JP2008292371A - Method for analyzing interaction by a plurality of components by fluorescence correlated spectroscopy and method for screening compound for controlling interaction - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To analyze interaction by a plurality of components using a fluorescence correlation spectroscopy method with high sensitivity. <P>SOLUTION: The analyzing method comprises the steps of labeling at least one of a plurality of components which interact by a fluorescent material, and analyzing a solution containing the plurality of components by fluorescence correlation spectroscopy method and detecting the number of particles of the fluorescent material, and/or a hydrodynamic radius. The solution containing the plurality of components has a viscosity regulator with molecular weight of 3,350 or greater so that the viscosity of the solution is 5 to 100 mPa s. This analyzing method can apply to a compound for controlling reaction of a plurality of components, for example effective screening of an inhibitor. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for analyzing the interaction of a plurality of components using fluorescence correlation spectroscopy, and more particularly to a method for analyzing intermolecular interactions and the like with high sensitivity by fluorescence correlation spectroscopy. Furthermore, the present invention relates to a method for screening a compound that controls multi-component interaction using the analysis method.

  Fluorescence Correlation Spectroscopy (FCS) is a method for analyzing physical changes in fluorescence intensity in a minute region and obtaining physical quantities related to the number, shape, and size of fluorescent particles involved in the fluorescence fluctuation ( Non-Patent Documents 1 and 2). In general, the range of information obtained by fluorescence correlation spectroscopy is wide, and in addition to information resulting from Brownian motion in sub-femtoliter microregions, for example, information on molecular structural fluctuations and chemical reactions. It reaches. This analysis method is based on the principle of single molecule measurement, and the volume of one sample may be very small, and is suitable for measurement using a highly difficult protein sample in which protein production is difficult. In addition, in the conventional measurement methods such as ELISA and surface plasmon resonance, it is necessary to immobilize the measurement sample. In fluorescence correlation spectroscopy, however, the sample solution only needs to be mixed and dispensed onto the plate. There is an advantage that preparation is simple and quick.

  Fluorescence correlation spectroscopy is a system that analyzes the translational diffusion time of a fluorescently labeled sample by single molecule fluorescence analysis. With a confocal laser, it is possible to measure a temporal change in fluorescence intensity with a high sensitivity of several molecules in a very small region of about 1 / 1,000 trillion. Therefore, the fluctuation of the fluorescence intensity caused by molecules entering and exiting the measurement region due to Brownian motion is observed. At this time, a small molecule becomes a fast fluctuation and a translational diffusion time becomes small, and a large molecule becomes a slow fluctuation and a translational diffusion time becomes large. It is known that the translational diffusion time is proportional to the third root of the molecular weight.

  This principle can be used to analyze two-component interactions such as interactions between proteins and interactions between proteins and nucleic acid molecules. For example, a fluorescent substance is bound to one of a nucleic acid molecule and a protein and labeled, and then the nucleic acid molecule and the protein are allowed to interact in a solution. And the fluorescence in the said area | region is measured by making the micro area | region in a solution into a measurement area | region. The fluctuation of the detected fluorescence intensity differs depending on whether the nucleic acid molecule interacts with the protein or not, and the interaction between the nucleic acid molecule and the protein based on the fluctuation of the fluorescence intensity. The presence or absence of can be detected. A method for analyzing a two-component interaction based on such a principle has been reported by the present applicant as, for example, Japanese Patent Application Laid-Open No. 2005-98766 (Patent Document 1), the contents of which are incorporated herein by reference. . As disclosed in the above-mentioned Patent Document 1, if the value of fluctuation (translational diffusion time) of fluorescence intensity of a large molecule can be measured larger, when the two components to be measured interact with each other, It is considered that the difference in measured value from the case where it exists is large, and the presence or absence of interaction can be detected with higher sensitivity.

  On the other hand, a compound that inhibits an interaction between proteins has recently attracted attention as a molecular target drug because it can specifically inhibit a protein network with only one pathway. One example is Nutlin3, which inhibits the interaction between tumor suppressor p53 and MDM2 (see, for example, Non-Patent Document 3). MDM2 protein (ACCESSION: Q00987) is known to bind to p53 protein and promote degradation of p53. This interaction is known to bind the N-terminal peptide (15-29) of p53 to the SWIB domain of the N-terminal (17-125) of MDM2, and its complex structure has also been reported (PDB) : 1 YCR). In many human tumors, enhancing p53 function is thought to provide an anti-tumor effect. Nutlin3 has been shown to activate the p53 pathway in tumor cells by binding to the p53 binding pocket of MDM2 and inhibiting these interactions and preventing p53 degradation.

JP 2005-98766 A “Protein Nucleic Acid Enzyme”, vol. 44, no. 9 (1999), p1431-1438 Eggeling C, Fries JR, Brand L, Guenther R, and Seidel CAM, Proc. Natl. Acad. Sci. USA 95, 1556-1561, 1998 Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, Kong N, Kammlott U, Lukacs C, Klein C, Fotouhi N, Liu EA, Science 2004, 303 (5659): 844-8

  The translational diffusion time determined by fluorescence correlation spectroscopy is in principle proportional to the third root of the molecular weight of the labeled sample. Therefore, for example, assuming that the binding of the MDM2 protein domain (molecular weight: 13.4 kDa) and the 16-residue fluorescently labeled peptide (molecular weight: 2347) bound thereto is detected, the measured value of the translational diffusion time is: When the inhibitor screening is carried out under the condition of 50% binding, the difference in the measured values is only 1.4 times. At this time, since the measurement of the translational diffusion time involves a large measurement error, screening for inhibitors is difficult unless the measurement accuracy is ensured by increasing the measurement time and the number of measurements.

  However, in order to efficiently screen for inhibitors, it is necessary to shorten the time required for measuring one candidate compound. For this reason, there is a need for a method for detecting a translational diffusion time with little error even in a short measurement time. In order to efficiently perform screening using fluorescence correlation spectroscopy, a technique capable of measuring a multi-component interaction such as the protein-protein interaction with high sensitivity is essential.

  The present invention has been made in order to solve the above-described problem. When measuring the translational diffusion time of a fluorescent substance by fluorescence correlation spectroscopy, the polymer fluorescent particles are adjusted by adjusting the viscosity in the measurement solution. It was found that the difference between the translational diffusion time and the translational diffusion time of small molecule fluorescent particles can be detected as a larger value with good reproducibility. It can be predicted from the Einstein-Stokes equation that the translational diffusion time is proportional to the viscosity of the solvent. However, when actually examined, it was found that the molecular weight of the viscous polymer (viscosity regulator) used and the molecular weight of the fluorescent particles had a very complicated effect on the translational diffusion time. If the viscosity is too high, the dispensing error of the solution may increase, or the binding affinity of the multi-component interaction may be affected. The present invention has been completed based on these findings.

  That is, in the first aspect, the multi-component interaction analysis method of the present invention includes a step of labeling at least one of a plurality of interacting components with a fluorescent substance, and a solution containing the plurality of components by fluorescence correlation spectroscopy. Analyzing and detecting the number and / or hydrodynamic radius of the phosphor particles. Here, the solution containing the plurality of components includes a viscosity modifier having a molecular weight of 3350 or more so that the viscosity of the solution is 5 to 100 mPa · s. In a preferred embodiment, the viscosity modifier includes any selected from polyalkylene glycol, polyvinyl alcohol, and polyvinyl pyrrolidone. More preferably, the polyalkylene glycol is polyethylene glycol having an average molecular weight of about 6000 to about 600,000. A more preferred range for the average molecular weight of the polyethylene glycol is from about 6000 to about 35000, and an even more preferred range is from about 6000 to about 20000.

  In another aspect, there is provided a method for screening a compound that controls a multi-component interaction using the above analysis method. That is, the screening method of the present invention includes a step of labeling at least one of a plurality of interacting components with a fluorescent substance, and the plurality of components in the presence or absence of a candidate compound for controlling the interaction. Analyzing the solution by fluorescence correlation spectroscopy and measuring the number of particles and / or translational diffusion time of the fluorescent material. Here, the solution containing the plurality of components contains polyethylene glycol having an average molecular weight of 3350 or more, whereby the viscosity of the solution is adjusted to 5 to 100 mPa · s. The average molecular weight of polyethylene glycol is preferably about 6000 to about 600,000. When the translational diffusion time in the presence of the candidate compound is smaller than the translational diffusion time in the absence of the candidate compound, the candidate compound inhibits the interaction of at least two components. In a preferred embodiment, at least one of the plurality of components is a protein or nucleic acid or an interaction region thereof, and the other is another protein, peptide, compound or nucleic acid that binds to the protein or nucleic acid or an interaction region thereof. It is characterized by that.

  According to the method of the present invention, the dependence of the translational diffusion time on the fluorescent particle molecular weight by fluorescence correlation spectroscopy can be enhanced, so that the detection sensitivity of multiple component interactions such as protein-protein interactions can be improved. Can do. Therefore, it is possible to quantitatively analyze the interaction between multiple components using this method, and it is possible to shorten the measurement time and effectively carry out screening for compounds that inhibit the interaction.

  In the interaction analysis of a plurality of components according to the present invention, the interaction between the components is analyzed using fluorescence correlation spectroscopy. In the present method, “multiple components” means two or more components that interact with each other, and each component itself may not be a single molecule. That is, each component in the plurality of components may be a complex composed of a plurality of components. Therefore, in the present method, the expression “multi-component interaction” means that the interaction includes two or more components. When three or more components interact, other components may interact competitively in the same site or region of one component, or other components in different sites or regions of one component May interact with each other. Furthermore, the part or area | region which interacts may overlap partially.

  Here, the constituent elements contributing to the interaction include proteins, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA) and their interaction regions (domains), and peptides and nucleic acid analogs. Examples thereof include nucleic acid molecules, low molecular compounds, other high molecular compounds excluding nucleic acid molecules and proteins. Accordingly, in the present method, the interaction of multiple components is not particularly limited as long as these components contribute, but for example, interaction between protein and protein, interaction between protein and peptide, nucleic acid and nucleic acid Interaction, nucleic acid-protein interaction, ligand-receptor protein interaction, and the like.

  In this method, first, a solution containing a plurality of components at least one of which is labeled with a fluorescent substance is prepared. Here, usable fluorescent substances include, but are not particularly limited to, substances that exist as proteins and emit light signals such as fluorescence and light emission by laser irradiation or their own energy. For example, green fluorescent protein (hereinafter referred to as GFP; Green Fluorescent Protein), CFP (Cyan Fluorescent Protein), YFP (Yellow Fluorescent Protein), and RFP (Red Fluorescent Protein) extracted from Aequorea jellyfish. Furthermore, the same fluorescent protein derived from Renilla may be used.

  Further, usable fluorescent substances include binding to the N-terminal and / or C-terminal of a peptide, the 5′-end and / or 3′-end of a nucleic acid molecule, a part of a specific compound, and the like to label the molecule. Examples thereof include TAMRA, Alexa488, Alexa633, Cy3, Cy5, FITC, Rhodamine, Texas Red, Rhodamine Green, GFP, YOYO1, TMR, EVOblue, Alexa647 and the like.

  For example, when one of a plurality of components to be analyzed is a protein and a fluorescent substance is bound to the protein, an expression having a recombinant gene encoding a fusion protein of the protein to be analyzed and the fluorescent protein described above A vector can be constructed, and a labeling substance can be bound to the protein using the expression vector.

  In addition, when the other one of the plurality of components to be analyzed is a peptide, a technique for specifically fluorescently labeling its N-terminal amino group or C-terminal carboxyl group is known. For example, the N-terminal α-amino group can be labeled with various fluorescent dyes using N-hydrosuccinimide ester or the like.

In the method of the present invention, the analysis by fluorescence correlation spectroscopy means detection of information such as the number of fluorescent particles and hydrodynamic radius in a minute region. Based on the obtained information, typically a translation diffusion time is measured. The translational diffusion time τ _D measured by fluorescence correlation spectroscopy is based on the following equation (1).
τ _D = ω ₀ ² / 4D (1)
This equation is based on the horizontal axis ω ₀ of the fluorescence intensity measurement region (confocal region) and the diffusion constant D of the molecule. The diffusion constant D of the molecule is expressed by equation (2).
D = K _B T / 6πηr (2)
Boltzmann constant K _B , absolute temperature T, solution viscosity (viscosity coefficient) η, and molecular radius (hydrodynamic radius) r when the molecule is assumed to be spherical are parameters.

From these equations, it can be seen that the translational diffusion time τ _D is approximately proportional to the third root of the molecular weight. On the other hand, when the measured translational diffusion time is a weighted average value of translational diffusion times of two or more fluorescent particles having different translational diffusion times, as in the following examples of the present specification, In fluorescence correlation spectroscopy, these are decomposed into two components from the analysis of fluorescence autocorrelation function, and the abundance ratio of each component (in the following examples, protein-labeled peptide complex and dissociated labeled peptide alone). Can be analyzed.

  In order to enhance the sensitivity of fluorescence correlation spectroscopy, a viscosity modifier having a molecular weight of 3350 or more is added to the solution containing the plurality of components, and the viscosity of the solution becomes 5 to 100 mPa · s, preferably 5 to 50 mPa · s. Adjust as follows. Examples of the viscosity modifier include homopolymers of polyalkylene oxides such as polyethylene glycol (PEG) and polypropylene glycol, polyoxyethylenated polyols, copolymers thereof, and block copolymers thereof. Alternatively, including but not limited to hydrophilic polyvinyl polymers such as polyvinyl alcohol and polyvinyl pyrrolidone. Preferred viscosity modifiers include polyethylene glycol, more preferably polyethylene glycol having an average molecular weight of about 6000 or more. More preferably, the average molecular weight of the polyethylene glycol is in the range of about 6000 to about 600000. In addition, polyethylene glycols having molecular weights of 35,000, 100,000, 200,000, 300,000, and 400,000 are commercially available. Those having a molecular weight of 100,000 or more are called polyethylene oxide. In a still further preferred embodiment, the average molecular weight of the PEG is about 10,000 to about 20,000, and the viscosity of the solution containing the two components is 7.5 to 25 mPa · s. For example, when PEG10000 is used, the concentration in the solution is adjusted to about 10% or more. Further, when PEG 20000 is used, it is about 5% to about 20%. High molecular weight polyethylene glycols and hydrophilic polyvinyl polymers such as polyvinyl alcohol and polyvinyl pyrrolidone can give higher viscosity at a lower concentration to the solution as the molecular weight becomes higher, such as 10,000 or more. This is useful because it can attenuate the additional effects. On the other hand, polyethylene glycol / polyethylene oxide having molecular weights of 900000, 1000000, 2000000, 4000000, 5000000, 7000000, 8000000, etc. are also commercially available, but problems such as poor water solubility and low purity of the commercial products arise.

  Polyethylene glycol (PEG) used in the method of the present invention is a linear water-soluble polymer obtained by polymerizing ethylene glycol having two terminal hydroxyl groups, and PEG is classified by their molecular weight. For example, PEG 5000 has an average molecular weight of about 5000 daltons, and PEG 10000 has an average molecular weight of about 10,000 daltons.

  In one embodiment of the present invention, there is provided a method of screening for an inhibitor of the interaction using the method for analyzing a multicomponent interaction. FIG. 1 is a schematic diagram showing a typical embodiment thereof. This screening method is an assay system that measures a complex formation / dissociation reaction between a protein interaction domain and a binding domain peptide of a partner protein that binds to the protein interaction domain. The binding region peptide of the protein is fluorescently labeled, and the translational diffusion times of the labeled peptide alone and the protein-labeled peptide complex are compared. Then, by allowing the candidate compound to coexist in this assay system, it is detected whether or not the protein-labeled peptide complex is dissociated. When one candidate compound binds to the protein interaction domain, that is, when it is an inhibitor, the labeled peptide is dissociated from the binding site, so that the molecular weight of the fluorescent substance is reduced, thereby reducing the translational diffusion time. Therefore, inhibitors can be screened by measuring this translational diffusion time in the presence or absence of a candidate compound.

  Candidate compounds for controlling the interaction that can be used in the screening method of the present invention are not particularly limited. For example, various known compounds (including peptides) registered in the chemical file, combinatorial A compound group obtained by a chemistry technique or a normal synthesis technique, or a random peptide group created by applying a phage display method or the like can be used. In addition, microbial culture supernatants, natural substances derived from plants or marine organisms, or animal tissue extracts can also be used as candidate compounds for screening. Furthermore, a compound obtained by chemically or biologically modifying a compound obtained by the screening method of the present invention can also be used.

  A compound that controls the interaction of a plurality of components in a living body typically includes an inhibitor of these interactions. For example, inhibitors of various tyrosine kinases, which are receptor proteins, have been developed as molecular target drugs for cancer and imatinib, which is already a therapeutic agent for chronic myeloid leukemia, and a therapeutic agent for lung cancer. A molecular targeted drug such as gefitinib is on the market. Therefore, the interaction control compound screened by the method of the present invention may be used, for example, as a pharmaceutical or diagnostic agent as an inhibitor of an in vivo signal transduction system.

[Reagents and methods]
0.05 M Tris-HCl (pH 8) and 0.05% Tween 20 were used as a buffer for fluorescence correlation spectroscopy measurement (FCS buffer). Examples of PEG added here include PEG1000 (50%, w / v), PEG3350 (50%, w / v), PEG6000 (50%, w / v), PEG8000 (50%, w / v) manufactured by Hampton Research. ), PEG 10000 (50%, w / v), PEG 20000 (30%, w / v) were purchased. The purchased stock solution was diluted with FCS buffer to final concentrations of 1.25, 2.5, 6.25, 12.5, 18.75, 25% (v / v) to prepare a 1.25-fold PEG buffer. .

  As the fluorescent dye, TAMRA (molecular weight: 528 Da) (standard reagent for MF20) was dissolved in FCS buffer and used. A 10-mer peptide (molecular weight: 1.5 kDa) was obtained from Kirk D. Beebe, P. Wang, G. Arabaci, D. Pei: Determination of the Binding Specificity of the SH2 Domains of Protein Tyrosine Phosphatase SHP-1. SHP-1 binding peptide sequence selected based on the Combinatorial Phosphotyrosyl Peptide Library. 39: 13251-13260, 2000), TAMRA-labeled (ITpYSLLKGGK-TAMRA), 16mer peptide (Molecular weight: 2.3 kDa) purchased a p53N terminal peptide (SQETFSDLWKLLPENK-TAMRA) labeled with TAMRA on the C-terminal side.

[Preparation of MDM2 protein sample]
The MDM2 (17-124) (13.4 kDa) sample was prepared by the following method. The protein is expressed by a reaction at 30 ° C. for 4 hours using an E. coli cell-free protein synthesis system (Kigawa et al., FEBS Lett., 442-1: 15-19, 1999) (9 ml of internal solution / 90 ml of external solution). It was. The expressed protein was dialyzed overnight at 4 ° C. against 20 mM Tris-HCl (pH 8.0) containing 4% PEG8000, 100 mM NaCl, and 20 mM imidazole.

  Since the obtained sample was cleaved with a TEV protease cleavage site and a histidine tag, affinity purification was performed using the tag. The sample was passed through a HisTrap column and eluted with 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 500 mM imidazole. The eluted sample was applied to a desalting column to remove imidazole and exchange the buffer with 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 2 mM DTT. To this sample, 4000 units of TEV protease was added and mixed by inversion at 4 ° C. overnight to cleave the histidine tag. The sample cleaved with the tag was separated into the cleaved tag and the target protein by affinity purification with HisTrap again. The obtained target protein was concentrated by ultrafiltration (Amicon Ultra-15 MWCO 5000). The concentrated final sample was quantified using the Bradford method and found to be 36 mg / ml. 350 μl of protein solution was obtained.

[PEG and viscosity]
As the viscometer, an SV type viscometer manufactured by A & D Co., Ltd. was used. Two-point calibration was performed with viscometer calibration standard solutions JS10 and JS100 (Nippon Grease Co., Ltd.) to correct the viscosity value (measurement temperature 22 ° C.). The prepared 1.25-fold PEG buffer was diluted to 1-fold concentration with FCS buffer. 10 ml of diluted PEG buffer was placed in a sample container, adjusted so that the liquid level of the sample was at the center of the neck of the vibrator, and placed in the PEG buffer for measurement (see Table 1 below). As shown in Table 1, the viscosity increased as the PEG% concentration increased. As the PEG molecular weight increases, the viscosity increases. As the PEG molecular weight increases, the size of the PEG% concentration affects the viscosity.

[Viscosity and apparent translational diffusion time]
For the measurement of the translational diffusion time, a 10-fold concentration fluorescently labeled sample was added to a solution obtained by mixing 1.25-fold PEG buffer and FCS buffer. TAMRA and TAMRA labeled peptide were added at a final concentration of 1 nM. For the measurement, a single molecule fluorescence analysis system (MF20) (manufactured by OLYMPUS) was used. These prepared samples were dispensed on a 384 plate for MF20 measurement 30 μl at a time and measured. Measurement of MF20 was performed 5 times at an excitation wavelength of 543 nm for 10 seconds. The PEG buffer for measurement was converted into viscosity and a graph was created (see FIGS. 2 and 3). FIG. 2 shows the viscosity and translational diffusion time for three fluorescent materials with different molecular weights. The vertical axis plots the relative value with the translational diffusion time without addition of PEG as 100 after calibrating the measured value with the standard labeling reagent. FIG. 3 compares the difference in viscosity and translational diffusion time due to the difference in molecular weight of PEG. After calibrating the actual measurement value with a standard labeling reagent, the relative value with the translational diffusion rate without addition of PEG as 100 is plotted. When PEG of any molecular weight was used, a positive correlation was found between viscosity and translational diffusion time. However, both are different from the theoretical values. 1) The translational diffusion time is smaller than the proportional value to the viscosity. 2) This anomalous effect was more prominent as the molecular weight of PEG was larger. 3) Also, the larger the molecular weight of PEG, the smaller the proportionality constant when the relationship between the two is approximated by a straight line. 4) The proportional constant of the straight line approximating both is larger as the molecular weight of the solute is larger if the solvent is the same. As estimated from FIGS. 2 and 3, when a solvent having a PEG molecular weight of 6000 or more and a viscosity of 10 mPa · s or more is used, the molecular weight difference of the solute is more greatly reflected in the translational diffusion time.

  Therefore, in order to examine the effect of viscosity on larger solute molecules, Alexa Fluor 647-labeled Goat anti-human IgG (molecular weight: about 150 kDa) was purchased, and the translational diffusion time in PEG buffer was determined at an excitation wavelength of 633 nm. The measurement results are shown in addition to the graph of FIG. From this result, it was expected that when the translational diffusion time was measured using a solvent having a PEG molecular weight of 10,000 or more and a viscosity of about 15 mPa · s or more, the translational diffusion time of a solute molecule having a large molecular weight could be greatly measured.

[Effect of PEG on dissociation constant]
The dissociation constant of the MDM2 p53 peptide complex was measured by fluorescence correlation spectroscopy. The p53 peptide was synthesized by synthesizing SQETFSDLWKLLPENK- (TAMRA) (16mer) labeled with TAMRA at the C-terminus of the peptide chain corresponding to the 15th to 29th residues from the N-terminus, which is the interaction site between p53 and MDM2. A solution dissolved in DMSO was used as a stock solution. DMSO was added to FCS buffer so that the final concentration was 0.5%, and 150 mM NaCl was added or not. PEG 20000 was adjusted to a 2.5-fold concentration with FCS buffer and added to the reaction solution so that the final concentration was 10%. TAMRA-labeled p53 peptide was added to this solution to a final concentration of 1 nM, and protein solution was added to a final concentration of 0, 0.1, 0.3, 1, 3, 10, 30 μM, and 1 at 25 ° C. Reacted for hours. 30 μl each of the reacted solution was dispensed onto a 384 plate for MF20 measurement, and measured 5 times at an excitation wavelength of 543 nm for 10 seconds. The obtained results were plotted and fitting was performed with data analysis / graph creation software Origin (OriginLab).

  First, the case where PEG 20000 was included in the FCS buffer and the case where PEG 20000 was not included were compared (see FIG. 4). In MDM2, the KD value was about 2 μM, and there was almost no difference in the KD value between the case where PEG was contained and the case where PEG was not contained.

  Therefore, the case where NaCl was not contained in the FCS buffer with MDM2 was compared (see FIG. 5). In the interaction of p53 peptide / MDM2, the KD value was about 250 nM when NaCl was contained, and the KD value was about 1.5 μM when NaCl was not contained. The addition of PEG slightly affects the dissociation constant of the protein / peptide complex, which is less than the effect of adding a salt to the solvent, and was not considered a major problem for screening. .

[Acceleration of measurement with MF20]
The measurement of MF20 is performed 5 times for 10 seconds. Under this condition, the measurement time of the 384 plate is about 7 hours. To perform screening, it is necessary to speed up the measurement. However, if the measurement time and the number of times are reduced, there is a problem that the reliability of data is reduced. Therefore, in order to speed up the measurement, the measurement error was examined under the condition where PEG was added to the buffer and the condition where it was not. In this case, when the viscosity of the solution exceeded 52 mPa · s, the handling of the solution became somewhat difficult, and when it exceeded 100 mPa · s, it became very difficult.

  Measurement under conditions without NaCl, 10 seconds 5 times, 5 seconds 5 times, 5 seconds 3 times under two conditions of PEG-free buffer and PEG 20000 added (12.2 mPa · s) buffer Was repeated four times. In the measurement, the translational diffusion rates of Free (1 nM TAMRA-labeled p53 peptide) and Complex (1 nM TAMRA-labeled p53 peptide, 200 nM without PEG, 2 μM MDM2 with PEG 20000), which are control values at the time of screening, were measured. It was measured. A two-sided test was conducted by ANOVA to determine whether the difference between the measured value group of TAMRA-labeled p53 peptide as Free and the measured value group of TAMRA-labeled p53 peptide / MDM2 complex as Complex was significant (see Table 2). .

  As a result, under the condition containing PEG, there was a significant difference between Free and Complex even if the speed was increased to 5 seconds 5 times. It was found that the conditions with PEG were more reliable when the speed was increased.

[Possibility for faster screening]
In order to examine the possibility of screening with a buffer containing PEG, a competitive inhibition experiment was conducted with and without PEG. TAMRA-labeled p53 peptide and MDM2 were used, and Nutlin3a was used as an inhibitor. Sample preparation was performed as described above. The conditions of PEG were molecular weight 20000, 12.2 mPa · s and molecular weight 10,000, 7.53 mPa · s. The measurement was performed 5 times for 10 seconds at an excitation wavelength of 543 nm (see Table 3).

As a result, the addition of PEG did not affect the inhibition of peptide-protein binding by the inhibitor. By including PEG, the difference in translational diffusion time between Free and Complex becomes large, and the difference in inhibition can be measured greatly.

It is the schematic diagram which showed the outline of the screening method of the interaction inhibitor by fluorescence correlation spectroscopy. It is the graph which showed the relationship between the viscosity of PEG buffer and the apparent translational diffusion time. It is the graph which showed the relationship between the viscosity of PEG buffer and the apparent translational diffusion time. It is the graph which showed the addition effect of PEG which gives to the dissociation constant of MDM2 and p53 peptide. It is the graph which showed the addition effect of NaCl which gives to the dissociation constant of MDM2 and p53 peptide.

Claims (10)

Labeling at least one of the interacting components with a fluorescent substance;
Analyzing the solution containing the plurality of components by fluorescence correlation spectroscopy and detecting the number of particles and / or the hydrodynamic radius of the fluorescent material, and analyzing the interaction of the plurality of components. ,
The solution containing a plurality of components includes a viscosity modifier having a molecular weight of 3350 or more so that the solution has a viscosity of 5 to 100 mPa · s.   The method according to claim 1, wherein the viscosity modifier comprises any selected from polyalkylene glycol, polyvinyl alcohol, and polyvinyl pyrrolidone.   The method of claim 2, wherein the polyalkylene glycol is polyethylene glycol having an average molecular weight of about 6000 to about 600,000.   The viscosity adjusting agent includes polyethylene glycol having an average molecular weight of about 10,000 to about 20,000, and the viscosity of the solution containing the plurality of components is adjusted to 7.5 to 25 mPa · s. the method of.   5. At least one of the plurality of components is a protein or nucleic acid or an interaction region thereof, and the other is another protein, peptide, compound or nucleic acid that binds to the protein or nucleic acid or an interaction region thereof. Any of the methods. Labeling at least one of the interacting components with a fluorescent substance;
A step of measuring the number of particles and / or translational diffusion time of the fluorescent substance by analyzing the solution containing the plurality of components by fluorescence correlation spectroscopy in the presence or absence of a candidate compound for controlling the interaction And including
Here, the solution containing the plurality of components includes polyethylene glycol having an average molecular weight of 3350 or more, whereby the viscosity of the solution is adjusted to 5 to 100 mPa · s, and the translational diffusion time in the presence of the candidate compound is A method for screening an interaction controlling compound, which shows that the candidate compound inhibits the interaction of at least two components when it is smaller than the translational diffusion time in the absence of the candidate compound.   The method of claim 6, wherein the multi-component solution comprises polyethylene glycol having an average molecular weight of about 6000 to about 600000.   The solution containing the plurality of components contains polyethylene glycol having an average molecular weight of about 10,000 to about 20,000, and the viscosity of the solution containing the plurality of components is adjusted to 7.5 to 25 mPa · s. The method described in 1.   9. At least one of the plurality of components is a protein or nucleic acid or an interaction region thereof, and the other is another protein, peptide, compound or nucleic acid that binds to the protein or nucleic acid or an interaction region thereof. Any of the methods.   The method according to any one of claims 6 to 9, wherein the concentration of each component in the solution containing the plurality of components is optimized according to the molecular weight and concentration of the viscosity modifier present in the solution.

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