC, so that multimer formation can be detected by detecting this fluorescence. It is possible to accurately observe the presence or absence of multimer formation by FIDA-FRET, which can measure the fluorescence intensity per molecule.

  Next, in order to confirm the inhibition state of the reaction between EGFR and fluorescently labeled EGF, an experiment in which an antibody against EGFR (Santa Cruz) was added in the above experiment was performed, and this concentration was changed. The results will be shown (Figure 2). As shown in FIG. 2, when the inhibitor concentration in the reaction system was low, the fluorescence intensity of tetramethylrhodamine (square) was high, and when the inhibitor concentration was high, it was low (diamond). Thus, it is considered that whether or not molecular association has occurred can be detected by observing FRET using fluorescence fluctuation analysis such as fluorescence correlation spectroscopy or fluorescence intensity distribution analysis.

Detection of intermolecular interaction As an experimental example, the case of analyzing the interaction of troponin, tropomyosin, and actin is described. Troponin was fluorescently labeled with rhodamine green, tropomyosin with tetramethylrhodamine, and actin with Cy5. Troponin, tropomyosin and actin are extracted from rabbit skeletal muscle, purified and fluorescently labeled.

  First, labeled troponin, tropomyosin, and actin are mixed. Next, irradiation with 488 nm laser light that excites rhodamine green is performed, and fluctuations in fluorescence of rhodamine green, tetramethylrhodamine, and CY5 are detected, and FCS analysis is performed. For example, as shown in the upper part (a) of FIG. 3, it is considered that fluorescence from rhodamine green is observed. Next, after adding and mixing a calcium chloride aqueous solution as a reaction initiator between these molecules, a laser beam of 488 nm is irradiated to perform FCS analysis. For example, as shown in the upper part (b) of FIG. 3, it is considered that the fluorescence from rhodamine green decreases and the fluorescence from tetramethylrhodamine is newly observed. The translational diffusion time calculated from the fluorescence from tetramethylrhodamine is longer than the translational diffusion time calculated from the fluorescence from rhodamine green. This phenomenon is caused by the interaction between troponin and tropomyosin, the distance between the fluorescent dyes labeled on each molecule is reduced, and the signal of tetramethylrhodamine is detected by the occurrence of FRET between the fluorescent dyes. Therefore, it is considered that the translational diffusion time becomes longer. Further, after a certain period of time, the fluorescence from rhodamine green further decreases, the fluorescence from tetramethylrhodamine decreases, and the fluorescence from Cy5 is newly observed as shown in the upper part (c) of FIG. it is conceivable that. In addition, it is considered that the translational diffusion time calculated from the fluorescence from tetramethylrhodamine is longer than the translational diffusion time calculated from the fluorescence from rhodamine green, and the translational diffusion time calculated from the fluorescence from Cy5 is further increased. This phenomenon is thought to be due to the occurrence of FRET between the three fluorescent dyes due to the bond where the interaction of the three molecules has occurred and the distance between the fluorescent dyes of the respective labeled molecules. Therefore, it will be clear that the presence of calcium ions is necessary for the reaction to occur, and the order in which the reactions occur is the order of troponin, tropomyosin and actin. Furthermore, if the occurrence of FRET can be detected, it becomes clear that the distance between the labeled rhodamine green and tetramethylrhodamine and the distance between tetramethylrhodamine and Cy5 are within the range where FRET occurs (about 10 nm).

  For example, if the troponin-actin-tropomyosin sequence interacts, FRET does not occur and only a rhodamine green signal is obtained. Moreover, it turns out that these do not react also from translational diffusion time.

  Even if molecular interaction has occurred, even if the distance between the fluorescent dyes that label the molecule does not reach the distance at which FRET occurs (about 10 nm), whether or not the reaction has occurred based on the diffusion rate by FCS analysis It is possible to evaluate. It is also possible to perform a cross-correlation analysis on the fluorescence signals that can be excited simultaneously using two types of lasers that excite each fluorescent dye, and calculate the ratio of molecules that interact with each other. From these measurements, it is possible to obtain various findings regarding molecular interactions.

Thus, knowledge about the structure of the molecule | numerator produced | generated and the interaction place between molecules can be acquired by devising the order of reaction of several molecular interaction, and the place to label to a molecule | numerator.

  An example of detecting molecular dissociation will be given.

  An example is the detection of the dissociation of G proteins present on the membrane. G protein is a subunit of a trimer composed of three subunits triggered by the binding of a G protein coupled receptor (GPCR), which is a membrane protein on the same membrane, to a ligand. (Α subunit) is released and transmits the stimulus into the cell. For example, in molecular biology, each subunit of this G protein is labeled with a different fluorescent protein that causes FRET. For example, an angiotensin receptor (angiotensin receptor) is used as GPCR. Specifically, a cultured cell (for example, CHO-K1 cell) in which an angiotensin receptor is expressed is used. Next, genes are introduced into the cells, and the genes are introduced so that fluorescent proteins having different spectral characteristics are expressed in each subunit (α, β, γ) when the G protein-coupled receptor is expressed. For example, CFP is expressed in the α subunit, GFP is expressed in the β subunit, and YFP is expressed in the γ subunit. The GPCR and G protein used in the experiment are used as a membrane fraction by disrupting cells with a hypotonic solution treatment and a homogenizer.

  When the GPCR in the membrane fraction is not activated, the G protein is thought to form a trimer with α, β, and γ subunits. The CFP labeling the α subunit is then excited at a wavelength of 450 nm (GFP is excited by this wavelength). Next, when CFP fluorescence, GFP fluorescence, and YFP fluorescence are observed, fluorescence is observed from the three fluorescent proteins. It is thought that FRET occurs between CFP and GFP or YFP. When angiotensin as a ligand is added to the membrane fraction solution and GPCR is activated, the α subunit of the G protein is released. When CFP is excited at a wavelength of 450 nm (CFP is excited by this wavelength) and the fluorescence of each fluorescent protein is observed, the fluorescence intensity of CFP is high, and the fluorescence from other fluorescent proteins is hardly observed. It is done. This phenomenon occurs because only the fluorescence of the α subunit is observed without FRET. FIG. 4 shows a model of the dissociation reaction. For example, in the state where the GPCR is not bound to the ligand as shown in the upper part of FIG. 4, since the GPCR is not activated, it is considered that all the subunits of the G protein are associated. Here, when CFP is irradiated with excitation light having a wavelength of 450 nm, fluorescence energy transfer occurs from CFP, and as a result, YFP fluorescence is observed. Next, when angiotensin which is a ligand is added, GPCR binds to the ligand and is activated, and the α subunit of the G protein is dissociated. Fluorescence energy transfer does not occur even when the CFP is irradiated with excitation light with a wavelength of 450 nm, and thus CFP fluorescence is observed. According to fluorescence correlation spectroscopy, the translational diffusion time is shortened and the α subunit is dissociated. Is clearly observed. In addition, when irradiating light with a wavelength that excites GFP, YFP fluorescence is observed, and the translational diffusion time by fluorescence correlation spectroscopy is shorter than when α, β, and γ subunits are bound. Longer than the translational diffusion time of a single γ subunit. Therefore, it can be clearly seen that the α subunit is dissociated, and the β and γ subunits are bound to each other (lower part of FIG. 4). In this way, it is also possible to detect molecular dissociation using FRET.

Detection of structural changes in molecules (corresponding to claim 4)
It is also possible to obtain knowledge about structural changes when troponin interacts with muscle proteins due to changes in calcium ion concentration.

  For example, using a molecular biological technique, a fluorescent protein is expressed at a site that is thought to undergo structural changes upon expression of troponin. In the three-dimensional structure of troponin, GFP is expressed at the TnT1 site and RFP is expressed at the C-TnT site. First, when light of a wavelength that excites GFP is irradiated, it is considered that a lot of fluorescence from GFP is observed as shown in the left of FIG. Next, when FRET analysis is performed after adding calcium ions and FRET is observed, the three-dimensional structure of the troponin molecule changes, and the two parts of the TnT1 site and the C-TnT site are in close proximity. Are known. Therefore, as shown in the left of FIG. 5, it is considered that the fluorescence intensity of RFP increases and the fluorescence intensity of GFP decreases. In this way, it is possible to know whether or not a three-dimensional structure change has occurred.

  Furthermore, by adding tropomyosin that reacts with troponin and performing FCS analysis, the translational diffusion times calculated before and after the addition can be compared to confirm the presence or absence of interaction between both molecules. The upper part of FIG. 6 shows the FCS measurement results before calcium addition, and the lower part of FIG. 6 shows the FCS measurement results after calcium addition. Thus, an increase in the fluorescence intensity of RFP is observed when calcium is added, and the translational diffusion time calculated by FCS analysis of the fluorescence signal of RFP when tropomyosin is added is observed. Can do.

  As a control, when tropomyosin is added to troponin to which calcium is not added, fluorescence of GFP is observed, and the translational diffusion time calculated by performing FCS analysis is also considered to indicate the diffusion time of troponin itself.

  Using such a technique, it is also possible to detect an interaction with another molecule due to an intramolecular structure change. For example, the reaction of calmodulin and calmodulin binding protein is taken as an example. Calmodulin has the property of activating the binding to calcium ions and greatly changing the structure. With this as a trigger, calmodulin-binding protein binds to calmodulin and a stimulus is transmitted. At this time, if the calmodulin is labeled with a different fluorescent dye and further labeled with a fluorescent dye that causes FRET on the calmodulin-binding protein, the molecular interaction can be detected using FRET.

Analysis of molecular structure (distance between molecules) involved in intermolecular interaction Intermolecular interaction occurs when a specific site of a molecule interacts closely. Therefore, in order to detect the interaction of these molecules by FRET, when analyzing the binding site in a given molecule, a fluorescent label may be provided in the vicinity of the binding site of each interacting molecule.

  When the binding sites of both molecules interact with each other and the two are in close proximity, FRET is considered to occur, and the molecular interaction can be detected by measuring this fluorescence emission.

. The efficiency of FRET has been shown to be inversely proportional to the sixth power of the distance r between the donor and the acceptor. In FIDA analysis, information about the fluorescence intensity per molecule can be obtained, so the FRET efficiency is calculated from the intensity ratio of the fluorescent dyes of each wavelength derived from both molecules, and converted to the distance between both molecules. Is possible. At this time, when the translational nucleic acid time is calculated, it becomes possible to clearly distinguish whether FRET does not occur due to dissociation of the molecule or whether it does not occur due to a structural change.

  For example, assuming that several binding sites are predicted for each molecule for analysis of intermolecular interactions, each binding site is fluorescently labeled and the presence of FRET is confirmed by FIDA analysis. To do. In addition, by calculating the ratio of fluorescence intensities and analyzing the time spent in the confocal region in FCS analysis, it is possible to confirm molecular interactions such as whether dissociation or binding has occurred. Further, as described above, it is also possible to know the degree of proximity between molecules from the ratio of fluorescence intensity. In other words, FRET has occurred, but from the above two data, it is possible to confirm whether interaction has occurred, whether there is interaction with the target binding site, and the distance between interacting molecules. . FIG. 8 shows the results assumed to be obtained for the donor-acceptor distance and the FRET efficiency.

The schematic diagram which shows the detection of the interaction with the epidermal growth factor receptor and its ligand by the method of this invention. The schematic diagram which shows the fluorescence intensity change (Q = arbitrary unit) by EGF inhibitor density | concentration. The figure which shows an example (Q = arbitrary unit) of the intermolecular interaction detected by the method of this invention. The figure which shows an example of the intermolecular interaction detected by the method of this invention. The schematic diagram which shows the detection (Q = arbitrary unit) of the interaction of the G protein receptor group by the method of this invention. The schematic diagram which shows the detection of the intramolecular structure change by the method of this invention. The figure which shows an example of the intermolecular interaction detected by the method of this invention.

Explanation of symbols

1, 8 ... Membrane fractionation
2, 9 ... EGFR
3 ・ ・ ・ FITC label EGF
4 ... TRITC sign EGF
5 ... Ch1 (Rhodamine Green-Troponin)
6 ... Ch2 (TAMRA-Tropomyosin)
7 ... Ch3 (Cy5-actin)
10 ... G protein DNA probe
11 ... CFP
12 ... GFP
13 ... YFP
14 ... Ligand

Claims (5)

A method of fluorescence spectroscopy for detecting the association or interaction of sample A and sample B, comprising:
A step of contacting the fluorescently labeled sample A and a sample B fluorescently labeled with a fluorescent substance such that the fluorescent energy of the fluorescent substance labeled on the sample A is an excitation energy;
Irradiating the sample A with light having a wavelength that excites the fluorescent substance labeled;
Detecting fluorescence generated from the fluorescent substances labeled on the sample A and the sample B;
Performing fluorescence correlation spectroscopy or fluorescence intensity distribution analysis based on the detected fluorescence, and detecting a reaction change process between the sample A and the sample B;
A method comprising the steps of: A method of fluorescence spectroscopy for detecting an association or interaction of sample A, sample B and sample C, wherein the method according to claim 1,
The step of contacting the sample A, which is fluorescently labeled with a fluorescent substance such that the fluorescent energy of the fluorescent substance labeled with the fluorescently labeled sample A, the fluorescent energy of the fluorescent substance labeled with the sample A is used as excitation energy;
Irradiating the sample A with light having a wavelength that excites the fluorescent substance labeled;
Detecting fluorescence generated from the fluorescent substances labeled on the sample A and the sample B;
Performing fluorescence correlation spectroscopy or fluorescence intensity distribution analysis based on the detected fluorescence, and detecting a reaction change process of the sample A, the sample B, and the sample C;
A method comprising the steps of: A method according to claim 2, comprising
Sample A labeled with the fluorescent material, Sample B fluorescently labeled with a fluorescent material that uses the fluorescent energy of the fluorescent material labeled with the sample A as excitation energy, and fluorescence energy of the fluorescent material labeled with the sample B A step of contacting the sample C fluorescently labeled with a fluorescent material such that is used as an excitation energy,
Irradiating the sample A with light having a wavelength that excites the fluorescent substance labeled;
Detecting fluorescence generated from the fluorescent substances labeled on the sample A, the sample B, and the sample C;
Performing fluorescence correlation spectroscopy or fluorescence intensity distribution analysis based on the detected fluorescence, and detecting a reaction change process of the sample A, the sample B, and the sample C;
A method comprising: A fluorescence spectroscopic method for detecting structural changes or association states of intramolecular domains in a sample, comprising:
A wavelength for exciting the first fluorescent material with respect to a sample fluorescently labeled with both the first fluorescent material and a second fluorescent material that uses the fluorescence energy of the first fluorescent material as excitation energy. Irradiating with light,
Detecting fluorescence generated from the first fluorescent material and the second fluorescent material;
Performing fluorescence correlation spectroscopy or fluorescence intensity distribution analysis based on the detected fluorescence, and detecting a structural change of the sample or an association state of intramolecular domains;
A method comprising the steps of:   The method according to any one of claims 1 to 4, wherein the sample A or the sample B or the sample is fixed to a cell membrane.

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