JP2003116583A - Aptamer capable of specifically adsorbing to bisphenol a and method for obtaining the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide both a method for obtaining a new affinity ligand, i.e., an aptamer capable of recognizing bisphenol A suspected to be an endocrine disrupter as a target molecule and specifically adsorbing to the bisphenol A and the aptamer. SOLUTION: This method is to obtain the aptamer capable of specifically adsorbing to the bisphenol A by using an in vitro selection method utilizing an affinity chromatography using an affinity column immobilizing the bisphenol A. In the method, an elution treatment in the affinity chromatography is carried out by using a buffer for antagonistic elution containing an amphoteric organic solvent. A single-stranded nucleic acid molecule which is the aptamer is obtained by the method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to bisphenol A.
TECHNICAL FIELD The present invention relates to a single-stranded nucleic acid molecule (aptamer) that can be specifically adsorbed on DNA and a method for obtaining the same.

[0002]

2. Description of the Related Art It is known that the release of certain chemical substances into the environment adversely affects the reproduction of humans and wild animals. Such chemical substances are called "endocrine disruptors" because they exhibit actions similar to the hormones of living organisms and are believed to disrupt the endocrine actions of wild animals and humans ("environmental hormones"). Also known as "."). Among the chemical substances suspected of being endocrine disrupting substances, bisphenol A is a typical one that is used in our daily lives.

Bisphenol A is a symmetrical dihydric phenol produced by a condensation reaction of phenol and acetone in the presence of an acidic catalyst, and is widely used as a raw material for general-purpose plastics such as polycarbonate resins and epoxy resins. Polycarbonate resin is used, for example, in baby bottles, tableware for lunch, compact discs (CDs), mobile phones, OA equipment and the like. Epoxy resin is a coating material for corrosion prevention of canned goods and water pipes, for example.
It is used for adhesives, wiring boards for electrical products, etc.

Although unreacted bisphenol A is contained in the above resin product, bisphenol A may be eluted into the environment depending on the usage of the resin product. Although the adverse effect on the living body of bisphenol A eluted from resin products is unknown at the present time, as a tool for studying bisphenol A suspected to be an endocrine disrupting substance, different low molecular weight organic compounds are used. It is very important to develop a high affinity ligand capable of selectively recognizing bisphenol A among compounds.

In recent years, due to the development of evolutionary molecular engineering, nucleic acid molecules having a high affinity for target molecules such as proteins from random oligonucleotide libraries,
That is, a technique for screening aptamers has been developed. This method is based on the in vitro selection method (in vit
ro selection method) or SELEX (Systema
tic evolution of ligands by exponential enrichmen
, etc., and many preparations of high-affinity ligands that are faster and easier than antibodies using this method have been reported (for example, Nature, 355: 564 (1992), International Patent Application WO92 / 14843). Publication, JP-A-8-252100 (E
P 0533838), JP-A-9-216895 (US 5780449), etc.).

However, in recognizing a low molecular weight organic compound, it is generally difficult to obtain an affinity ligand because the molecular weight is small and the epitope portion is limited. For bisphenol A, it is still difficult to obtain it. No high-affinity aptamer that can be selectively recognized has been obtained.

[0007]

The object of the present invention is to obtain a novel affinity ligand, ie, an aptamer, capable of specifically recognizing and adsorbing bisphenol A, which is suspected as an endocrine disruptor, as a target molecule. And to provide aptamers.

[0008]

The present inventors have completed the present invention as a result of intensive research to solve the above problems. The present invention is as follows. (1) A method for obtaining an aptamer capable of specifically adsorbing bisphenol A by an in vitro selection method using affinity chromatography using a carrier on which bisphenol A is immobilized, which comprises a buffer for antagonistic elution containing an amphoteric organic solvent A method comprising performing an elution treatment in the above affinity chromatography using a liquid. (2) Using a washing buffer containing an amphoteric organic solvent,
The method according to (1) above, which comprises performing a washing treatment in the affinity chromatography. (3) The method according to (1) above, which comprises using an affinity column filled with a carrier on which bisphenol A is immobilized. (4) A competitive elution buffer containing the amphoteric organic solvent,
The method according to (1) above, which contains 2% to 50% of an amphoteric organic solvent. (5) The washing buffer containing the amphoteric organic solvent is 2%
The method according to (2) above, which contains -50% of an amphoteric organic solvent. (6) The amphoteric organic solvent is at least one selected from dimethyl sulfoxide, dioxane, N, N-dimethylformamide, tetrahydrofuran, and ethanol, and the above (1) or (2).
The method described. (7) A single-stranded nucleic acid molecule that is an aptamer obtained by the method described in (1) above. (8) A method for obtaining an aptamer capable of being specifically adsorbed to bisphenol A by an in vitro selection method using affinity chromatography using an affinity column having bisphenol A immobilized, wherein the amphoteric organic compound is 2% to 50%. A method comprising performing elution treatment in the above affinity chromatography using a competitive elution buffer containing a solvent. (9) The method according to (8) above, wherein the washing treatment in the affinity chromatography is performed using a washing buffer containing 2% to 50% of an amphoteric organic solvent. (10) The amphoteric organic solvent is dimethyl sulfoxide,
The method according to (8) or (9) above, which is at least one selected from dioxane, N, N-dimethylformamide, tetrahydrofuran and ethanol. (11) A single-stranded nucleic acid molecule that is an aptamer obtained by the method according to any one of (8) to (10) above. (12) A single-stranded nucleic acid molecule, which is an aptamer capable of specifically adsorbing to bisphenol A and contains the base sequence of any of the following (a) to (l). (A) The base sequence represented by base numbers 38 to 96 in the base sequence represented by SEQ ID NO: 1 in the sequence listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. (B) The base sequence represented by base numbers 38 to 96 in the base sequence represented by SEQ ID NO: 2 in the sequence listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. (C) The base sequence represented by base numbers 38 to 91 in the base sequence represented by SEQ ID NO: 3 in the sequence listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. (D) The base sequence represented by base numbers 38 to 95 in the base sequence represented by SEQ ID NO: 4 in the sequence listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. (E) The base sequence represented by base numbers 38 to 94 in the base sequence represented by SEQ ID NO: 5 in the sequence listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. (F) The base sequence represented by base numbers 38 to 96 in the base sequence represented by SEQ ID NO: 6 in the sequence listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. (G) The base sequence represented by base numbers 38 to 95 in the base sequence represented by SEQ ID NO: 7 in the sequence listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. (H) The base sequence represented by base numbers 38 to 87 in the base sequence represented by SEQ ID NO: 8 in the sequence listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. (I) The base sequence represented by base numbers 38 to 96 in the base sequence represented by SEQ ID NO: 9 in the Sequence Listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. ) (J) Base sequence represented by base numbers 38 to 86 in the base sequence represented by SEQ ID NO: 10 in the Sequence Listing (however, when the nucleic acid molecule is RNA, T in the sequence is U) (k) The base sequence represented by base numbers 38 to 97 in the base sequence represented by SEQ ID NO: 11 (however, when the nucleic acid molecule is RNA, T in the sequence is U) (l) Above (a) to ( The single-stranded nucleic acid molecule according to (12) above, wherein the sequence (13) in which one or several bases are deleted, substituted, inserted or added in the base sequence of any one of k) is DNA.

As used herein, the term "in vitro selection method" refers to a randomly synthesized single-stranded oligonucleotide library,
A selection process of separating a single-stranded oligonucleotide having a specific function (for example, capable of specifically adsorbing to a target substance), amplifying the single-stranded oligonucleotide, and further separating the single-stranded oligonucleotide having the specific function It refers to a method of obtaining a nucleic acid molecule having a specific function by repeatedly performing. When it is desired to obtain a nucleic acid molecule (aptamer) that can be specifically adsorbed to a specific target substance from a random oligonucleotide library, as a method for separating the nucleic acid molecule having the adsorption ability, for example, a target substance is immobilized. Affinity chromatography using an affinity column can be mentioned. In addition, the term “aptamer” used herein refers to a single-stranded nucleic acid molecule having a specific adsorption ability for a specific target substance. The aptamers in the present specification are not limited to those obtained by the above-mentioned in vitro selection method.

In the present specification, "affinity chromatography" refers to a separation method utilizing a specific interaction (affinity) exhibited by a biological substance, and the separation means is not particularly limited, and is usually carried out in the field. Various techniques are used. Specific examples include affinity chromatography using an affinity column, and the method is an affinity column packed with a carrier on which a target substance is immobilized (hereinafter, may be referred to as an affinity column on which a target substance is immobilized for convenience), Treatment for applying a substance that can be specifically adsorbed to the target substance and / or substance that cannot be adsorbed, and treatment for separating the adsorbable substance and the non-adsorbable substance by washing the column with a washing buffer after applying (Washing treatment) and a treatment for elution of the adsorbable substance by weakening the binding force between the adsorbable substance and the target substance immobilized on the column with an elution buffer after the washing treatment (elution treatment). Shall be contained at least. Here, examples of the carrier for immobilizing the target substance include known carriers used in affinity chromatography, particularly affinity column chromatography.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
FIG. 1 is a flow chart for explaining a processing operation of a preferred example of the method for obtaining an aptamer capable of specifically adsorbing bisphenol A according to the present invention. First, in step s1, using a DNA / RNA automatic synthesizer, a single-stranded oligonucleotide containing a random region of a predetermined length of about 30 to about 80 bases (hereinafter referred to as s
It may be called sNt. ) Library is created. The library preferably contains 10 ¹³ to 10 ¹⁴ kinds or more of ssNt.

PC in steps s2 and s3 described later
To facilitate R amplification, each ssNt has a common priming site at each end of the random region (ie, a sequence homologous to the sense primer at the 5 ′ end and a sequence complementary to the antisense primer at the 3 ′ end). ("Sense" and "antisense" primers are primers for amplifying the original ssNt and its complementary strand, respectively). Such priming sites are each about 15 bases to about 40 bases, preferably about 15 bases to about 30 bases, and PCR primers corresponding to them are preferably designed so as to satisfy general conditions of suitable primers. . Further, when the aptamer after selection needs to be subcloned into an appropriate vector, the priming site may include an appropriate restriction enzyme recognition site in order to facilitate the cloning. However, as in the example shown in FIG. 1, when amplification is performed by asymmetric PCR so that most single-stranded oligonucleotides are contained, the aptamer sequence can be directly determined without subcloning.

In the following step s2, the obtained ssNt
Using the library as a template and sense and antisense primers corresponding to priming sites at both ends of ssNt, double-stranded oligonucleotide (hereinafter referred to as dsNt
In some cases. ) Is amplified. This amplification of dsNt can be performed by PCR according to a conventional method.

In the example shown in FIG. 1, in the subsequent step s3, asymmetric PCR using only the sense primer is performed on the dsNt library amplified by the above PCR, and a pool of ssNt in which only the sense strand (ie, the original ssNt) is amplified. To prepare. It is believed that the aptamer is a single-stranded nucleic acid molecule capable of forming a unique secondary structure, based on the structural and sequence characteristics, and has a specific adsorption ability to a target substance, Therefore, it must exist in a single-stranded state having a predetermined secondary structure before the bisphenol A column affinity chromatography in steps s4 to s6 described later.

The ssNt amplified by asymmetric PCR can be purified by agarose or polyacrylamide gel electrophoresis. If desired, the PCR product can be ethanol precipitated and concentrated prior to electrophoresis. The gel portion containing the band corresponding to the desired ssNt is recovered, and ssNt is purified by a conventional method. Prior to affinity chromatography, 90 ssNt was added.
It is denatured at a temperature of ℃ or above and allowed to cool to room temperature to form an appropriate secondary structure.

In the present invention, in place of the PCR of step s2 and the asymmetric PCR of step s3 in the above example, a sense primer is added to the antisense primer in a large excess (for example, about 50 to 100: 1) and P is added.
By performing CR, only the sense strand was amplified.
The sNt pool may be prepared.

The subsequent steps s4, s5 and s6 are a series of processes of affinity chromatography using an affinity column on which bisphenol A is immobilized. That is, in step s4, ssN having the appropriate secondary structure obtained in step s3
A process of applying t to an affinity column on which bisphenol A is immobilized. In step s5, the affinity column is washed to remove nucleic acid molecules that are not adsorbed on bisphenol A (hereinafter, may be referred to as "non-adsorbed nucleic acid molecules"). In the separation process (washing process), in step s6, a process (elution process) of eluting the nucleic acid molecule specifically adsorbed to bisphenol A (hereinafter, also referred to as “adsorbed nucleic acid molecule”) from the affinity column is performed. Thus, the in vitro selection method of the present invention utilizes affinity chromatography with an affinity column having bisphenol A immobilized thereon to separate adsorbed nucleic acid molecules from non-adsorbed nucleic acid molecules.
As the affinity column to which ssNt is applied in step s4, a column in which bisphenol A is immobilized in advance or a gel solid phase is packed by a conventionally known method may be used.

What is important in the present invention is that at least step s6 is performed using a buffer solution containing an ampholytic organic solvent.
Is to perform the elution treatment of. Preferably, at least the elution treatment of step s6 is performed using a buffer solution containing 2% to 50%, preferably 5% to 40% of an amphoteric organic solvent. The above-mentioned "amphoteric organic solvent" refers to an organic solvent having both acidity and basicity, and neither of them is prominent among active solvents. In the present invention, for example, dioxane, dimethyl sulfoxide (DM
SO), N, N-dimethylformamide (DMF), tetrahydrofuran (THF), ethanol, methanol and the like are used. Among the above, it is preferable to use an amphoteric organic solvent selected from dioxane, DMSO, DMF, THF and ethanol from the viewpoint of the solubility of bisphenol A and the resistance of the column matrix. The amphoteric organic solvent may be a mixture of different amphoteric organic solvents exemplified above. Here, bisphenol A, which is a target substance in the present invention, is a chemical substance having a symmetrical structure having an aromatic ring, and has a property of being insoluble in water. Therefore, although water is also an amphoteric solvent, it is not used in the present invention, and bisphenol A can be dissolved in order to competitively elute the adsorbed nucleic acid molecule specifically adsorbed to bisphenol A immobilized on the affinity column. The above amphoteric organic solvent is used as a buffer for elution.

When the concentration of the amphoteric organic solvent in the buffer solution for competitive elution in step s6 is less than 2%, there arises a problem that the selection efficiency of the aptamer is lowered.
Further, if the concentration of the amphoteric organic solvent in the buffer solution for competitive elution exceeds 50%, the column matrix is adversely affected such as precipitation of nucleic acids and denaturation of the column resin. The above concentration is the ratio of the volume of the amphoteric organic solvent to the volume of the buffer solution (volume% (v / v)).
Further, when the amphoteric organic solvent is a mixture, the above concentration refers to the final concentration of the entire mixture.

In the present invention, further step s
Also in the washing treatment of 5, it is preferable to use a buffer containing an amphoteric organic solvent, preferably a buffer containing 2% to 50%, preferably 5% to 30% of an amphoteric organic solvent. Examples of the amphoteric organic solvent used for the washing buffer include those similar to the above-mentioned antagonistic elution buffer, and among them, dioxane, DMSO, DM from the viewpoint of solubility of bisphenol A and resistance of the column matrix.
It is preferable to use an amphoteric organic solvent selected from F, THF and ethanol. The amphoteric organic solvent in the washing buffer and the amphoteric organic solvent in the competitive elution buffer may be the same or different.

If the concentration of the amphoteric organic solvent in the washing buffer in step s5 is less than 2%, the aptamer selection efficiency tends to be low, which is not preferable. Further, if the concentration of the amphoteric organic solvent in the washing buffer exceeds 50%, it tends to adversely affect the column matrix such as denaturation of the column resin, which is not preferable.

The adsorbed nucleic acid molecule obtained through steps s4 to s6 as described above is subjected to the above-mentioned PCR amplification (step s2), asymmetric PCR (step s3), and affinity chromatography using a bisphenol affinity column (steps s4 to s4). The aptamer of the present invention can be obtained by carrying out the cycle of s6) at least 5 times, preferably about 7 to 15 times.

The obtained aptamer is made into a double-stranded chain according to a conventional method, and then, using a restriction enzyme recognition site constructed in the priming site, blunt-ended, or TA cloning method in a suitable vector. And the nucleotide sequence can be determined by the Maxam-Gilbert method or the dideoxy method. Alternatively, the obtained single-stranded aptamer can be directly sequenced without subcloning.

According to the method of the present invention as described above, an aptamer capable of specifically recognizing and adsorbing bisphenol A which is a water-insoluble low molecular weight organic compound as a target substance, which has been difficult to obtain in the past. Can be obtained efficiently.

The aptamer of the present invention capable of specifically adsorbing bisphenol A is a single-stranded DNA or RNA, preferably a single-stranded DNA. The length is not particularly limited, but is preferably about 30 bases to about 120 bases.

In a preferred embodiment of the aptamer of the present invention, the base sequence represented by base numbers 38 to 96 in the base sequence shown in SEQ ID NO: 1 in the sequence listing and the base sequence in base sequence represented by SEQ ID NO: 2 in the sequence listing 38 to 96, the base sequence shown in SEQ ID NO: 3 in the sequence listing, the base sequence shown in base numbers 38 to 91 in the base sequence, and the base sequence shown in SEQ ID NO: 4 in the base sequence shown in base numbers 38 to 95 Nucleotide sequence, SEQ ID NO: 6 in the sequence listing, SEQ ID NO: 6 in the nucleotide sequence shown in SEQ ID NO: 5
In the base sequence shown in SEQ ID NO: 38 to 96 in the base sequence shown in SEQ ID NO: 7, the base sequence shown in SEQ ID NO: 7 in the base sequence shown in SEQ ID NO: 7 in the base sequence shown in SEQ ID NO: 8 in the sequence list In the base sequence represented by base numbers 38 to 87, the base sequence represented by base numbers 38 to 96 in the base sequence represented by SEQ ID NO: 9 in the sequence listing, and the base sequence 38 to 86 in the base sequence represented by sequence number 10 in the sequence listing The base sequence shown, the base sequence selected from the group consisting of the base sequences represented by base numbers 38 to 97 in the base sequence shown in SEQ ID NO: 11 of the sequence listing (provided that, when the nucleic acid molecule is RNA, T is U.). The term “substantially includes” as used herein includes any one of the above base sequences, or one or several bases are deleted, substituted, inserted or added in any of the above base sequences, and It is meant to include a sequence having a bisphenol A-specific adsorption ability.

The single-stranded nucleic acid molecule (aptamer) substantially containing the above-mentioned base sequence does not have to be prepared by the above-mentioned method of the present invention, and can be prepared by any method. It is preferably prepared by the method of the present invention described above.

[0028]

The present invention will be described in more detail with reference to the following examples, but these are merely examples and do not limit the scope of the present invention.

Example 1 [1] Preparation of amplified single-stranded DNA (ssDNA) library (1) Using an automatic DNA synthesizer, the following template DNA having a 59mer random region and sense (P1) A primer and an antisense (P2) primer were synthesized (step s1). Mold: 5'-TAGGGAATTCGTCGACGGATCC-N ₅₉ -CTGCAGGTCGACGC
ATGCGCCG-3 '(SEQ ID NO: 12) P1: 5'-TAATACGACTCACTATAGGGAATTCGTCGACGGAT-3'
(SEQ ID NO: 13) P2: 5'-CGGCGCATGCGTCGACCTG-3 '(SEQ ID NO: 14) (2) The above template DNA was amplified by PCR using P1 and P2 primers (step s2). The composition of the reaction solution and the reaction conditions are as follows. Reaction solution composition Distilled water 73.5 μl 10 × PCR buffer * 10 μl 20 mM dNTPs 1 μl 10 μM P1 primer 5 μl 10 μM P2 primer 5 μl 1 μg / ml template DNA 5 μl Ex Taq ^™ DNA polymerase 0.5 μl (2.
5 units) * 10 × PCR buffer composition 100 mM Tris-HCl (pH 8.5) 500 mM KCl 20 mM MgCl ₂ Reaction conditions First denaturation: 94 ° C., 1 minute denaturation: 94 ° C., 15 seconds Annealing: 55 ° C., 15 seconds 10 cycles Extension: 72 ° C., 15 seconds Final extension: 72 ° C., 6 minutes (3) Asymmetric PCR was performed using the PCR product as a template and P1 alone as a primer (step s3).
Finally, 2 ml of PCR product was prepared (100 μl × 20). The composition of the reaction solution and the reaction conditions are as follows. Reaction solution composition Distilled water 78.5 μl 10 × PCR buffer 10 μl 20 mM dNTPs 1 μl 10 μM P1 primer 5 μl 1 μg / ml template DNA 5 μl Ex Taq ^™ DNA polymerase 0.5 μl (2.
5 units) Reaction conditions First denaturation: 94 ° C., 1 minute Denaturation: 94 ° C., 15 seconds Annealing: 55 ° C., 15 seconds 40 cycle extension: 72 ° C., 15 seconds Last extension: 72 ° C., 6 minutes

400 μl of the PCR reaction solution was dispensed into 5 microtubes, 80 μl of 10 M ammonium acetate and 1 ml of 99.5% ethanol were added to each well, mixed gently, and allowed to stand at −80 ° C. for 20 minutes. 15,000
Centrifuge at rpm for 15 minutes, rinse with 70% ethanol,
After centrifugation at 15,000 rpm for 10 minutes, the precipitate was dried under reduced pressure. After adding 20 μl of sterile distilled water and vortexing vigorously to dissolve the precipitate, gel loading buffer (95% formamide, 0.5 mM EDTA (pH
8.0), 0.025% STS, 0.025% xylene cyanol, 0.025% bromophenol blue)
20 μl was added and vortexed further. This was treated at 90 ° C for 3 minutes for denaturation, quenched in ice, and then electrophoresed on a polyacrylamide gel (150 V, 50 V).
Minutes). After dipping in an ethidium bromide solution for about 5 minutes and then washing with water, the band was detected on a transilluminator, and the gel portion containing the desired band was cut out and crushed. Elution buffer (0.5M ammonium acetate, 1
0 mM magnesium acetate, 1 mM EDTA (pH 8.
0), 0.1% STS) (800 μl) was added, and the mixture was shaken for 3 hours and then passed through a filter to collect the filtrate.

[2] Affinity column chromatography (1) The DNA obtained in the above [1] was precipitated with ethanol, dried under reduced pressure, and dissolved in 100 μl of distilled water to obtain 2 × binding buffer (200 mM Tris-HCl, 400 m).
M NaCl, 50 mM KCl, 20 mM MgCl
₂ (pH 8.0), 10% dioxane (pH 8.0))
100 μl was added and mixed well, and the absorbance at 260 nm was measured. The DNA solution was treated at 90 ° C. for 5 minutes to denature it, and then allowed to stand by spontaneous cooling as it was. The change in absorbance confirmed the formation of secondary structure. (2) Immobilization of bisphenol A on the column was performed as follows. First, using potassium carbonate as a basic catalyst, a coupling reaction between bisphenol A and 4-bromo-n-butyric acid ethyl ester was performed. The reaction conditions were such that stirring was continued in dimethylformamide at room temperature for 4 hours. After the reaction, the product was confirmed by thin layer chromatography, and then the product was extracted with ether. The reaction product was purified by Silica gel 60 (manufactured by MERCK) to remove by-products. Next, alkaline hydrolysis of the obtained compound was performed. The reaction conditions were such that reflux was continued for 2 hours in the presence of sodium hydroxide in 95% ethanol. The sample after the reaction was subjected to thin film chromatography to confirm that the substrate was completely hydrolyzed. Finally, the bisphenol A derivative synthesized by the coupling reaction using carbodiimide was immobilized on EAH Sepharose 4B (manufactured by Amersham Pharmacia). As a result of immobilization, 7.96 μmol of bisphenol A was bound per 1 ml of gel. The immobilized resin was packed in a column of 8 mm x 5 mm, and
Wash with twice the amount of water, and further about 20 times the amount of 1 × binding buffer (100 mM Tris-HCl, 200 mM Na.
Cl, 25 mM KCl, 10 mM MgCl ₂ , 5%
It was equilibrated with dioxane (pH 8.0) (the liquid coming out at this time was used as a baseline). (3) After applying the DNA sample obtained in (1) above to the column, the cock was opened to receive the eluate into the microtube, and the sample was applied again to the column (step s4). After repeating this operation 3 times, it was left at room temperature for 30 minutes. 1 x binding buffer (washing buffer) 5 m
1 was poured and the cock was opened, and 6 fractions of about 12 drops (about 650 μl) were collected in a microtube (step s5). After closing the cock once, elution buffer (100 mM Tris-HCl, 200 mM NaC
1, 25 mM KCl, 10 mM MgCl ₂ , 30%
Dioxane (pH 8.0) and 35.1 mM bisphenol A (competitive elution buffer) were poured and the cock was opened. The eluate was received in a microtube and returned to the column again. By repeating this operation three times, the buffer was replaced with the elution buffer. The cock was opened again, and 3 fractions of 12 drops were dispensed into a microtube (step s6). The eluate was divided into 400 μl aliquots, 2 μl of glycogen was added to each, and ethanol was precipitated and dried under reduced pressure. The precipitate was well dissolved in 15 μl of water.

[3] Identification of bisphenol A-specific DNA aptamer Each operation of the above [1] (2) to [2] (3) (step s
Each operation from 2 to s6) was repeated 12 times. The nucleotide sequences of 13 kinds of bisphenol A-specific single-stranded DNA aptamers selected by the operation were determined by the dideoxy method. The base sequence of the determined random region of each clone was as follows. Clone 1: 5'-TGGTCGTTGGTCGTTCGCGTTTCTGGATTTTTTAT
TTCTGGGGTTCAGTTCTTTTTTGT-3 '(base numbers 38 to 96 of SEQ ID NO: 1) Clone 2: 5'-CAAGGGCCGAGCGTACCTGGTTTGCTCGTTTTTTG
TCGAATTTTTGGCGCCTTATATTT-3 '(base numbers 38 to 96 of SEQ ID NO: 2) Clone 3: 5'-TTGTGTAGGATTTAGGGGATATTTTTTATCTTATT
CTTTGACGCGCAAATTCTA-3 '(base number 38 of SEQ ID NO: 3
~ 91) Clone 4: 5'-AAAGTGGCCTGCAATCCCTCGGTATTTTAGTCTTT
TGTTTTTGCTGTATTCCTTTCAT-3 '(base numbers 38 to 95 of SEQ ID NO: 4) Clone 5: 5'-GGCCTGTATGGCATGCTGCGCTATTTTCACTCACA
TGTTCTTTTTATTCTTTTGGTT-3 '(base number 3 of SEQ ID NO: 5
8-94) Clone 6: 5'-GGTCCATTCAGCCTCTATTAATCCCCTAGTCTACT
ACTTTTCTCGTCTGGTTTTCTTTC-3 '(base numbers 38 to 96 of SEQ ID NO: 6) Clone 7: 5'-GGTGAATCAGTCTCTTATCATTTTTTCGATTCTTA
GCCGGATTAACAATTCTTTACTC-3 '(base numbers 38 to 95 of SEQ ID NO: 7) clone 8: 5'-GGATGTGGTCTTTATTTTTGTATCCTCGGCATCCT
CCTCCGGCCCGTTCC-3 '(base numbers 38 to 8 of SEQ ID NO: 8
7) Clone 9: 5'-TCTCGAATATTATTTTCCCGTAAACTCTTCGGAGG
GTAGCCATTTTTCCTCGTTGAGTA-3 '(base numbers 38 to 96 of SEQ ID NO: 9) Clone 10: 5'-GATATTTAGGGCGCGTCCGGCACCTTTTATTTT
TTCTTGATTGGTTTTT-3 '(base number 38 to SEQ ID NO: 10 to
86) Clone 11: 5'-GATTGTTGCGGAGTTCTGTTTTCTTTGGCGGTT
ATTTTTTCTATTTCTTAGCAGGTCGAC-3 '(base numbers 38 to 97 of SEQ ID NO: 11)

Comparative Example 1 In place of the elution buffer (competitive elution buffer) in the above [2] (3), 100 mM Tris-HCl, 2
00 mM NaCl, 25 mM KCl, 10 mM M
The same procedure as in Example 1 was carried out except that a buffer consisting of gCl ₂ , 0.5% dioxane (pH 8.0) and bisphenol A was used. However, since bisphenol A was not dissolved, the aptamer could not be recovered from the column matrix. Met.

Comparative Example 2 In place of the elution buffer (competitive elution buffer) in the above [2] (3), 100 mM Tris-HCl, 2
00 mM NaCl, 25 mM KCl, 10 mM M
gCl ₂ , 60% dioxane (pH 8.0) and 3
The same procedure as in Example 1 was carried out except that a buffer consisting of 5.1 mM bisphenol A was used. As a result, the nucleic acid molecules aggregated and precipitated during the elution treatment, and the column could not be eluted, and the aptamer could not be recovered. there were.

[0035]

As is apparent from the above description, the present invention provides a novel affinity ligand capable of recognizing bisphenol A as a target molecule and specifically adsorbing it, that is, a method for obtaining an aptamer and an aptamer. be able to. Since the aptamer of the present invention can specifically recognize and adsorb bisphenol A, it can be used for the detection and quantification of bisphenol A, and the study of the effect of bisphenol A suspected of being an endocrine disruptor on the living body. It can be applied to and is extremely useful.

[0036]

[Sequence list free text]

SEQ ID NO: 1 Single-stranded DNA against bisphenol A screened by in vitro selection method
Aptamer. SEQ ID NO: 1: Single-stranded DNA for bisphenol A screened by in vitro selection method
Aptamer. SEQ ID NO: 3: Single-stranded DNA against bisphenol A, screened by in vitro selection method
Aptamer. SEQ ID NO: 4: Single-stranded DNA against bisphenol A, screened by in vitro selection method
Aptamer. SEQ ID NO: 5: Single-stranded DNA against bisphenol A, screened by in vitro selection method
Aptamer. SEQ ID NO: 6: Single-stranded DNA against bisphenol A, screened by in vitro selection method
Aptamer. SEQ ID NO: 7: Single-stranded DNA against bisphenol A, screened by in vitro selection method
Aptamer. SEQ ID NO: 8: Single-stranded DNA against bisphenol A, screened by in vitro selection method
Aptamer. SEQ ID NO: 9: Single-stranded DNA against bisphenol A, screened by in vitro selection method
Aptamer. SEQ ID NO: 10: Single chain DN against bisphenol A screened by in vitro selection method
A aptamer. SEQ ID NO: 11: Single chain DN against bisphenol A screened by in vitro selection method
A aptamer. SEQ ID NO: 12: single-stranded D containing a 59mer random region flanked by A, G, C or TPCR priming sites
NA. SEQ ID NO: 13: oligo DNA designed to act as a PCR primer (sense) for amplifying the DNA sequence of SEQ ID NO: 12. SEQ ID NO: 14: oligo DNA designed to act as a PCR primer (antisense) for amplifying the DNA sequence of SEQ ID NO: 12.

[0037]

[Sequence list] SEQUENCE LISTING <110> Nitto Denko Corporation <120> APTAMER CAPABLE OF SPECIFICALLY ADSORBING TO BISPHENOL A AND METH OD FOR OBTAINING THE APTAMER <130> A5514 <150> JP 2001-203862 <151> 2001-07-04 <160> 16 <210> 1 <211> 118 <212> DNA <213> Artificial Sequence <220> <223> Single strand DNA aptamer to bisphenol A, screened by in vitro sel ection method. <400> 1 taatacgact cactataggg aattcgtcga cggatcctgg tcgttggtcg ttcgcgtttc 60 tggatttttt atttctgggg ttcagttctt ttttgtctac aggtcgacgc atgcgccg 118 <210> 2 <211> 118 <212> DNA <213> Artificial Sequence <220> <223> Single strand DNA aptamer to bisphenol A, screened by in vitro sel ection method. <400> 2 taatacgact cactataggg aattcgtcga cggatcccaa gggccgagcg tacctggttt 60 gctcgttttt tgtcgaattt ttggcgcctt atatttctgc aggtcgacgc atgcgccg 118 <210> 3 <211> 113 <212> DNA <213> Artificial Sequence <220> <223> Single strand DNA aptamer to bisphenol A, screened by in vitro sel ection method. <400> 3 taatacgact cactataggg aattcgtcga cggatccttg tgtaggattt aggggatatt 60 ttttatctta ttctttgacg cgcaaattct actgcaggtc gacgcatgcg ccg 113 <210> 4 <211> 117 <212> DNA <213> Artificial Sequence <220> <223> Single strand DNA aptamer to bisphenol A, screened by in vitro sel ection method. <400> 4 taatacgact cactataggg aattcgtcga cggatccaaa gtggcctgca atccctcggt 60 attttagtct tttgtttttg ctgtattcct ttcatctgca ggtcgacgca tgcgccg 117 <210> 5 <211> 116 <212> DNA <213> Artificial Sequence <220> <223> Single strand DNA aptamer to bisphenol A, screened by in vitro sel ection method. <400> 5 taatacgact cactataggg aattcgtcga cggatccggc ctgtatggca tgctgcgcta 60 ttttcactca catgttcttt ttattctttt ggttctgcag gtcgacgcat gcgccg 116 <210> 6 <211> 118 <212> DNA <213> Artificial Sequence <220> <223> Single strand DNA aptamer to bisphenol A, screened by in vitro sel ection method. <400> 6 taatacgact cactataggg aattcgtcga cggatccggt ccattcagcc tctattaatc 60 ccctagtcta ctacttttct cgtctggttt tctttcctgc aggtcgacgc atgcgccg 118 <210> 7 <211> 117 <212> DNA <213> Artificial Sequence <220> <223> Single strand DNA aptamer to bisphenol A, screened by in vitro sel ection method. <400> 7 taatacgact cactataggg aattcgtcga cggatccggt gaatcagtct cttatcattt 60 tttcgattct tagccggatt aacaattctt tactcctgca ggtcgacgca tgcgccg 117 <210> 8 <211> 109 <212> DNA <213> Artificial Sequence <220> <223> Single strand DNA aptamer to bisphenol A, screened by in vitro sel ection method. <400> 8 taatacgact cactataggg aattcgtcga cggatccgga tgtggtcttt atttttgtat 60 cctcggcatc ctcctccggc ccgttccctg caggtcgacg catgcgccg 109 <210> 9 <211> 118 <212> DNA <213> Artificial Sequence <220> <223> Single strand DNA aptamer to bisphenol A, screened by in vitro sel ection method. <400> 9 taatacgact cactataggg aattcgtcga cggatcctct cgaatattat tttcccgtaa 60 actcttcgga gggtagccat ttttcctcgt tgagtactgc aggtcgacgc atgcgccg 118 <210> 10 <211> 108 <212> DNA <213> Artificial Sequence <220> <223> Single strand DNA aptamer to bisphenol A, screened by in vitro sel ection method. <400> 10 taatacgact cactataggg aattcgtcga cggatccgat atttagggcg cgtccggcac 60 cttttatttt ttcttgattg gtttttctgc aggtcgacgc atgcgccg 108 <210> 11 <211> 119 <212> DNA <213> Artificial Sequence <220> <223> Single strand DNA aptamer to bisphenol A, screened by in vitro sel ection method. <400> 11 taatacgact cactataggg aattcgtcga cggatccgat tgttgcggag ttctgttttc 60 tttggcggtt attttttcta tttcttagca ggtcgacctg caggtcgacg catgcgccg 119 <210> 12 <211> 103 <212> DNA <213> Artificial Sequence <220> <221> unsure <222> (23) .. (81) <223> A, G, C or T <220> <223> Single strand DNA containing 59mer random region flanked with PCR priming sites. <400> 12 tagggaattc gtcgacggat ccnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnnnnnnnnn nctgcaggtc gacgcatgcg ccg 103 <210> 13 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Oligo-DNA designed to act as PCR primer (sense) for amplification of DNA sequence of SEQ ID NO: 12. <400> 13 taatacgact cactataggg aattcgtcga cggat 35 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Oligo-DNA designed to act as PCR primer (antisense) for amplificat ion of DNA sequence of SEQ ID NO: 12. <400> 14 cggcgcatgc gtcgacctg 19

[Brief description of drawings]

FIG. 1 is a flow chart for explaining a processing operation of a preferred example of the method for obtaining an aptamer capable of specifically adsorbing bisphenol A according to the present invention.

Continued front page    (72) Inventor Akio Kobayashi             1-10-22 Uenosaka, Toyonaka City, Osaka Prefecture (72) Inventor Eiichiro Fukusaki             4-2-2 Satakedai, Suita City, Osaka Prefecture (72) Inventor Tadashi Yanagihara             14-37-105 Yamadakita, Suita City, Osaka Prefecture (72) Inventor Go Nakanishi             1-9-15 Yoko Garden, Yao City, Osaka Prefecture Hidewa Cash Register             Dens 204 F-term (reference) 4B024 AA11 CA05 HA14                 4D017 AA09 BA07 CA14 DA03 DB02                 4G066 AB03B AB06B CA54 EA02                       GA11 GA35

Claims (13)

[Claims] 1. A method for obtaining an aptamer capable of specifically adsorbing bisphenol A by an in vitro selection method using affinity chromatography using a carrier on which bisphenol A is immobilized, the method comprising competitive elution containing an amphoteric organic solvent. A method of performing an elution treatment in the above affinity chromatography using a buffer for use. 2. The method according to claim 1, wherein the washing treatment in the affinity chromatography is performed using a washing buffer containing an amphoteric organic solvent. 3. The method according to claim 1, wherein an affinity column prepared by packing a carrier having bisphenol A immobilized thereon is used. 4. The method according to claim 1, wherein the competitive elution buffer containing the amphoteric organic solvent contains 2% to 50% of the amphoteric organic solvent. 5. The method according to claim 2, wherein the washing buffer containing the amphoteric organic solvent contains 2% to 50% of the amphoteric organic solvent. 6. The method according to claim 1 or 2, wherein the amphoteric organic solvent is at least one selected from dimethyl sulfoxide, dioxane, N, N-dimethylformamide, tetrahydrofuran and ethanol. 7. A single-stranded nucleic acid molecule which is an aptamer obtained by the method according to claim 1. 8. A method for obtaining an aptamer capable of specifically adsorbing bisphenol A by an in vitro selection method using affinity chromatography using an affinity column on which bisphenol A is immobilized, which comprises 2% to 50%. A method comprising performing an elution treatment in the above affinity chromatography using a competitive elution buffer containing an amphoteric organic solvent. 9. The method according to claim 8, wherein the washing treatment in the affinity chromatography is performed using a washing buffer containing 2% to 50% of an amphoteric organic solvent. 10. The amphoteric organic solvent is dimethyl sulfoxide, dioxane, N, N-dimethylformamide,
9. It is at least one selected from tetrahydrofuran and ethanol.
Or the method according to 9. 11. A single-stranded nucleic acid molecule which is an aptamer obtained by the method according to any one of claims 8 to 10. 12. A single-stranded nucleic acid molecule, which is an aptamer capable of specifically adsorbing to bisphenol A, and which comprises any of the following nucleotide sequences (a) to (l). (A) The base sequence represented by base numbers 38 to 96 in the base sequence represented by SEQ ID NO: 1 in the sequence listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. (B) The base sequence represented by base numbers 38 to 96 in the base sequence represented by SEQ ID NO: 2 in the sequence listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. (C) The base sequence represented by base numbers 38 to 91 in the base sequence represented by SEQ ID NO: 3 in the sequence listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. (D) The base sequence represented by base numbers 38 to 95 in the base sequence represented by SEQ ID NO: 4 in the sequence listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. (E) The base sequence represented by base numbers 38 to 94 in the base sequence represented by SEQ ID NO: 5 in the sequence listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. (F) The base sequence represented by base numbers 38 to 96 in the base sequence represented by SEQ ID NO: 6 in the sequence listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. (G) The base sequence represented by base numbers 38 to 95 in the base sequence represented by SEQ ID NO: 7 in the sequence listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. (H) The base sequence represented by base numbers 38 to 87 in the base sequence represented by SEQ ID NO: 8 in the sequence listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. (I) The base sequence represented by base numbers 38 to 96 in the base sequence represented by SEQ ID NO: 9 in the Sequence Listing (provided that the nucleic acid molecule is R
In the case of NA, T in the sequence is U. ) (J) Base sequence represented by base numbers 38 to 86 in the base sequence represented by SEQ ID NO: 10 in the Sequence Listing (however, when the nucleic acid molecule is RNA, T in the sequence is U) (k) The base sequence represented by base numbers 38 to 97 in the base sequence represented by SEQ ID NO: 11 in the sequence table (however, when the nucleic acid molecule is RNA, T in the sequence is U). (L) Above (a) to ( Sequence in which one or several bases have been deleted, substituted, inserted or added in any of the base sequences of k) 13. The single-stranded nucleic acid molecule according to claim 12, wherein the nucleic acid is DNA.