JP2006145470A - Interaction measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of analyzing an interaction as a dynamic function of an objective dimer, and of analyzing competitive interaction with a plurality of coexisting dimers. <P>SOLUTION: A biomolecule A and biomolecule B are under a relation of forming the hetero-dimer by the interaction, and the interaction with a biomolecule C is measured by changing a ratio of the biomolecule A to the biomolecule B to form a complex, and by controlling an existence ratio of a homo-dimer to the hetero-dime. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a dimer biomolecule obtained by changing the mixing ratio of two different types of monomers and a method for measuring the interaction between the biomolecules.

  Many biomolecules may interact with certain other substances to exert structural stability and function. For example, among transcription factors that are proteins that control gene expression, there are molecules that require metal molecules and molecules that function by binding to proteins other than themselves.

  As a transcription factor, there is a transcription factor that requires a zinc molecule in order to stably interact with DNA. This structure is called a Zn finger structure and is one of typical DNA binding motifs of transcription factors. A cysteine residue or histidine residue in a protein's own amino acid sequence and a zinc molecule can form a chelate and interact with DNA.

  Among transcription factors, there are substances that form a dimer via a protein binding site in a transcription factor because a dimer can bind to DNA more stably than a single monomer. A leucine zipper structure, a helix-turn-helix structure, a helix-loop-helix structure, etc. can be mentioned as structures (motifs) involved in dimer formation of transcription factors. Not only do dimers form with homologous transcription factors (homodimers), but they also form dimers with transcription factors that have the same motif (heterodimers). Thus, various transcription factors can be combined, and it is considered that diversity and specificity are imparted to gene expression (Academic Literature 1).

  The b-Zip structure is one of the structures often found in DNA-binding proteins as a motif that forms a dimer and binds to DNA. This structure consists of a DNA binding site rich in basicity and a leucine zipper involved in dimer formation. The leucine zipper has a repeating structure of leucine residues every 7 amino acids in the primary structure, and when an α-helix structure is formed, the leucine residues are arranged on the same plane. Α-helices in the b-Zip structure of the two transcription factors are aligned in parallel, leucine residues are linked to each other, form a dimer, stabilize the structure, and interact accurately with DNA . Since a heterodimer can be easily formed with a transcription factor having a leucine zipper structure, it is widely applicable to control of gene expression in response to various external stimuli.

The functional analysis of transcription factors has been progressed mainly in search of DNA binding sequences using gel shift method, footprint method and the like. In recent years, analysis of interaction with DNA by bond dissociation rate has been regarded as important from the viewpoint of functional analysis of proteins (Non-patent Document 2).
When adjusting a heterodimer by in vitro studies such as the above method, dimers (such as homodimers) other than the target may be formed depending on the mixing ratio of the monomers. . Since they bind to DNA antagonistically, it has been difficult to analyze the interaction as a dynamic function of the heteroprotein (Non-patent Document 3).

Kataoka et al. Mol. Cell. Biol. 15 (1995) 2180-2190 Kyo et al. Gene to Cell 9 (2004) 153-164. Kataoka et al. Mol. Cell. Biol. 14 (1994) 700-712

  An object of the present invention is to analyze an interaction as a dynamic function of a target dimer, and to analyze a competitive interaction of a plurality of dimers that coexist.

As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means.
1. A complex obtained by mixing the biomolecule (A) and the biomolecule (B) is reacted with a biomolecule (C) capable of interacting with the complex, and the complex and the biomolecule (C) are interacted with each other. A method for measuring an action, which comprises controlling the abundance ratio of a homodimer and a heterodimer contained in the complex.
2. By changing the mixing ratio of the biomolecule (A) and the biomolecule (B), the homodimer and the heterodimer contained in the complex obtained by mixing the biomolecule (A) and the biomolecule (B) The interaction measurement method according to 1, wherein the abundance ratio of the monomer is controlled.
3. The complex obtained by mixing the biomolecule (A) and the biomolecule (B) includes at least a heterodimer comprising the biomolecule (A) and the biomolecule (B) and / or the biomolecule (A). A method for measuring interaction between 1 and 2, wherein a homodimer is included.
4). 4. The interaction measurement method according to any one of 1 to 3, wherein the heterodimer comprising the biomolecule (A) and the biomolecule (B) interacts with the biomolecule (C).
5. The interaction measurement method according to any one of 1 to 3, wherein the homodimer comprising the biomolecule (A) interacts with the biomolecule (C).
6). The interaction measurement method according to any one of 1 to 3, wherein the homodimer comprising the biomolecule (B) does not interact with the biomolecule (C).
7). The interaction measurement method according to any one of 1 to 3, wherein the biomolecule (B) alone does not form a homodimer.
8). The interaction measuring method according to any one of 1 to 7, wherein the biomolecule (A) and / or the biomolecule (B) is a protein.
9. The interaction measurement method according to any one of 1 to 8, wherein the biomolecule (A) and / or the biomolecule (B) is a transcription factor.
10. The method for measuring an interaction according to any one of 1 to 9, wherein the biomolecule (C) is a nucleic acid molecule.
11. The interaction measuring method according to any one of 1 to 10, wherein the biomolecule (C) is immobilized on a solid substrate.

  According to the present invention, it is possible to measure the interaction between a biomolecule and a homo- and hetero-dimer obtained by changing the mixing ratio of two different types of biomolecules.

  The present invention is described in detail below. The present invention is a method for measuring the interaction between a dimer obtained by changing the mixing ratio of two different types of biomolecules and a biomolecule that can be recognized by the dimer.

  In the present invention, two different types of biomolecules (A) and (B) can form a dimer by interaction. In general, the interaction of biomolecules is an equilibrium reaction. Even if (A) and (B) are mixed in equimolar amounts, (A) and (B) existing in the system all interact. , (A) and (B) heterodimers are not formed.

Therefore, in the present invention, by changing the mixing ratio of the two biomolecules (A) and (B), the conditions under which the heterodimer is formed are changed, and the composition and biomolecule (C ) Interaction is observed.

  The biomolecules (A) and (B) may interact with each other to form a homodimer or not. For example, when the biomolecule (A) has the ability to interact with itself and can form a homodimer and the biomolecule (B) cannot interact with itself, the biomolecules (A) and (B) In the mixture, the heterodimer of (A) and (B), the homodimer of (A), the monomer of (A), and the monomer of (B) exist in equilibrium. Become. In the present invention, the interaction between the mixture and the biomolecule (C) is observed. In this case, it is an advantage of the present invention that the contribution of each substance to the interaction can be estimated by measuring the interaction while changing the mixing ratio.

  The mixing method of the biomolecules (A) and (B) is not particularly limited, but the biomolecule (A) and the biomolecule (B) are mixed in a solution. It is preferable to change the mixing ratio by keeping the concentration of the biomolecule (A) constant and changing the concentration of the biomolecule (B). This is because, by keeping the concentration of the biomolecule (A) constant, the ratio of forming only the heterodimer can be easily understood.

  The biomolecules (A) and (B) that can constitute the heterodimer are preferably proteins. This is because the protein has a complex higher-order structure and is often non-covalently bound between protein molecules. Non-covalent bonds include hydrogen bonds, van der Waals forces, electrostatic bonds, and the like.

  The biomolecules (A) and (B) are more preferably transcription factors. This is because there are many transcription factors that function as dimers regardless of whether they are homozygous or heterozygous. It is preferable that the transcription factor has a motif for forming a dimer. Examples of the motif include a leucine zipper, a helix-turn-helix structure, and a helix-loop-helix structure. The molecular weight of the transcription factor is preferably in the range of 1 to 100 kDa, more preferably 10 to 50 kDa. If it is below this range, the above-mentioned motif may not be included, and if it is above this range, a non-specifically bound dimer may be formed.

  The biomolecule (C) is preferably a nucleic acid molecule. This is because transcription factors can interact. The nucleic acid molecule includes not only DNA and RNA, but also artificial nucleic acids such as PNA (peptide nucleic acid) and LNA (Locked nucleic acid), and derivatives thereof. The nucleic acid molecule also includes a nucleic acid molecule synthesized by a PCR method. The length of the nucleic acid molecule is not particularly limited.

  The biomolecule (C) is preferably immobilized on a solid substrate. In order to immobilize the substrate, the biomolecule (C) is preferably modified with a functional group or a binding group. Examples of functional groups and binding groups include amino groups, thiol groups, and biotin. Among them, thiol groups are most preferable because they can be covalently bonded and the reaction proceeds specifically. The thiol group can react with the maleimide group, epoxy group, thiol group, and disulfide group formed on the surface. The thiol group can also be directly bonded to the (111) plane of the solid gold surface.

The solid phase is preferably a metal. This is because the surface of the metal substrate is easy and can be used for various optical measurement methods and crystal oscillator methods. It is also advantageous in that it has excellent thermal stability and high drug resistance. Examples of the form include a flat substrate and beads containing nanoparticles, but a flat substrate is preferred because of the advantage that it can be applied to an array.

  The metal substrate to be fixed is preferably a thin gold layer formed on a transparent substrate. A gold-sulfur bond can be used on the gold surface to immobilize biomolecules directly or indirectly on the surface. Directly, thiol-terminated biomolecules can be immobilized on a gold surface. Indirectly, after introducing a functional group to the gold surface, it is possible to directly or indirectly bind to a functional group or binding group at the end of the nucleic acid molecule. As a method for introducing a functional group onto the gold surface, a method in which a bifunctional group type alkane is closely packed on the gold surface is preferable. Although it is not limited as a bifunctional type alkane, for example, an alkanethiol having an amino group, a carboxyl group, an aldehyde group, a succinimide group, a vinyl group, etc. at the terminal and a thiol group at the terminal on the opposite side, or Examples thereof include disulfides which are dimers thereof.

  After introducing a functional group on the surface using a bifunctional alkane, the biomolecule is immobilized on the surface directly or indirectly. Indirectly, it is particularly preferable that a biomolecule is immobilized through a spacer because mobility is imparted to the immobilized nucleic acid molecule. Examples of the spacer include synthetic hydrophilic polymers such as polyethylene glycol, natural hydrophilic polymers such as dextran, and DNA repeat sequences of thymine and adenine.

  As the means for observing the interaction, there are a method of detecting with a label and a method of detecting without a label. Examples of the labeling method include a method of labeling and detecting a protein, a method of detecting a protein with a labeled antibody, and a method of detecting with a secondary antibody. The labeling method is not particularly limited, and examples thereof include a method of forming a fusion protein with a fluorescent protein such as GFP, and a method of forming a conjugate with a radioisotope. As label-free methods, label-free detection means include surface plasmon resonance (SPR), localized plasmon resonance (LPR), crystal oscillator (QCM), ellipsometry, two-plane polarization interference, sum frequency generation (SFG), second harmonic generation (SHG), and the like. Among them, SPR is preferable because a plurality of points can be measured simultaneously by operating the optical system. The SPR imaging method is a method in which a p-polarized light beam is irradiated over a wide area of the chip and the reflected image is photographed with a CCD camera, and is more preferable because it can be analyzed in an array format. It is possible to measure the interaction between a nucleic acid molecule having a plurality of sequences immobilized thereon and a protein.

  The type of buffer used in the interaction analysis is not particularly limited, and phosphate buffer, HEPES buffer, PIPES buffer, Tris buffer, and the like are used. Moreover, although salt concentration is not specifically limited, Usually, 100-300 mM NaCl is added to a buffer solution. Substances such as a surfactant such as Tween and a blocking agent such as bovine serum albumin may be added to the buffer solution.

  In the interaction analysis, the sample liquid may be constantly flown or may be stopped. When flowing, the flow rate is not particularly limited, but is generally selected in the range of 5 to 500 μl / min.

  Another embodiment of the present invention is a method of observing the interaction by a gel shift method. In this case as well, the mixing ratio of the biomolecules (A) and (B) is changed, and the interaction with the biomolecule (C) is observed. The composition obtained by mixing (A) and (B) and (C) are mixed, moved in the gel, and the ratio of the interacted molecules can be obtained by the difference in the moving speed.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples. By changing the mixing ratio of the transcription factors Nrf2 and MafG, the abundance ratio of homo-MafG and hetero-Nrf2 / MafG was changed, and the interaction between the formed dimer and DNA was measured. This time, SPR imaging method and gel shift method were used as the interaction method.

(Protein constituting heterodimer)
It has been clarified that the transcription factor Nrf2 does not function alone in the leucine zipper and the DNA binding region, forms a heterodimer with small Maf groups such as MafG and MafK, and interacts with DNA. MafG, like Nrf2, has a leucine zipper and a DNA binding site. MafG can form homo and hetero dimers via leucine zippers, but it suppresses transcription in homo dimers compared to MARE, which is a binding sequence of Maf group factors, and hetero dimers. It will be activated. This time, Nrf2 and MafG used both leucine zipper and Nrf2CT and MafG1-123 having a DNA binding site.

(DNA sequence)
Six types of binding sequences MARE of existing Maf groups were examined. Furthermore, a MARE consensus sequence (CEN0) and a non-recognition sequence (MARE23, thiolB (SPR method only)) were added, and a total of 8 or 9 types of sequences were examined. Detailed sequences are shown in Table 1.

(Creation of SPR measurement chip)
The DNA chip used in SPR imaging was a MultiSPRinter NH ₂ chip (manufactured by Toyobo Co., Ltd.). This chip has 8 × 12 = 96 spot areas of 500 μm square into which NH ₂ groups are introduced. Polyethylene glycol that suppresses non-specific adsorption is immobilized in a region that is not a spot region (background region).
In the spot region, 5 mg / ml of a crosslinker MAL-dPEG ₁₂ -NHS (manufactured by Quanta BioDesign) having maleimide group and NHS group at both ends of PEG was reacted for 2 hours to introduce maleimide group on the surface.

(Preparation of DNA solution for SPR)
One of the DNA strands was designed such that each sequence was inserted after 15 bases of thymine as a spacer from the 5 ′ thiol end.

  Prepare a solution of x5 SSC (75 mM sodium citrate, 750 mM NaCl, pH 7.0) / 1 mM TCEP (tris (2-carboxyethyl) phosphine) so that the 5 ′ thiol terminal DNA is 50 μM and its complementary DNA is 100 μM. DNA was hybridized. Furthermore, this reaction solution was diluted with x5 SSC / 1 mM TCEP to prepare a 5 μM double-stranded DNA solution.

  Although the protecting group for the thiol group has been removed in advance, oxidation of the thiol group is prevented by TCEP.

(Immobilization of DNA on SPR chip)
Each DNA solution was spotted on a chip on the surface of the maleimide group using an automatic spotter (MultiSPRinter: manufactured by Toyobo Co., Ltd.) and reacted at room temperature for 16 hours to immobilize the DNA on the surface.

(SPR chip surface cleaning, installation in SPR equipment)
The surface on which the double-stranded DNA was immobilized was washed with 5 × SSC / 0.1% SDS for 15 minutes, 0.2 × SSC / 0.1% SDS for 15 minutes, and 0.2 × SSC for 15 minutes. 1 mM thiol-terminated PEG was reacted for 1 hour to block unreacted maleimide groups.

Set in the flow cell of an SPR imaging device (MultiSPRinter: manufactured by Toyobo Co., Ltd.), SPR transcription factor measurement buffer (20 mM HEPES, 300 mM (homo) / 250 mM (hetero) NaCl, 4 mM MgCl ₂ , 1 mM EDTA, 0.005 % Tween 20, 1 mM TCEP, pH 7.9) was flowed into the flow cell.

(Preparation of SPR method protein solution)
The transcription factor protein was adjusted by mixing with the aforementioned SPR transcription factor measurement buffer at the mixing ratio shown in Table 2.

(SPR measurement)
After confirming that the signal from the SPR was stabilized, it was injected into the cell prepared above at a rate of 100 μl / min for 15 minutes, and a buffer solution containing no transcription factor was allowed to flow. The presence or absence of interaction was judged by the presence or absence of signal rise.

(Chip playback)
The measured chip was regenerated by peeling the bound protein from the DNA. Regeneration was performed by passing a 5 × SSC / 0.1% SDS solution for 5 minutes, and the chip surface was washed with a buffer solution, and used for measurement of heteroproteins with the following mixing ratios changed.

(Preparation of gel shift reaction solution)
250 pg of DNA strand radiolabeled with γ- ³² P-ATP on one DNA strand and 50 ng of dimer protein were added to a buffer for measuring transcription factor for gel shift (20 mM HEPES, 20 mM KCl, 4 mM MgCl ₂ , 1 mM EDTA, 0. 005% Tween 20, 5 mM dithiothreitol, 400 μg / ml poly (dI-dC), 100 μg / ml bovine serum albumin, pH 7.9) were mixed and reacted. The mixing ratio of the proteins is shown in Table 3.

(Gel shift electrophoresis)
The adjusted solution was electrophoresed on a 4% acrylamide gel. As the running buffer, TBE (89 mM Tris-borate, 2 mM EDTA, pH 8.3) was used.

(Measurement result)
From the results of SPR and gel shift (FIG. 1), the sequences that interact with homodimer MafG (hereinafter referred to as homo) and heterodimer Nrf2 / MafG (hereinafter referred to as hetero) are CEN0 (FIG. 1-A), hOPSIN. (FIG. 1-D), hNQO1 (FIG. 1-E) and mGSTY (FIG. 1-G), sequences that interact only heterozygously are MARE23 (FIG. 1-B) and hBgIHS4 (FIG. 1-F), homo-only interactions The sequence to be found was mG2cryst. Common to all sequences, homo showed a slow affinity for dissociation and hetero showed a fast affinity for both dissociation. Moreover, from the result of SPR, homo and hetero are mixed in MafG: Nrf2 = 1: 1 (FIG. 1-2), and only hetero is present in MafG: Nrf2 = 1: 10 (FIG. 1-3). I understood that.

  This sequence interacts with both homo and hetero, and the interaction differs depending on the sequence even at the same mixing ratio. CEN0 and hOPSIN interact with homo and hetero when MafG: Nrf2 = 1: 1 and with hetero only when 1:10. However, hNQO1 is 1: 1 and does not interact with homo. Since homo and hetero are mixed, it is considered that DNA selectively interacts with both homo and hetero, or only hetero.

  In vivo, even when homozygous or heterozygous binds to the same sequence, the transcriptional activation mechanism and affinity are different, resulting in different amounts of protein to be finally expressed. As described above, MafG activates transcription in the heterodimer but represses it in the homodimer. Transcription factors that form dimers have different interactions with DNA depending on their abundance, but the interaction can be measured by using the method of the present invention.

[Comparative example]
Nrf2CT modified with His tag and MafG were mixed to form a heterodimer. In order to remove excess MafG, purification with an affinity column yielded only the heterodimer. The interaction between the purified heterodimer and DNA was measured by SPR imaging. Although homo-MafG should not exist, a homo-MafG-like signal was seen (see FIG. 2). In this method, the abundance ratio of each transcription factor could not be controlled.

  According to the present invention, it is possible to measure the interaction between a biomolecule and a homo- and hetero-dimer obtained by changing the mixing ratio of two different types of biomolecules. For example, the present invention makes it possible to analyze the contribution of each interaction between a transcription factor homo- or hetero-dimer and a nucleic acid molecule. In an actual cell, it is assumed that various transcription factors coexist, and it is speculated that gene expression control by transcription is performed by combining them in competition. Stimuli from the outside world are ultimately transmitted to transcription factors and exhibit various phenotypes such as proliferation, differentiation, development, regeneration, canceration, apoptosis, and maintenance of homeostasis. Therefore, developments such as functional analysis of transcription factors and approaches to searching for new drugs are expected according to the present invention, which greatly contributes to the industry.

Results of SPR imaging and gel shift (Example) Heterodimer interaction (comparative example)

Claims (13)

  A complex obtained by mixing the biomolecule (A) and the biomolecule (B) is reacted with a biomolecule (C) capable of interacting with the complex, and the complex and the biomolecule (C) are interacted with each other. A method for measuring an action, which comprises controlling the abundance ratio of a homodimer and a heterodimer contained in the complex.   By changing the mixing ratio of the biomolecule (A) and the biomolecule (B), the homodimer and the heterodimer contained in the complex obtained by mixing the biomolecule (A) and the biomolecule (B) The interaction measurement method according to claim 1, wherein the abundance ratio of the mer is controlled.   The complex obtained by mixing the biomolecule (A) and the biomolecule (B) includes at least a heterodimer comprising the biomolecule (A) and the biomolecule (B) and / or the biomolecule (A). The interaction measuring method according to claim 1, wherein the homodimer is included.   The interaction measuring method according to any one of claims 1 to 3, wherein the heterodimer composed of the biomolecule (A) and the biomolecule (B) interacts with the biomolecule (C).   The interaction measuring method according to any one of claims 1 to 3, wherein the homodimer comprising the biomolecule (A) interacts with the biomolecule (C).   4. The interaction measuring method according to claim 1, wherein the homodimer comprising the biomolecule (B) does not interact with the biomolecule (C).   4. The interaction measurement method according to claim 1, wherein the biomolecule (B) alone does not form a homodimer.   The biomolecule (A) and / or biomolecule (B) is a protein, The interaction measuring method according to any one of claims 1 to 7.   The biomolecule (A) and / or biomolecule (B) is a transcription factor, The interaction measuring method according to any one of claims 1 to 8.   The method for measuring interaction according to any one of claims 1 to 9, wherein the biomolecule (C) is a nucleic acid molecule.   The method according to claim 1, wherein the biomolecule (C) is immobilized on a solid substrate.   The method for measuring an interaction according to claim 1, wherein the method for observing the interaction is a surface plasmon resonance method.   The method for measuring an interaction according to any one of claims 1 to 12, wherein the method for observing the interaction is a gel shift method.