EP1403641B1 - Verfahren zum Kalkulieren von Assoziations- und Dissoziationskonstanten mit einem Polymerchip zum Auffinden von ionischen Polymeren - Google Patents

Verfahren zum Kalkulieren von Assoziations- und Dissoziationskonstanten mit einem Polymerchip zum Auffinden von ionischen Polymeren Download PDF

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EP1403641B1
EP1403641B1 EP20030019932 EP03019932A EP1403641B1 EP 1403641 B1 EP1403641 B1 EP 1403641B1 EP 20030019932 EP20030019932 EP 20030019932 EP 03019932 A EP03019932 A EP 03019932A EP 1403641 B1 EP1403641 B1 EP 1403641B1
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dna
protein
probe
association
double
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EP1403641A1 (de
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Yoshifumi Yokohama City University Nishimura
Hiroshi Hitachi Software Eng. Co. Ltd. Murai
Motonao Hitachi Software Eng. Co. Ltd. Nakao
Toshiki Hitachi Software Eng. Co. Ltd. Morita
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Hitachi Software Engineering Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding

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  • the present invention relates to a method of calculating the association constant and the dissociation constant between a probe and a protein, a method of searching for the functionality of proteins using the calculation method, and a method of identifying proteins using the calculation method.
  • the invention relates to a method of searching for the functionality of proteins by calculating the DNA-binding property of the proteins, and a method of identifying the proteins.
  • DNA-binding proteins are considered to be among the important substances controlling or adjusting the expression of gene functions, the important substances controlling or adjusting the expression of gene functions, and clarification of their DNA binding properties is an important task.
  • a DNA is a polymer formed by a sequence of four kinds of bases, and two DNAs can specifically bind to one another through their sequences. Because the nucleic acids of both such DNAs are negatively charged, hybridization is carried out in a highly ionic solvent. Conventional biochips take advantage of this fact to detect a sample DNA by immobilizing a probe DNA on a substrate.
  • a DNA-binding protein cannot be identified by a conventional biochip. This is because a protein as a sample may be specifically adsorbed on a probe DNA on a biochip by its complementary DNA portion, and the protein may also be nonspecifically adsorbed on the probe DNA even though this is not supposed to happen, in light of the DNA. Because of such specific and nonspecific adsorptions that apparently occur on the probe DNA, it has been virtually impossible to identify sample proteins using a conventional biochip.
  • association constant and the dissociation constant between ionic polymers are calculated based on a binding curve or dissociation curve obtained by monitoring the adsorption of a substance using an apparatus for measuring the strength of bonding between ionic polymers, such as a surface plasmon resonance measuring apparatus (see JP Patent Publication (Kohyo) No. 7-507865, for example).
  • a ligand is immobilized on a sensor surface, and a sample containing a substance that acts on the ligand is added via a microchannel system. Minute mass changes due to the association or dissociation of molecules on the sensor surface are then monitored in real time in terms of changes in a surface plasmon resonance signal.
  • This technique is effective in studying the interaction between biomolecules or the correlation between structure and function.
  • Surface plasmon resonance is being used in a wide variety of areas including basic research, protein engineering, and the screening of new drugs.
  • surface plasmon resonance is utilized include research into the mechanism of AIDS or cancer development, immunologic response, signal transduction, the ways in which receptors and ligands bind to one another, and the mechanism of gene expression control (see Chong L et al. A human telomeric protein. Science 1995 Dec 8; 270 (5242): 1663-7, for example).
  • WO 02/18648 discloses a method of analysis of binding interactions, wherein a protein is bound to a nucleic acid chip. Fluorescence values are normalized versus the highest fluorescence intensity on a spot for the particular measurement. Selective washing steps to increase the specificity of binding are not disclosed.
  • US 6,355,428 discloses a method for determining the relative binding affinity between DNA immobilized on a chip and DNA-binding proteins, wherein the second ionic polymer is labeled. Again, selective washing steps for increasing the specificity are not disclosed.
  • the method using an ionic-polymer identifying chip makes it possible to determine the presence or absence of binding between double-stranded DNA and DNA-binding proteins or the relative association strengths for a large number of proteins simultaneously.
  • This method is not capable of calculating the association and dissociation constants, which are absolute values.
  • the invention was made upon the realization that the calculation of rough values of association and dissociation constants of many kinds of double-stranded DNA samples, which used to take a long time, can be made in a short time by using a specific protein identifying chip.
  • the aforementioned need is solved by a method according to claim 1.
  • the first to third ionic polymers may be selected from the group consisting of proteins, polyamino acids, DNA, RNA, and synthetic polymers.
  • the invention provides a method of calculating association and dissociation constants between a probe and a protein, comprising:
  • Examples of the method of measuring the association strength between a double-stranded DNA and a protein include a surface plasmon resonance method and a gel shift assay method.
  • the surface plasmon resonance method is superior in accuracy and operability, and is suitable for the method of calculating the association and dissociation constants according to the invention.
  • the invention also provides a method of searching for the functionality of proteins using the method of calculating association and dissociation constants as described above, wherein the binding property between the probe in which the first and second DNA are complementarily bound and the protein is evaluated.
  • the material and shape of the substrate used in the present invention are not particularly limited, and the materials and shapes typically used for the substrates for biochips can be used.
  • the material is selected from the group consisting of glass, silica gel, polystyrene, polypropylene, and membrane.
  • the shape is preferably either plate-like or bead-like.
  • the marker to be provided to the second DNA is selected from those typically used in the field of biochips. Particularly, a fluorescent substance is preferable from the viewpoint of optical processes in a read step.
  • a fourth, sample polymer with an opposite ionic property should preferably be added so that they can be firmly immobilized, followed by a step of washing them with a nonionic solvent.
  • the number of base sequences in the DNA should preferably be between 4 to 13 for each polymer.
  • the complementary binding of the DNA comprising the first and second DNA is made stronger, and further the specific binding between the DNA chip comprising these polymers and the DNA-binding proteins as samples based on their chemical and physical structures is made stronger.
  • the search for and identification of the functionality of the sample proteins can be made more easily and accurately.
  • the nonionic solvent used in the invention is not particularly limited.
  • it is a mixture of one or more kinds of substances selected from the group consisting of pure water, alcohol, acetone, and hexane.
  • the ionic solvent is not particularly limited either. It may be an aqueous solution of sodium chloride or potassium chloride.
  • Biopolymers such as DNA and proteins have a charge, which is negative in the case of DNA due to the phosphate group and either positive or negative in the case of proteins depending on the kind of amino acids.
  • the double strand of DNA normally has a negative ion and is therefore repulsive, so that it does not hybridize in pure water. However, it can hybridize by hydrogen bonding in a system where a positive ion exists, as an Na + ion chelates with the phosphor group of DNA, for example. Proteins with a positive charge bind with DNA having a negative charge at the charge portion.
  • the polymers with the same charge are dissociated when the ionic strength is low, whereas the polymers with opposite charges strongly bind to one another.
  • the ionic strength is high, even polymers with the same charge can bind to one another, while polymers with different charges lose their association strength and are dissociated.
  • the DNA-binding proteins as used herein refer to proteins that have affinity for DNA and that specifically or nonspecifically bind to base sequences.
  • examples of such proteins include, mainly, 1) double-stranded DNA-binding proteins that control gene expression by causing changes in DNA structure, 2) single-stranded DNA-binding proteins that are indispensable for the process of duplication, recombination, or repair of DNA, 3) proteins involved with the maintenance of higher-order structures of chromosomes, 4) ATP-dependent Dnase, and 5) topoisomerases forming the DNA superhelix structure.
  • proteins 1 are transcription factors, for which several structural motifs are known, such as helix-turn-helix that was found in the structures of lambda phage Cro protein and cAMP receptor proteins, zinc finger in which cysteine and histidine chelate a zinc ion, and leucine zipper in which two molecules of proteins comprising an alpha helix with leucine disposed on one side which are combined like a zipper.
  • the proteins 2) are called SSB (single-stranded DNA-binding proteins), and they are found in various life forms ranging from bacteriophage to higher forms of life.
  • the proteins 3) are represented by the histone proteins that exist in the chromosomes of eukaryotes, and they form a nucleosome structure.
  • the proteins 4 include helicase that promote DNA replication (DnaB proteins, for example), recombination (RecA and RecBC proteins), and the unwinding of double-stranded DNA. Further, in the present invention, not only the above-mentioned proteins that inherently have DNA binding property but also proteins to which a DNA binding property has been given by physical or chemical treatments may be used.
  • the above-mentioned various DNA-binding proteins can be used.
  • the invention includes cases using not only one kind of DNA-binding proteins but two or more kinds of DNA-binding proteins.
  • DNA-binding proteins characteristically have a positive charge at the DNA binding domain and bind to DNA or RNA with negative charges either specifically or nonspecifically.
  • the double-stranded DNA to which these DNA-binding proteins have bound has increasing association strength with decreasing ionic strength.
  • Fig. 1 shows a protein identifying DNA chip as an example of the polymer chip for identifying ionic polymers according to the invention.
  • a first DNA 2 having a base sequence GCTA is immobilized on a substrate 1.
  • a second DNA 3 labeled with a fluorescent substance and having a base sequence CGAT is added to an ionic solvent in the presence of the first DNA 2, they complimentarily bind to each other and form a double strand.
  • a second DNA 6 labeled with the same fluorescent substance and having a base sequence GGTT is added to another DNA 5 having a base sequence CCAA, they complimentarily bind to each other and form a double strand.
  • the protein 4 As a protein 4 that has not been identified is added to the DNA chip, the protein 4 binds to the double strand with the complementary binding of DNA 2 and DNA 3. However, the protein 4 does not bind to the double strand with the complementary biding of DNA 5 and DNA 6. Such phenomena can be measured with a fluorescence intensity meter and then analyzed in order to identify the protein.
  • Fig. 2 shows a chart illustrating the process of identifying proteins using the protein identifying DNA chip shown in Fig. 1.
  • a DNA chip is prepared, which comprises a substrate 1 on which DNA 2 and DNA 5 are immobilized (a).
  • DNA 3 and DNA 6 that are labeled with a fluorescent substance are caused to complimentarily bind on the DNA chip in the presence of an ionic solvent (b).
  • unidentified proteins 4 are added.
  • the proteins 4 specifically bind to the double strands of DNAs 2 and 3 as they should, and they also nonspecifically bind to the double strands of DNAs 5 and 6 although they should not (c).
  • the DNA chip is washed with an ionic solvent to remove the nonspecifically bound proteins (d).
  • the DNA chip is further washed with a nonionic solvent to remove DNA 6 that is labeled with fluorescent substance (e).
  • the remaining fluorescence is then read, and the protein is identified based on the result of reading (f).
  • Fig. 3 shows the principle of measurement carried out by a surface plasmon resonance measuring apparatus available from Biacore.
  • a gold thin film is formed at the bottom of a prism to a thickness of 50 nm, and the interface is irradiated with polarized light of wavelength 760 nm. This produces an energy wave called an evanescent wave on the thin film. Because this evanescent wave is utilized for the resonance of a free electron wave or a plasmon wave on the gold thin film, a loss of energy is observed at a certain angle of the reflected light. When the intensity of the reflected light is measured with a photodiode array, an optical valley is recognized, as indicated by I. This optical phenomena is the surface plasmon resonance, in which the loss angle varies depending on changes in the concentration of the solvent on the gold film surface.
  • a synthesized oligo DNA is spotted on a substrate, and a complementary oligo DNA with a labeled terminal is hybridized, thus manufacturing a double-stranded DNA chip.
  • a substance of which the association constant is to be measured is added onto the double-stranded DNA chip as a sample.
  • the complementary DNA is dissociated if the sample has not bound to the double-stranded DNA.
  • it can be determined to what degree the sample to be measured has bound to the double-stranded DNA.
  • the values of dissociation constants KD for three or more kinds of samples on the chip are determined using surface plasmon measuring equipment, and an analytical curve is made.
  • the dissociation constant KD values can be determined from the fluorescence intensity ratio using the analytical curve.
  • TRF1 is introduced in the above-mentioned Non-Patent Document 1 (Chong L et al. A human telomeric protein. Science 1995 Dec 8; 270(5242): 1663-7).
  • a biochip was fabricated as follows.
  • probe DNA to be immobilized on a chip Four kinds of probe DNA to be immobilized on a chip were provided, and their respective 5' terminals were biotinylated. They were then spotted on six locations of an avidin-coated slide glass for each kind of probe using SPBIO (available from Hitachi Software Engineering Co., Ltd.). In the present example, four kinds of probe DNA were used, so that 24 spots were made.
  • the sequences of the chip-immobilized probe DNA were 5'-GTTAGGGTTAGGG-3, 5'-GTTAAGGTCAGGG-3, 5'-GTTAAGGTTAGGG-3, and 5'-GTTAGGGCTAGGG-3. In the following, they will be referred to as an AGTT probe, an AATC probe, an AATT probe, and an AGCT probe.
  • the concentration of the spotted probe DNA was adjusted to 1 mM with pure water.
  • An example of the thus spotted biochip is shown in Fig. 4.
  • the 5'-terminal was labeled with a fluorescent pigment Cy5.
  • a 5xSSC solution was prepared containing the sample DNA to a concentration of 100 nM, and 10 ⁇ l of the solution was added onto the prepared biochip to effect hybridization for 30 minutes in a humid environment so as to avoid drying.
  • the chip was washed with 1xSSC, dried, and then read using a fluorescent scanner CRBIO2 (available from Hitachi Software Engineering Co., Ltd.).
  • FIG. 5 An example of an image that has been read is shown in Fig. 5.
  • darkened spots indicate the locations where the Cy5-labeled sample exists. Thus, the formation of a double-stranded DNA can be confirmed.
  • TRF1 protein sample solution prepared with 5 mM of KPB (phosphoric acid) and 30 mM of NaCl to a TRF1 protein concentration of 110 ⁇ M was added onto the hybridized biochip, and hybridization was effected for 10 minutes.
  • Fig. 6 shows an image that has been read after processing in Fig. 5.
  • the double-stranded DNA hybridized with TRF1 does not dissociate even in pure water.
  • the difference in fluorescent intensity among the four kinds of probes shown in Fig. 6 corresponds to the difference in association strength between the TRF1 protein and the double-stranded DNA chip.
  • Fig. 7 shows a table of the fluorescent intensity ratios for the individual probe sequences.
  • the above-described double-stranded DNA probes were then immobilized on an avidin-coated chip using the surface plasmon resonance measuring apparatus.
  • the avidin coat was Sensor Chip SA available from Biacore.
  • the immobilization buffer employed 10 mM HEPES pH7.4 + 3M EDTA + 150 mM NaCl.
  • the flow volume during immobilization was 5 ⁇ l/min.
  • the TRF1 protein was caused to flow on the immobilized chip in five different concentrations of 10 nM, 25 nM, 50 nM, 75 nM, and 100 nM.
  • the TRF 1 binding amount was monitored using the surface plasmon measuring apparatus to prepare an association curve and a dissociation curve, based on which the dissociation constant (KD value) was calculated.
  • the coupling buffer employed 10 mM HEFES pH 7.4 + 3M EDTA + 50 mM NaCl, and the flow volume was 20 ⁇ l/min.
  • the accurate dissociation constants of the four kinds of probe are shown in the table of Fig. 7.
  • FIG. 8 shows an analytical curve prepared based on the probes AGTT, AATC, and AATT.
  • the vertical axis indicates the dissociation constant (KD value), while the horizontal axis indicates the fluorescent intensity ratio (T/H).
  • the fluorescent intensity can read from the relevant spot on the biochip and compared with the analytical curve in order to calculate a rough dissociation constant.
  • the fluorescent intensity of the AGCT probe is 0.437, as shown in Fig. 7, so that it can be expected that the dissociation constant lies roughly at the latter half of 10 -8 , based on the analytical curve of Fig. 8.
  • the accurate dissociation constant KD value
  • the association constant (KA value) and the dissociation constant (KD value), which are indicators of the structure and function of the mutation, can be comprehensively and effectively estimated by a simple method.
  • the invention makes it possible to calculate rough association and dissociation constants in a number of mutants in which single base substitution such as SNP has occurred in an easy and quick manner.
  • the invention can provide tools for drug development and/or medical examinations.

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Claims (4)

  1. Verfahren zum Berechnen von Assoziations- und Dissoziationskonstanten zwischen einer Sonde und einem Protein, umfassend:
    einen ersten Schritt der Herstellung eines DNA-Chip zum Identifizieren von Proteinen, wobei der DNA-Chip ein Substrat umfaßt, auf dem eine erste DNA immobilisiert ist, wobei eine zweite DNA, die markiert ist, an die erste DNA in einem ionischen Lösungsmittel komplementär gebunden ist, um eine Sonde mit einer doppelsträngigen DNA zu bilden, wobei die Sonde dann getrocknet wird;
    einen zweiten Schritt, bei dem ein Protein als eine Probe dazu gebracht wird, auf der Sonde zu adsorbieren,
    einen dritten Schritt der Berechnung von genauen Assoziations- und Dissoziationskonstanten zwischen der Sonde mit einer doppelsträngigen DNA, bestehend aus der ersten und zweiten DNA, und dem Protein, unter Verwendung eines Verfahrens zum Messen der Assoziationsstärke zwischen einer doppelsträngigen DNA und einem Protein, wie etwa Oberflächenplasmonresonanzverfahren und Gel-Shift-Assays, und
    einen vierten Schritt der Herstellung einer analytischen Kurve, basierend auf einer Korrelation zwischen der relativen Assoziationsstärke zwischen der doppelsträngigen DNA und dem Protein, erhalten in dem zweiten Schritt, und den genauen Assoziations- und Dissoziationskonstanten zwischen der doppelsträngigen DNA und dem Protein, erhalten in dem dritten Schritt, um die Assoziations- und Dissoziationskonstanten zwischen der Sonde mit der doppelsträngigen DNA, in der die erste und zweite DNA komplementär gebunden sind, und dem Protein zu berechnen,
    dadurch gekennzeichnet,
    daß in dem zweiten Schritt, nachdem ein Protein als eine Probe dazu gebracht wird, auf der Sonde zu adsorbieren, der DNA-Chip dann mit einem ionischen Lösungsmittel gewaschen wird, um Proteine zu entfemen, die nicht-spezifisch gebunden sind, und daß der DNA-Chip dann mit einem nicht-ionischen Lösungsmittel gewaschen wird, um die zweite markierte DNA zu entfemen, außer da, wo Proteine spezifisch gebunden sind, und daß die Menge an verbleibender Markierung abgelesen wird.
  2. Verfahren zum Berechnen von Assoziations- und Dissoziationskonstanten nach Anspruch 1, wobei das Verfahren zum Messen der Assoziationsstärke zwischen einer doppelsträngigen DNA und einem Protein ein Oberflächenplasmonverfahren oder ein Gel-Shift-Assay-Verfahren ist.
  3. Verfahren zum Untersuchen der Funktionalität von Proteinen unter Verwendung des Verfahrens zum Berechnen von Assoziations- und Dissoziationskonstanten nach einem der Ansprüche 1-2, wobei die Bindungseigenschaft zwischen der Sonde, in der die erste und zweite DNA komplementär gebunden sind, und dem Protein bewertet wird.
  4. Verfahren zum Identifizieren von Proteinen unter Verwendung des Verfahrens zum Berechnen von Assoziations- und Dissoziationskonstanten nach einem der Ansprüche 1-2, wobei die Bindungseigenschaft zwischen der Sonde, in der die erste und zweite DNA komplementär gebunden sind, und dem Protein bewertet wird.
EP20030019932 2002-09-26 2003-09-02 Verfahren zum Kalkulieren von Assoziations- und Dissoziationskonstanten mit einem Polymerchip zum Auffinden von ionischen Polymeren Expired - Lifetime EP1403641B1 (de)

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JP2002281530A JP2004117201A (ja) 2002-09-26 2002-09-26 イオン性高分子同定用高分子チップを用いた結合定数および解離定数算出方法
JP2002281530 2002-09-26

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JP4520873B2 (ja) * 2005-02-02 2010-08-11 セイコーインスツル株式会社 解離定数測定装置、解離定数測定方法、及び解離定数測定プログラム
CN101419214B (zh) * 2007-10-23 2012-07-04 中国科学院上海药物研究所 基于分层原子加和模型的分子酸碱解离常数的预测方法

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US6420109B1 (en) * 1998-09-11 2002-07-16 Genelabs Technologies, Inc. Nucleic acid ligand interaction assays
WO2002018648A2 (en) * 2000-08-25 2002-03-07 President And Fellows Of Harvard College Analysis of binding interactions
JP2003284552A (ja) * 2002-03-28 2003-10-07 Hitachi Software Eng Co Ltd イオン性高分子同定用高分子チップ及びイオン性高分子の同定方法

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