JP2004264027A - Bio-molecule interaction measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To secure mobility for an immobilized molecule, to restrain nonspecific adsorption, and to analyze precise interaction kinetics. <P>SOLUTION: This biomolecule interaction measuring method has a process for immobilizing the biomolecule on a solid surface, using a bifunctional hetero group type hydrophilic polymer as a crosslinker, and for measuring an interaction between the immobilized molecule and a biomolecule or a biomolecule aggregate. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for immobilizing a biomolecule on a solid surface and measuring an interaction between the immobilized biomolecule and the biomolecule or an aggregate thereof.
[0002]
[Prior art]
Conventionally, biomolecule interactions have been examined as a means for examining the function of biomolecules. As a means for examining the interaction, a method of immobilizing one molecule on a solid surface, bringing the target substance into contact, and detecting whether or not the target substance is adsorbed has become common. When measuring the interaction, it is preferable that non-specific adsorption be suppressed while securing the mobility of the immobilized molecule.For this purpose, it is necessary to use a spacer to secure a space between the immobilized molecule and the surface. Existence is considered essential. For example, in a method for immobilizing a nucleic acid molecule by a covalent bond, a method is disclosed in which a thymine 16 base or a polyethylene glycol spacer is provided in the nucleic acid molecule (for example, see Patent Document 1). However, in this method, the mobility of the molecule is insufficient because the cross-linking agent has no effect of the spacer, and the target substance for the interaction analysis is limited.
[0003]
On the other hand, a method using a polymer as a spacer has also been reported (for example, see Patent Document 2). However, the polymer used in this method is a polyfunctional polymer having a large number of amino groups, and the distance from the surface cannot be sufficiently taken, and the effect is insufficient. Further, if both ends have the same functional group, both ends will react with each other, and the reaction efficiency with the biological substance will be deteriorated.
[0004]
The US patent discloses a method in which a hydrogel is formed on the surface and a functional group is provided in the gel to secure the mobility of the immobilized molecule and suppress nonspecific adsorption (for example, Patent Document 3). reference). Although effective, the permeation and diffusion rates of the target substance are different between the inside and the outside of the gel, which is inappropriate as a method for strictly measuring real-time interaction kinetics.
[0005]
In addition, a molecule having a functional group for immobilizing biomolecules at the end of the spacer having a hydrophilic polymer and a molecule having a functional group capable of directly binding to the solid surface at the other end has been designed. It has also been reported to do so (see Non-Patent Documents 1 and 2). However, it is difficult to densely pack this substance alone on the surface, which results in the mixing of other hydrophilic group terminal substances at a high ratio in order to suppress non-specific adsorption, and the biomolecules are immobilized on the surface. And the measurement sensitivity is low.
[0006]
[Patent Document 1]
WO 00/67028 [0007]
[Patent Document 2]
JP 2000-146976 A
[Patent Document 3]
US Patent 5,436,161
[0009]
[Non-patent document 1]
Sigal et. al. Anal. Chem. 68 (1996) 490-497
[0010]
[Non-patent document 2]
Jung et. al. Langmuir 16 (2000) 9421-9432
[0011]
[Problems to be solved by the invention]
It is a main object of the present invention to provide a method for securing the mobility of an immobilized molecule, suppressing nonspecific adsorption, and enabling strict interaction kinetic analysis.
[0012]
[Means for Solving the Problems]
The present inventors have conducted intensive studies with the main object of solving the above problems. As a result, the interaction between biomolecules is measured by using a heterobifunctional hydrophilic polymer as a cross-linking agent and measuring the interactions between biomolecules using a substrate on which biomolecules are immobilized on a solid surface. The inventors have found that the measurement can be performed appropriately, and further studied, and completed the present invention.
[0013]
That is, the present invention relates to the following matters.
[0014]
1. As a crosslinking agent, a heterobifunctional hydrophilic polymer represented by the general formula XRY (where X represents a functional group on a solid surface or a functional group bonded to a functional group introduced on the solid surface). Y represents a functional group that binds to the biomolecule (A); R represents a repeating unit of a polymer), and a biomolecule (A) is immobilized on a solid surface using a biomolecule (A). A biomolecule interaction measurement method comprising the step of measuring the interaction between A) and a biomolecule (B) or an aggregate thereof.
[0015]
2. Item 2. The method according to Item 1, wherein the heterobifunctional hydrophilic polymer has a molecular weight of 200 to 20,000.
[0016]
3. R of the heterobifunctional hydrophilic polymer is represented by the following repeating unit-(— O—R ₁ —) n—
(Here, R ₁ represents an alkylene group having 2 to 5 carbon atoms. N is 4 to 450.)
Item 3. The method according to Item 1 or 2, which has a structure represented by:
[0017]
4. The functional groups X and Y of the heterobifunctional hydrophilic polymer are an amino group, a carboxyl group, a succinimide group, a sulfonated succinimide group, a maleimide group, a thiol group, an aldehyde group, a vinyl group, an isocyanate group, an epoxy group, and a hydrazide group. 4. The method according to any one of Items 1 to 3, which is any two selected from the group consisting of an azide group.
[0018]
5. 5. The method according to claim 1, wherein the solid surface is a flat substrate.
[0019]
6. Item 6. The method according to any one of Items 1 to 5, wherein the solid surface is a substrate having a thin gold layer on the surface.
[0020]
7. The solid surface is a compound represented by the general formula X′-R′-Y ′ (where X ′ represents a functional group that reacts with a thin gold layer. Y ′ binds to a heterobifunctional hydrophilic polymer) 7. The method according to item 6, wherein the functional group is introduced to the surface of the thin gold layer using R.
[0021]
8. Item 8. The method according to any one of Items 1 to 7, wherein the substrate on which the biomolecules (A) are immobilized is a substrate on which a plurality of types of biomolecules (A) are immobilized in an array.
[0022]
9. Item 9. The method according to any one of Items 1 to 8, wherein the substrate on which the biomolecule (A) is immobilized is a substrate on which 10 or more types of biomolecules (A) are immobilized.
[0023]
10. Item 10. The method according to any one of Items 1 to 9, wherein the biomolecule (A) is a nucleic acid.
[0024]
11. Item 11. The method according to any one of Items 1 to 10, wherein the interaction between the biomolecule (A) and the biomolecule (B) or an aggregate thereof is measured using a surface plasmon resonance method.
[0025]
12. Item 11. The method according to any one of Items 1 to 10, wherein the interaction between the biomolecule (A) and the biomolecule (B) or an aggregate thereof is measured using surface plasmon resonance imaging.
[0026]
13. Item 13. The method according to any one of Items 1 to 12, wherein the biomolecule (B) is a protein.
[0027]
14． Item 14. The method according to Item 13, wherein the protein is a transcription factor.
[0028]
The present invention preferably provides a heterobifunctional hydrophilic polymer represented by the general formula XRY as a crosslinking agent (where X is a functional group on a solid surface or a functional group introduced on a solid surface) Y represents a functional group that binds to the biomolecule (A), and X and Y represent an amino group, a carboxyl group, a succinimide group, a sulfonated succinimide group, a maleimide group, a thiol group, and an aldehyde. And any two selected from the group consisting of a vinyl group, a vinyl group, an isocyanate group, an epoxy group, a hydrazide group, and an azide group, provided that X ≠ Y.R represents a repeating unit of a polymer. The present invention relates to a biomolecule interaction measurement method including a step of measuring an interaction between a biomolecule (A) and a biomolecule (B) or an aggregate thereof using a substrate having a biomolecule (A) immobilized on a surface.
[0029]
More preferably, a compound represented by the general formula X′-R′-Y ′ (where X ′ represents a functional group that reacts with a thin gold layer. Y ′ binds to a heterobifunctional hydrophilic polymer) R ′ represents an organic group), and a heterobifunctional hydrophilic compound represented by the general formula X—R—Y is used as a cross-linking agent on a solid surface obtained by introducing a functional group on the surface of the thin gold layer. A polymer (where X represents a functional group on the solid surface or a functional group that binds to a functional group introduced to the solid surface; Y represents a functional group that binds to the biomolecule (A); and X and Y Is an amino group, a carboxyl group, a succinimide group, a sulfonated succinimide group, a maleimide group, a thiol group, an aldehyde group, a vinyl group, an isocyanate group, an epoxy group, a hydrazide group, or any two selected from the group consisting of azide groups. Where X ≠ Y.R is a polymer The interaction between the biomolecule (A) and the biomolecule (B) or an aggregate thereof is determined by the surface plasmon resonance method or the surface The present invention relates to a biomolecule interaction measuring method including a step of measuring using a plasmon resonance imaging method.
[0030]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
[0031]
Heterobifunctional hydrophilic polymer The present invention ensures the mobility of immobilized biomolecules, suppresses nonspecific adsorption, and enables strict interaction kinetics analysis. A method for immobilizing a biomolecule on a solid surface is characterized in that a heterobifunctional hydrophilic polymer is used as a crosslinking agent.
[0032]
By this method, it is possible to immobilize a biomolecule while keeping a certain space from the surface, and it is possible to analyze the interaction kinetics (kinetics) of the biomolecule in real time while securing the mobility of the biomolecule. Observations can be made.
[0033]
The heterobifunctional hydrophilic polymer used as a crosslinking agent is represented by the general formula XRY. Here, X represents a functional group on the solid surface or a functional group bonded to a functional group introduced on the solid surface. Y represents a functional group that binds to the biomolecule (A). R represents a polymer repeating unit. Here, the polymer refers to a polymer having three or more repeating units.
[0034]
The two heterofunctional functional groups which are the ends of the polymer serving as the crosslinking agent of the present invention are functional groups in which the functional group X at one end is bonded to the solid surface or the functional group introduced to the solid surface, and the other is The terminal functional group Y reacts with the biomolecule (A) and is composed of different functional groups. That is, X ≠ Y.
[0035]
The functional group Y is preferably a functional group that does not react with the solid surface. When the functional group Y reacts with the surface, a loop is formed on the surface, the number of functional groups for immobilizing biomolecules decreases, and the reaction efficiency deteriorates, which is not preferable.
[0036]
Examples of the functional groups X and Y include an amino group, a carboxyl group, a succinimide group, a sulfonated succinimide group, a maleimide group, a thiol group, an aldehyde group, a vinyl group, an isocyanate group, an epoxy group, a hydrazide group, and an azide group. Can be Two different functional groups as X and Y are selected from such a group of functional groups, and are configured in combination.
[0037]
In particular, as the combination of X and Y, a combination of an amino group and a carboxyl group and a combination of a succinimide group and a maleimide group, which are different from each other, are preferable.
[0038]
When the biomolecule (A) is DNA, the functional group Y to be introduced into the terminal is preferably an amino group, a thiol group or biotin.
[0039]
The molecular weight of the heterobifunctional hydrophilic polymer is 200 or more, preferably 1000 or more, more preferably 1500 or more, 20,000 or less, preferably 10,000 or less, and more preferably 6,000 or less.
[0040]
In terms of the repeating unit, the number of repeating structural units is 4 or more, preferably 20 or more, more preferably 30 or more, 450 or less, preferably 225 or less, and more preferably 130 or less.
[0041]
The heterobifunctional hydrophilic polymer of the present invention not only functions as a crosslinking agent but also plays a role of a spacer. If the molecular weight is too small, it will not function as a sufficient spacer, which is not preferable. On the other hand, if the molecular weight is too large, the number of grams of the crosslinking agent required for immobilization, which requires a certain molar concentration, is not preferred.
[0042]
In the present invention, the polymer constituting the crosslinking agent needs to be hydrophilic in order to suppress non-specific adsorption. Here, the hydrophilicity means that a polymer serving as a crosslinking agent is water-soluble.
[0043]
Although the repeating unit R of the polymer is not particularly limited, the following repeating unit — (— O—R ₁ —) n—
(Here, R ₁ represents an alkylene group having 2 to 5 carbon atoms. N is 4 to 450.)
It is preferable to have a structure represented by
[0044]
The R, _{specifically, - (- CH 2 -CH 2} -O -) - or _{- (- CH 2 -CH 2 -CH} 2 -O -) - or linear alkylene group such as - _{(-CH (CH 3) -CH 2} -O -) - or the like, such as an alkylene group branched in. _{Also, - (- CH 2 -CH 2} -O-) p - (- CH (CH 3) -CH 2 -O-) q- block copolymers such as are included.
[0045]
Among them, those having a repeating unit of ethylene glycol — (— CH ₂ —CH ₂ —O —) — are preferable in view of hydrophilicity and chain flexibility.
[0046]
Method for immobilizing biomolecules The method for immobilizing biomolecules using a cross-linking agent is not particularly limited. For example, after immobilizing one end X of the cross-linking agent by reacting it with the surface, Of immobilizing a biomolecule using another functional group Y existing at the end of the biomolecule.
[0047]
Specifically, when a heterobifunctional polymer having an amino group at one end and a carboxyl group at the other end is used as a cross-linking agent, the solid surface having the carboxyl group introduced thereto is treated with carbodiimide and N-hydroxyl. After activation with succinimide, it reacts with an amino group at one end to immobilize the crosslinking agent on the surface. Then, the biomolecule (A) is immobilized on the surface using the carboxyl group introduced on the surface via the cross-linking agent.
[0048]
When a heterobifunctional polymer having a succinimide group at one end and a maleimide group at the other end is used as a cross-linking agent, the succinimide group of the cross-linking agent reacts with the solid surface into which the amino group has been introduced. Let it. Next, the biomolecule is immobilized on the surface using the maleimide group introduced on the surface via the cross-linking agent.
[0049]
The method of binding the biomolecule (A) to the heterobifunctional hydrophilic polymer as a cross-linking agent is not particularly limited, and it may be bound directly or indirectly via another substance. You may. The type of the bond is not particularly limited. For example, the bond can be formed by a covalent bond, an ionic bond, a chelate bond, a hydrogen bond, or the like.
[0050]
Introduction of a functional group or substance that binds to a biomolecule after using the heterobifunctional crosslinking agent of the present invention is also included in the present invention. For example, a method of immobilizing a nitrilotriacetic acid (NTA) group with a heterobifunctional cross-linking agent, then chelating the NTA group with a histidine tag by Ni chelate, and introducing a histidine tag protein to the surface, biotin or biotin or the like. A method is conceivable in which, for example, a method in which streptavidin is reacted with the functional group Y of the cross-linking agent and introduced, and then the introduced biotin or streptavidin is reacted with the biomolecule (A).
[0051]
As the solid surface on which the biomolecules are immobilized, a planar substrate is preferable because it is suitable for interaction analysis.
[0052]
As the planar substrate, a metal substrate suitable for interaction analysis by surface plasmon resonance (SPR) is preferable. Examples of the metal include gold, silver, copper, aluminum, and chromium. Of these, a transparent substrate having a thin gold layer on the surface is particularly preferred.
[0053]
The reason why the gold thin layer surface is preferable is that a functional group can be introduced into the surface by using a gold-sulfur bond.
[0054]
As a method of introducing a functional group to the surface by using a gold-sulfur bond, for example, a compound represented by the general formula X′-R′-Y ′ (where X ′ reacts with a thin gold layer, Represents a functional group having a sulfur atom, Y ′ represents a functional group that binds to a heterobifunctional hydrophilic polymer, and R ′ represents an organic group. A method of immobilizing the biomolecule (A) on the surface using a heterobifunctional hydrophilic polymer or the like can be given.
[0055]
Examples of the functional group X ′ include a thiol group, a sulfide group and a disulfide group. Examples of Y ′ include an amino group, a carboxyl group, an aldehyde group, an azido group, an N-hydroxysuccinimide group, an epoxy group, a carbonyldiimidazole group, and an isocyanate group. Examples of R ′ include an alkylene group. The number of carbon atoms of the alkylene group is not particularly limited, but is preferably 5 or more and 18 or less. If the number of carbon atoms is less than 4, the hydrophobic bonds between the alkane chains are not sufficient, and the self-assembled surface lacks stability. If it is 19 or more, the alkane chain has a strong hydrophobic property, which makes it difficult to react and immobilize a hydrophilic substance. In addition, since the sensor surface has a strong hydrophobic property, non-specific adsorption during measurement may be a concern. You.
[0056]
Specific examples of the compound represented by the general formula X'-R'-Y 'include 8-amino-1-octanethiol (Y-amino-octanethiol) wherein Y' is an amino group, and Y ' Is a carboxyl group, and 7-carboxy-1-heptanethiol (7-Carboxy-1-heptanethiol).
[0057]
As the biomolecule (A), a single substance may be used, or a plurality of types of substances may be used. When a plurality of types of substances are used, high-throughput analysis becomes possible. By performing analysis at high throughput, an extremely large number of compounds can be efficiently screened. In particular, it is preferable to use 8 or more, preferably 10 or more, more preferably 12 or more, and still more preferably 24 or more biomolecules. The upper limit is not particularly limited, but is usually about 96 types. In order to immobilize a plurality of substances, it is preferable to immobilize the substances in an array. This makes it possible to analyze the interaction at each immobilization site.
[0058]
The type of the biomolecule (A) is not particularly limited, and for example, nucleic acids, proteins, peptides, sugar chains, and the like can be used. Among them, nucleic acids are preferable because they are stable and many types can be easily obtained.
[0059]
Since the biomolecule (A) immobilized by the present invention is excellent in mobility and has an effect of suppressing nonspecific adsorption, it is necessary to measure the interaction with the biomolecule (B) or an aggregate thereof in an appropriate state. Can be.
[0060]
The type of the biomolecule (B) is not particularly limited, and includes, for example, nucleic acids, proteins, peptides, sugar chains, and the like. Also, an aggregate of biomolecules (B) such as a dimer of a protein can be used.
[0061]
The present invention is particularly suitable for measuring a protein having a complex structure such as a transcription factor. This is because many transcription factors have a steric special structure as seen in b-zip and zinc fingers, and if the mobility of the nucleic acid molecule to be bound is insufficient, the transcription factor cannot be easily accessed. . In addition, since the transcription factor has a remarkably high nonspecific adsorption amount on the surface, it is impossible to analyze the transcription factor without suppressing the nonspecific adsorption. In the present invention, since the mobility of the immobilized molecule can be ensured and nonspecific adsorption can be suppressed, it can be suitably used for analysis of interaction evaluation of biomolecules having a special structure such as a transcription factor.
[0062]
The type of the transcription factor is not particularly limited, and for example, the NF1 family, the Maf family, the GATA family, and the like can be applied. In particular, the Maf family is known to form not only homodimers but also heterodimers. In the present invention, the interaction of such an aggregate of biomolecules can be appropriately analyzed, and by using the method of the present invention, the difference in binding behavior due to various combinations of heterodimers is evaluated. It is possible to perform very significant measurements such as
[0063]
Examples of the interaction that can be measured in the present invention include protein-protein interaction, nucleic acid-protein interaction, nucleic acid-nucleic acid interaction, protein-peptide interaction, protein-sugar chain interaction, antigen-antibody interaction, and the like. Is mentioned.
[0064]
Measurement method As a method for measuring the interaction between the immobilized biomolecule (A) and the biomolecule (B) or an aggregate thereof, it is preferable to use a surface plasmon resonance (SPR) method. Further, among the SPR methods, the SPR imaging method is particularly preferable.
[0065]
The SPR method is a label-free and real-time measurement method. Therefore, it is particularly suitable when at least one of the biomolecules (A) and (B) is a protein. The reason is that the protein may change its structure or lose its activity by labeling, but the SPR method is an interaction analysis method that does not require labeling (labeling) of biomolecules. It is possible to perform appropriate measurement while maintaining the activity. In addition, the method enables real-time measurement and analyzes not only the equilibrium state but also the rates of binding and dissociation. From these results, valuable information for knowing the function of biomolecules is obtained. Obtainable.
[0066]
The SPR imaging method irradiates the entire array with parallel polarized light and captures the reflected light with a CCD camera. It is possible to know the surface plasmon resonance angular displacement at a point on the array by the change in reflected light intensity. It is possible. Therefore, it is possible to measure the interaction analysis between a double-stranded oligonucleotide array having a plurality of immobilized sequences and a biomolecule in a label-free and real-time manner. It can be suitably used for analysis of molecular interaction.
[0067]
【Example】
Hereinafter, the present invention will be described specifically with reference to Examples and Comparative Examples, but the present invention is not limited to Examples.
[0068]
Example 1
After depositing 3 nm of chromium on a transparent glass substrate made of SF10 having a thickness of 1 mm and 18 mm × 18 mm, gold was deposited to a thickness of 45 nm. The thickness of the evaporation was monitored with a crystal oscillator. The substrate on which gold was deposited was immersed in a 1 mM ethanol solution of 8-Amino-1-Octanethiol, Hydrochloride (8-AOT, manufactured by Dojindo Laboratories) for 16 hours to form a self-assembled surface of 8-AOT. Was. Heterobifunctional polyethylene glycol (NHS-PEG-MAL, manufactured by Sheawater Polymers) having a succinimide (NHS) group and a maleimide (MAL) group at the terminal having a molecular weight of 3,400 was added to a phosphate buffer (20 mM phosphoric acid, 150 mM NaCl). , PH 7.2) at 10 mg / ml and reacted with 8-AOT on the gold surface for 2 hours. Since the amino group of 8-AOT reacted with the NHS group of NHS-PEG-MAL and the MAL group remained unreacted, a maleimide group could be introduced to the surface via PEG.
[0069]
Two types of 5 ′ thiol-terminal DNAs were hybridized to the obtained surface using a Hand spotter from Greiner bio-one, and then spotted. The DNA sequence is a Maf recognition sequence (MARE25) and a Maf non-recognition sequence (MARE23), and the detailed sequence is shown in FIG. It is designed so that the target sequence is inserted after the thymine 15 base spacer from the 5 'thiol end.
[0070]
A solution was prepared in an X5 SSC solution (75 mM sodium citrate, 750 mM NaCl, pH 7.0) so that the 5 ′ thiol-terminal DNA was 25 μM and its complementary DNA was 100 μM, and the mixture was heated at 0 ° C. for 5 minutes in a boiling bath. The DNA was hybridized by quenching for 15 minutes, followed by incubation at 37 ° C. for 3 hours. The hybridized double-stranded DNA (dsDNA) was spotted and reacted for 15 hours to immobilize the dsDNA on the surface.
[0071]
After the surface on which the dsDNA was immobilized was washed with a phosphate buffer, it was set on an SPR imaging device (SPRImager: manufactured by GWC Instruments), and 10 mM Hepes, 300 mM NaCl, 4 mM MgCl ₂ , 1 mM EDTA, 100 μg / ml bovine serum albumin And a buffer for measuring transcription factors having a pH of 7.9 was flowed into the flow cell.
[0072]
After confirming that the signal from the SPR was stable, a homodimer of the transcription factor MafG was dissolved in the above-mentioned buffer for the transcription factor measurement at a concentration of 1 μg / ml, and the solution was placed in the cell at a rate of 100 μl / min for 10 minutes. After injection, a buffer containing no transcription factor was further flowed, and changes in SPR signals for binding and dissociation were observed.
[0073]
Observation was carried out at three points: an immobilized site of MARE25 and MARE23 and a portion where no DNA was immobilized (background site). A graph showing the change in the SPR signal is shown in FIG. It can be observed that MafG homodimer binds only to the MARE25 sequence and hardly binds to MARE23 and the background site. The binding rate constant is 2.22 × 10 ⁵ (M ⁻¹ s ⁻¹ ) and the dissociation rate constant is 8 ^{.80 × 10 -4 (s -1)} , to obtain a binding equilibrium constant ^{2.52 × 10 8 (M -1)} .
[0074]
An image photographed before the MafG homodimer was injected into the cell and an image photographed 10 minutes after the injection were photographed, and the difference between the two was subjected to arithmetic processing using image processing software NIH Image. The result is shown in FIG. It is clear that MafG is bound only to the MARE25 sequence.
[0075]
Comparative Example 1
In the same manner as in the example, a self-assembled surface of 8-AOT is formed on a gold vapor-deposited transparent substrate, and a heterobifunctional cross-linking agent Sulfo-SMCC (Sulfosuccinimidyl 4 [) having a succinimide (NHS) group and a maleimide (MAL) group is formed. N-maleimidemethyl] -cyclohexane-1-carboxylate (Pierce) was dissolved at a concentration of 1 mM in a phosphate buffer (pH 7.0) and reacted with 8-AOT. Sulfo-SMCC is a low molecular weight substance having a molecular weight of 436.37, and has no repeating unit.
[0076]
In the same manner as in Example 1, the sequences of MARE25 and MARE23 having a thiol at the 5 'end were converted to dsDNA, immobilized on the surface, and SPR imaging measurement was performed.
[0077]
Although the interaction of the MafG homodimer was observed in the same manner as in Example 1, it was non-specifically adsorbed to MARE25, MARE23, and all of the background sites, and the specificity of the transcription factor MafG could not be observed (FIG. 5). ).
[0078]
【The invention's effect】
According to the present invention, a biomolecule can be immobilized on a solid surface while maintaining mobility, activity, and function. By using the method of the present invention, the interaction between a biomolecule or a biomolecule assembly and a biomolecule can be improved. A rigorous and appropriate assessment of the action kinetics is possible.
[Brief description of the drawings]
FIG. 1 is a drawing showing the nucleotide sequence of MARE25.
FIG. 2 is a drawing showing the nucleotide sequence of MARE23.
FIG. 3 is a drawing showing a binding dissociation curve of MafG homodimer (Example).
FIG. 4 is a drawing showing the difference between images before and after MafG homodimer binding (Example).
FIG. 5 is a drawing showing a binding dissociation curve of MafG homodimer (comparative example).

Claims (14)

A heterobifunctional hydrophilic polymer represented by the general formula XRY as a cross-linking agent (where X represents a functional group on a solid surface or a functional group bonded to a functional group introduced on the solid surface). Y represents a functional group that binds to the biomolecule (A); R represents a repeating unit of a polymer), and a biomolecule (A) is immobilized on a solid surface by using a biomolecule (A). A method for measuring the interaction between biomolecules, comprising the step of measuring the interaction between A) and a biomolecule (B) or an aggregate thereof. The method according to claim 1, wherein the molecular weight of the heterobifunctional hydrophilic polymer is from 200 to 20,000. R of the heterobifunctional hydrophilic polymer is represented by the following repeating unit-(— O—R ₁ —) n—
(Here, R ₁ represents an alkylene group having 2 to 5 carbon atoms. N is 4 to 450.)
The method according to claim 1, having a structure represented by: The functional groups X and Y of the heterobifunctional hydrophilic polymer are an amino group, a carboxyl group, a succinimide group, a sulfonated succinimide group, a maleimide group, a thiol group, an aldehyde group, a vinyl group, an isocyanate group, an epoxy group, and a hydrazide group. The method according to any one of claims 1 to 3, which is any two selected from the group consisting of, and an azide group. 5. The method according to claim 1, wherein the solid surface is a flat substrate. The method according to any one of claims 1 to 5, wherein the solid surface is a substrate having a thin gold layer on the surface. The solid surface is a compound represented by the general formula X′-R′-Y ′ (where X ′ represents a functional group that reacts with a thin gold layer. Y ′ binds to a heterobifunctional hydrophilic polymer) The method according to claim 6, wherein the surface is a solid surface obtained by introducing a functional group into the surface of the gold thin layer using a functional group (R 'represents an organic group). The method according to any one of claims 1 to 7, wherein the substrate on which the biomolecules (A) are immobilized is a substrate on which a plurality of types of biomolecules (A) are immobilized in an array. The method according to any one of claims 1 to 8, wherein the substrate on which the biomolecule (A) is immobilized is a substrate on which 10 or more types of biomolecules (A) are immobilized. The method according to any one of claims 1 to 9, wherein the biomolecule (A) is a nucleic acid. The method according to any one of claims 1 to 10, wherein an interaction between the biomolecule (A) and the biomolecule (B) or an aggregate thereof is measured using a surface plasmon resonance method. The method according to any one of claims 1 to 10, wherein an interaction between the biomolecule (A) and the biomolecule (B) or an aggregate thereof is measured using surface plasmon resonance imaging. The method according to any one of claims 1 to 12, wherein the biomolecule (B) is a protein. 14. The method according to claim 13, wherein the protein is a transcription factor.

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