JP2000350597A - Obtaining of single-stranded nucleic acid binding to endocrine disrupting substance and single-stranded nucleic acid binding to endocrine disrupting substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject single-stranded nucleic acid by mixing an endocrine disrupting substance with a nucleic acid mixture comprising many kinds of single stranded nucleic acids having mutually different base sequences, forming a complex of the single-stranded nucleic acid with the endocrine disrupting substance, separating the resultant complex and dissociating the complex into the single-stranded nucleic acid and the endocrine disrupting substance. SOLUTION: An endocrine disrupting substance is mixed with a nucleic acid mixture comprising many kinds of single-stranded nucleic acids having mutually different base sequences by a method for feeding a solution containing the nucleic acid mixture to an affinity column filled with a packing material bound to the endocrine disrupting substance or it derivative and a complex of the above single-stranded nucleic acid having the ability to bind to the endocrine disrupting substance and the endocrine disrupting substance is formed when the endocrine disrupting substance is mixed with the nucleic acid mixture. The resultant complex is then separated from the free endocrine disrupting substance and the free single-stranded nucleic acids and the separated complex is then dissociated into the above single-stranded nucleic acid and the above endocrine disrupting substance. Thereby, the single-stranded nucleic acid capable of specifically recognizing the endocrine disrupting substance and binding to the objective endocrine disrupting substance is obtained in a short period.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an endocrine disruptor,
In particular, the present invention relates to a single-stranded nucleic acid binding to dioxins and a method for obtaining the same.

[0002]

BACKGROUND OF THE INVENTION In recent years, it has been suggested that chemicals may disrupt the endocrine system of animals, either as agonists or antagonists of hormones or by other mechanisms of action. And the chemical substance which shows such an action is called "endocrine disrupting substance" or "environmental hormone". As properties common to the above endocrine disrupting substances, many have a benzene ring structure in their molecular skeleton, and it is known that there are many structural isomers for each endocrine disrupting substance.

Here, the following chemical substances are known as endocrine disrupting substances having a benzene ring structure in the molecular skeleton. Octyl phenol, nonyl phenol,
There are pentyl phenol, butyl benzyl phthalate, butyl phthalate and bisphenol A, and some whose use is prohibited such as polychlorinated biphenyls (PCB). Those used as insecticides include o, p '
-Dichlorodiphenyltrichloroethane (DDT),
There are p, p'-DDT, p, p'-dichlorodiphenyldichloroethylene (DDE), p, p'-dichlorodiphenyldichloroethane (DDD), methoxychlor, monophenol methoxychlor, bisphenolmethoxychlor, dicophor and permethrin. In addition, anol, dihydrobenzanthracene and the like are known. In addition to the above-mentioned compounds, dioxins which are unintentionally produced in association with industrial activities and the like are particularly attracting attention as endocrine disrupting substances. Dioxins are polychlorodibenzo-para-
Dioxin and a general term for compounds including analogous compounds having the same toxicity as polychlorodibenzo-para-dioxin. Examples of the analogous compound include polychlorodibenzofuran, coplanar PCB, tetrabromodibenzothianthrein, and tetrabromodibenzo-para-.
Dioxin, tetrachlorobiphenylene, tetrachloroazobenzene, hexachlorobiphenyl and the like.

[0004] As endocrine disrupting substances having no benzene ring structure, heptachlor, kepon (chlordecon), dieldrin, endosulfan and hexachlorocyclohexane are known.

[0005] The effects of the above-mentioned endocrine disrupting substances on living organisms and ecosystems are still in the research stage, and in order to understand the effects, a sensitive analysis method capable of recognizing the endocrine disrupting substances at the isomer level is also underway. It is important to establish a method. Here, as a conventional method, a method using liquid chromatography is known. As for dioxins, a method of biochemically detecting the dioxins by using an immunoassay method using an anti-dioxin antibody is also known. Further, Japanese Patent Application Laid-Open
As described in JP-A-274654, a biosensor in which an antibody that specifically binds to the endocrine disrupting substance is immobilized on the surface of a piezoelectric vibrator has also been proposed.

Here, in the immunoassay and the biosensor, an antibody that specifically recognizes a target molecule is prepared as follows. The endocrine disrupting substance,
As such, they are often not immunogenic (so-called haptens). Therefore, after obtaining a hapten-carrier complex in which the endocrine disrupting substance is bound to a carrier having immunogenicity, this is inoculated into experimental animals such as mice,
Immunize. Then, antibody-producing cells (B lymphocytes) are collected from the immunized experimental animal, and the B lymphocytes are fused with tumor cells to prepare immortalized fused cells (hereinafter, referred to as "hybridomas"). I do. Furthermore, hybridomas producing anti-endocrine disrupting substance antibodies are selected from the hybridomas according to auxotrophy, the presence or absence of antibody-producing ability, etc., and each single clone is isolated.
The hybridoma derived from each single clone produces an antibody that recognizes only a specific antigen recognition site, and the antibody produced by the hybridoma is referred to as a “monoclonal antibody”.

[0007]

According to the above-mentioned conventional method for analyzing endocrine disrupting substances, in liquid chromatography, the pretreatment of the sample is complicated and the separation of structural isomers is very difficult. The analysis of the endocrine disrupting substances, particularly dioxins whose toxicity is largely determined among the isomers, had problems in terms of accuracy and sensitivity. On the other hand, in the method for analyzing an endocrine disrupting substance using the monoclonal antibody, in order to obtain immunized B lymphocytes, a laboratory animal serving as a source for collecting the B lymphocytes over a long period of time in a sanitary environment. Must be bred and managed. Therefore, there has been a problem that a large amount of labor and investment are required for raising the animals. In addition, there is a problem that a complicated operation and a long period of time are required for selecting and isolating a hybridoma producing the desired monoclonal antibody from the hybridoma. Furthermore, the method using the monoclonal antibody has a problem in that, although the ability to recognize an endocrine disrupting substance is improved as compared with the liquid chromatography, the detection sensitivity is insufficient.

Accordingly, an object of the present invention is to provide a substance capable of specifically recognizing the endocrine disrupting substance,
It is to provide a method for obtaining in a short time.

[0009]

The feature of the method for obtaining a single-stranded nucleic acid that binds to an endocrine disrupting substance according to the present invention for achieving this object is that an endocrine disrupting substance and a multi-stranded nucleic acid having different base sequences from each other are used. A mixing step of mixing a nucleic acid mixture comprising a species of single-stranded nucleic acid, a complex forming step of forming a complex between the single-stranded nucleic acid having an ability to bind to the endocrine disrupting substance and the endocrine disrupting substance, A separation step of separating the complex from a free endocrine disrupting substance and a free single-stranded nucleic acid, and a dissociation step of dissociating the complex into the single-stranded nucleic acid and the endocrine disrupting substance. In the mixing step, a solution containing the nucleic acid mixture is supplied to an affinity column filled with a filler having an endocrine disrupting substance or a derivative thereof as a reactive group,
In the complex forming step, a complex of the single-stranded nucleic acid capable of binding to the endocrine disrupting substance and the endocrine disrupting substance or a derivative thereof is formed, and in the separation step, a washing solution is supplied to the affinity column. do it,
The complex is separated from a free endocrine disrupting substance and a free single-stranded nucleic acid, and in the dissociation step, an eluate for dissociating the complex into the single-stranded nucleic acid and the endocrine disrupting substance is supplied to the affinity column. Dissociating the complex into the single-stranded nucleic acid and the endocrine disrupting substance, and recovering a single-stranded nucleic acid capable of binding to the endocrine disrupting substance. Is single-stranded DNA
In addition, the endocrine disrupting substance is an endocrine disrupting substance having a benzene ring structure,
The endocrine disrupting substance having the benzene ring structure is a dioxin. Further, the characteristic configuration of the single-stranded nucleic acid binding to the endocrine disrupting substance according to the present invention is characterized in that the endocrine disrupting substance is mixed with a nucleic acid mixture composed of many kinds of single-stranded nucleic acids having different base sequences from each other, After forming a complex between the single-stranded nucleic acid having an ability to bind to an endocrine disrupting substance and the endocrine disrupting substance, the complex is separated from a free endocrine disrupting substance and a free single-stranded nucleic acid,
The nucleic acid mixture is further obtained by dissociating the complex into the single-stranded nucleic acid and the endocrine disrupting substance, and further comprising: Is supplied, and after forming a complex between the single-stranded nucleic acid capable of binding to the endocrine disrupting substance and the endocrine disrupting substance or a derivative thereof, the affinity column is provided with a free endocrine disrupting substance. And supplying a washing solution for separating free single-stranded nucleic acid, and further supplying an eluate for dissociating the complex into an endocrine disrupting substance and a single-stranded nucleic acid, thereby forming the complex into the single-stranded nucleic acid. The nucleic acid and the endocrine disrupting substance are obtained by dissociation, furthermore, the single-stranded nucleic acid is a single-stranded DNA, and further, the endocrine disrupting substance has a benzene ring structure. And the endocrine disrupting substance having a benzene ring structure is a dioxin. And these effects are as follows.

The inventors have found that the base sequence and structure of single-stranded nucleic acids can be easily analyzed, that they can be easily obtained by chemical synthesis or cloning, and that low-molecular-weight compounds (eg, ATP, glycolipids, etc.) ) Has been reported to have binding ability and can be used as a tool for molecular recognition, and to search for a single-stranded nucleic acid capable of binding to the endocrine disrupting substance. Arriving,
The present invention has been completed.

[0011] In particular, the advantage of using a nucleic acid as a molecular species capable of binding to the endocrine disrupting substance is that storage is easy, and once the base sequence is known, the nucleic acid having that sequence is not necessarily used. It does not need to be stored and is newly available by chemical synthesis. In the case of the monoclonal antibody, when the monoclonal antibody-producing hybridoma dies, it becomes unavailable. here,
The present inventors have noted that single-stranded nucleic acids have a higher degree of freedom in the range that can take a three-dimensional structure than double-stranded nucleic acids, and have adopted single-stranded nucleic acids instead of double-stranded nucleic acids. Nucleic acids having the ability to bind to the endocrine disrupting substance were searched. Double-stranded DNA usually has a very stable helical structure in which nucleic acids having complementary base sequences form regular hydrogen bonds. When the double-stranded DNA constituting the helical structure interacts with a low molecular weight compound such as the endocrine disrupting substance, sequence non-specific intercalation often occurs. On the other hand, the three-dimensional structure of a single-stranded DNA does not have a specific structure, and can take various three-dimensional structures due to various changes depending on the sequence of bases constituting the single-stranded DNA and the environment surrounding the molecule. . Therefore, when searching for a nucleic acid having the ability to bind to the endocrine disrupting substance, it is more efficient to use a single-stranded nucleic acid.

[0012] Specifically, a single-stranded nucleic acid capable of binding to the endocrine disrupting substance is obtained as follows. First, the endocrine disrupting substance is mixed with a nucleic acid mixture comprising a large number of single-stranded nucleic acids having different base sequences from each other, and the single-stranded nucleic acid having a binding ability to the endocrine disrupting substance, Form complexes with endocrine disruptors. Here, in the case of the monoclonal antibody, usually, a non-proteinaceous low-molecular compound such as the endocrine disrupting substance cannot be directly recognized, so that a hapten-carrier complex with a carrier having immunogenicity is prepared. There is a need to. However, the single-stranded nucleic acid itself directly binds to a target molecule.
At this stage, since the complex is mixed with the free endocrine disrupting substance and the free single-stranded nucleic acid, the unnecessary free endocrine disrupting substance and the free single-stranded nucleic acid are separated from the complex. And remove. Further, when the separated complex is dissociated into the single-stranded nucleic acid and the endocrine disrupting substance, a single-stranded nucleic acid capable of binding to the endocrine disrupting substance can be obtained.

[0013] More specifically, the above operation can be performed by affinity chromatography.
That is, a packing material to which the endocrine disrupting substance is bound is prepared, an affinity column filled with the packing material is prepared, and a single-stranded nucleic acid having a binding ability to the endocrine disrupting substance is separated using the same. Is what you do. If it is difficult to bind the intended endocrine disrupting substance itself to the filler, etc., the endocrine disrupting substance to which a chain structure or the like has been added to facilitate the binding with the filler. May be used. Supplying a solution containing the nucleic acid mixture to the affinity column to form a complex of the endocrine disrupting substance bound to the filler and a single-stranded nucleic acid capable of binding to the endocrine disrupting substance; Thereafter, a washing solution is supplied to the affinity column to separate the complex from free endocrine disrupting substances and free single-stranded nucleic acid. When the eluate for dissociating the complex into the single-stranded nucleic acid and the endocrine disrupting substance is supplied to the affinity column, the complex dissociates into the single-stranded nucleic acid and the endocrine disrupting substance, A single-stranded nucleic acid capable of binding to a disturbing substance is included in the eluate passed through the affinity column, and may be collected.

The single-stranded nucleic acid is a single-stranded DNA.
Is used, because of its excellent chemical stability, the handling during the separation operation is easy, and the handling of the single-stranded nucleic acid having a binding ability to the endocrine disrupting substance obtained by the method is also easy. Becomes

Among the endocrine disrupting substances, those having a benzene ring structure are liable to generate structural isomers, and it is difficult to accurately distinguish each isomer by the liquid chromatography or the like. Here, the single-stranded nucleic acid having an ability to bind to an endocrine disrupting substance according to the present invention is excellent in that these isomers can be accurately distinguished.

Among the endocrine disrupting substances having a benzene ring structure, molecules capable of recognizing dioxins whose toxicity is significantly different among isomers at the isomer level are those which have high utility in the analysis and the like. It is.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to a single-stranded DNA capable of binding to dioxins.

[Construction of Single-Stranded DNA Pool] First, a single-stranded DNA having a binding ability to a target dioxin.
A single-stranded DNA pool to be a source of NA (hereinafter, referred to as "dioxin-bound single-stranded DNA") is prepared.
The single-stranded DNA pool may use a commercially available DNA library, or may be prepared by itself. Here, a single-stranded DNA pool derived from an organism uses a chemically synthesized single-stranded DNA pool in consideration of variations in the nucleotide sequence because the combination of the nucleotide sequences is limited depending on the species and the state of cells. Is preferred.
The chemically synthesized single-stranded DNA pool can be constituted by a mixture of many types of single-stranded DNAs having different base sequences from each other so as to have substantially all possible combinations of the base length. Here, in order to further enrich the variation of the base sequence,
As the DNA, a DNA obtained by substituting a naturally-modified base as a component of a nucleic acid with a chemically modified derivative may be used. Further, when each single-stranded DNA constituting the single-stranded nucleic acid pool is prepared so as to include a linker sequence or a PCR primer binding sequence serving as a recognition sequence of a restriction enzyme at an end thereof, PCR amplification / cloning described later is performed. Operation becomes easy. The single-stranded DN
When the A pool is constructed by chemical synthesis, the total length of the base sequence of the single-stranded DNA is 13
It is preferable to design so as to be within 0 bases.

[Selection of Dioxin-Binding Single-Stranded DNA] A dioxin derivative to which a structure (for example, an amino group, a carboxyl group, etc.) necessary for binding to a filler is added is prepared, and the dioxin derivative is subjected to a conventional method. Fillers (eg, agarose, sepharose (pha)
rmacia)) to obtain a filler for use in an affinity column capable of specifically adsorbing dioxins. Then, after supplying the solution containing the single-stranded DNA pool to a column packed with a packing material to which the dioxin derivative is bound, a washing solution (eg, Tris buffer solution or the like) is passed through the column, and the dioxin The single-stranded DNA not bound to the derivative is removed. Next, the single-stranded DNA bound to the dioxin derivative
An eluate (for example, an alkaline aqueous solution (pH 12) or the like) is applied to the column so that is dissociated from the dioxin derivative. The eluate (dioxin-bound fraction) that has passed through the column contains single-stranded DNA bound to the dioxin derivative, and is collected and subjected to the following operation.

[DN obtained from dioxin-bound fraction
A) Amplification of polymerase using single-stranded DNA contained in the dioxin-bound fraction (if necessary, the single-stranded DNA may be purified from the dioxin-bound fraction and used as a template) as a template (Hereinafter, referred to as “PCR”) to amplify the single-stranded DNA. Here, it is known that the complementary strand of the single-stranded DNA that specifically binds to a certain substance does not necessarily bind to the substance.
And mixing with the single-stranded DNA bound to the dioxins. For this reason, asymmetric PCR for mainly amplifying the target single-stranded DNA is performed, or a primer for replicating the single-stranded DNA is labeled with biotin,
It is preferable to take measures such as separating and recovering with a column packed with a packing material containing avidin (or streptavidin).

Since the amount of dioxin-bound single-stranded DNA obtained by the above-mentioned selection is very small in many cases, even if the amplification / selection operation is performed a plurality of times for the convenience of the analysis operation described later. good. At this time, by repeating the selection or amplification operation while strictly selecting the above-described selection or amplification conditions, the single-stranded D having a higher binding ability can be obtained.
It is also possible to select NA.

[Cloning and base sequence determination] In order to stably store the dioxin-bonded single-stranded DNA and to make it readily available, the dioxin-bonded single-stranded DN is used.
Preferably, A is cloned into a virus, plasmid or the like. The analysis of the base sequence of the dioxin-bound single-stranded DNA selected through the above-mentioned operation can be performed according to a standard method such as an enzymatic method. The dioxin-binding single-stranded DN thus isolated and identified
A can be obtained easily and in large quantities by chemical synthesis or by preparing from plasmid clones, phage clones, etc. obtained by the above cloning.

The single-stranded DN whose base sequence has been determined
Prepare a filler to which A is bound, flow a solution containing the target dioxins into the affinity column filled with the filler, and bind the single-stranded nucleic acid with the target dioxins. After confirmation, it is preferable to provide the information to the examples described later.

According to the method for obtaining a dioxin-bound single-stranded DNA according to the present invention described above, it is theoretically possible to identify the dioxin-bound single-stranded DNA in about one month. Therefore, a molecule that can recognize the dioxins can be obtained in a shorter time than in the case of preparing the monoclonal antibody. In addition, it is not necessary to breed animals to obtain the dioxin-bound single-stranded DNA, so that labor and investment can be reduced. Furthermore, since the isolation operation is performed in an inanimate system except for cloning, it is considered that reproducibility is good. Further, when the dioxin-bonded single-stranded DNA according to the present invention is used for the analysis of dioxins, the influence of impurities contained in the sample is small and easy as compared with the liquid chromatography method described above. It is considered possible to recognize the dioxin at the structural isomer level. Therefore,
It is considered that an analytical method superior to the above-mentioned liquid chromatography can be provided in terms of accuracy and sensitivity.

[0025]

DESCRIPTION OF THE PREFERRED EMBODIMENTS [Device for removing dioxins] FIGS. 1 (a) and 1 (b) show a dioxin-bound single-stranded DNA according to the present invention.
1 shows a dioxin removing apparatus 1 utilizing the above. As shown in FIG. 1 (a), the dioxin removing apparatus 1 is used to discharge a water supply hole 11 for supplying a solution to be treated and a treated solution to a casing 10 made of stainless steel or FRP. The inside of the casing 10 is provided with a filter 2 having glass or plastic as a base material and carrying the dioxin-bonded single-stranded DNA on its surface (see the above description). The substrate may be coated with avidin to fix the dioxin-bound single-stranded DNA to the substrate.) The dioxin-removing device 1 is different from the filter 2 carrying the dioxin-bonded single-stranded DNA in that
It may be configured to incorporate therein a particulate filler carrying A.

When a solution containing dioxins is injected into the casing 10 through the water supply hole 11, the solution passes through the filter 2 and is discharged from the drain hole 12. Here, the dioxins 3 are:
As shown in FIG. 1 (b), the dioxin-bonded single-chain D
It is adsorbed by NA22 and removed from the solution. Therefore, the solution discharged from the drain hole 12 does not contain the dioxins 3. The filter 2 can be collected after use and the dioxins 3 can be decomposed and disposed of, and the eluate can dissociate the dioxins 3 from the dioxin-bound single-stranded DNA 22. It can be regenerated and reused by washing with.

The dioxin removing apparatus 1 is considered to be applicable to equipment for purifying a solution containing dioxins, for example, a sewage treatment facility, an industrial wastewater treatment facility, or a leachate treatment facility in a landfill. As shown in FIG. 2, the leachate treatment facility 4 may be configured by connecting a pH adjusting device 40, a biological treatment device 41, a coagulation / sedimentation treatment device 42, and the dioxin removal device 1 in the stated order. I can do it. The leachate is sent to the biological treatment device 41 after being adjusted to a pH range suitable for the growth of microorganisms by the pH adjustment device 40. The organic matter and the like of the pH-adjusted leachate is decomposed by microorganisms fixed to the membrane. Then, in the coagulation / precipitation treatment device 42,
A coagulant is added to the leachate that has undergone the biological treatment to coagulate and remove contaminants remaining in the leachate by gravity sedimentation. The leachate thus treated is finally discharged through the dioxin removal device 1 to remove dioxins and then to the outside of the landfill.

[Another Embodiment] Another embodiment will be described below. As an example utilizing the binding ability between the single-stranded nucleic acid capable of binding to the endocrine disrupting substance according to the present invention and the endocrine disrupting substance, the dioxin removing device 1 is exemplified. It can be used for detection and quantification of Further, the single-stranded nucleic acid capable of binding to the endocrine disrupting substance and the endocrine disrupting substance bound thereto can be easily dissociated, so that the single-stranded nucleic acid is used to concentrate or recover the endocrine disrupting substance. It is considered possible.

In order to select dioxin-bound single-stranded DNA from the DNA pool, affinity chromatography was used.
DNA that binds to the dioxin may be selected using a crystal oscillator or a surface plasmon resonance biosensor.

Although the embodiment of the present invention has been described by exemplifying a dioxin-bound single-stranded DNA, the method for obtaining a single-stranded nucleic acid that binds to an endocrine disrupting substance according to the present invention comprises the steps of: It is not based on the special interaction observed only with DNA, but on the interaction that can widely occur between the endocrine disrupting substance and single-stranded nucleic acid. Accordingly, by using the method for obtaining a single-stranded nucleic acid that binds to the endocrine disrupting substance, it is possible to obtain a single-stranded nucleic acid that binds to the endocrine disrupting substance having a benzene ring structure described above or the endocrine disrupting substance having another structure. Is thought to be possible. Here, when a single-stranded RNA is used as the single-stranded nucleic acid, it is necessary to convert the RNA into a DNA form when the nucleic acid is amplified by the PCR. Therefore, it is necessary to transcribe single-stranded RNA into DNA, subject it to PCR, and select the product obtained by the PCR after reverse transcription into RNA. Therefore, for the convenience of these operations, it is preferable that the single-stranded RNA is designed to include a recognition base sequence of an enzyme that catalyzes the transcription reaction and the reverse transcription reaction.

[Brief description of the drawings]

FIG. 1 is a cross-sectional view of an apparatus for removing dioxins using single-stranded DNA capable of binding to dioxins according to the present invention.

FIG. 2 is a schematic diagram of a leachate treatment facility provided with the dioxin removal device.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 Dioxin processing apparatus 2 Filter 22 Single chain D which has binding ability to dioxins
NA

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 30/48 G01N 30/48 R 30/88 30/88 E // C12N 15/09 33/566 G01N 33 / 566 C12N 15/00 A (72) Inventor Nobuyuki Nishiguchi 1-1-1, Hama, Amagasaki-shi, Hyogo Prefecture Inside Kubota Research Institute of Technology (72) Inventor Toshio Nakanishi 1-1-1, Hama, Amagasaki-shi, Hyogo Co., Ltd. F-term in Kubota Technology Development Laboratory (reference) 4B024 AA20 CA01 CA09 HA12 4B063 QA01 QQ42 QR32 QR54 QS02 QS15 4D017 AA01 BA04 CA11 DA03 4D024 AA04 AB11 BA17 4G066 AB30B CA01 CA20 CA33 DA08 EA02 FA11 FA40

Claims (10)

[Claims] A mixing step of mixing an endocrine disrupting substance with a nucleic acid mixture comprising a large number of single-stranded nucleic acids having different base sequences, wherein the single-stranded nucleic acid having a binding ability to the endocrine disrupting substance A complex forming step of forming a complex with the endocrine disrupting substance, a separating step of separating the complex from free endocrine disrupting substance and free single-stranded nucleic acid, and separating the separated complex into the single A method for obtaining a single-stranded nucleic acid binding to an endocrine disrupting substance, comprising a dissociation step of dissociating the strand nucleic acid with the endocrine disrupting substance. 2. In the mixing step, a solution containing the nucleic acid mixture is supplied to an affinity column filled with a filler to which an endocrine disrupting substance or a derivative thereof is bound, and in the complex forming step, the endocrine disrupting substance is supplied. And forming a complex of the single-stranded nucleic acid capable of binding to the endocrine disrupting substance or a derivative thereof, and in the separation step, supplying a washing solution to the affinity column to release the complex into free endocrine disrupting substance. Separating from the substance and free single-stranded nucleic acid, in the dissociation step, supplying an eluate for dissociating the complex into the single-stranded nucleic acid and the endocrine disrupting substance to the affinity column, 2. The method according to claim 1, wherein the nucleic acid is dissociated into a single-stranded nucleic acid and the endocrine disrupting substance, and a single-stranded nucleic acid capable of binding to the endocrine disrupting substance is recovered.
2. A method for obtaining a single-stranded nucleic acid that binds to an endocrine disrupting substance according to item 1. 3. The method for obtaining a single-stranded nucleic acid that binds to an endocrine disrupting substance according to claim 1, wherein the single-stranded nucleic acid is a single-stranded DNA. 4. The method for obtaining a single-stranded nucleic acid which binds to an endocrine disrupting substance according to claim 1, wherein the endocrine disrupting substance is an endocrine disrupting substance having a benzene ring structure. 5. The method for obtaining a single-stranded nucleic acid binding to an endocrine disrupting substance according to claim 4, wherein the endocrine disrupting substance having a benzene ring structure is a dioxin. 6. An endocrine disrupting substance is mixed with a nucleic acid mixture comprising a large number of single-stranded nucleic acids having different base sequences from each other, and the single-stranded nucleic acid having a binding ability to the endocrine disrupting substance is mixed with the single-stranded nucleic acid. After forming a complex with the endocrine disrupting substance, the complex is separated from free endocrine disrupting substance and free single-stranded nucleic acid, and the complex is further separated from the single-stranded nucleic acid and the endocrine disrupting substance. A single-stranded nucleic acid which binds to an endocrine disrupting substance obtained by dissociation into a nucleic acid. 7. A solution containing the nucleic acid mixture is supplied to an affinity column packed with a filler to which an endocrine disrupting substance or a derivative thereof is bound, and a single-stranded nucleic acid capable of binding to the endocrine disrupting substance is provided. After forming a complex with the endocrine disrupting substance or a derivative thereof, a washing solution for separating a free endocrine disrupting substance and a free single-stranded nucleic acid is supplied to the affinity column, and the complex is further purified. The endocrine disrupting substance is bound to the endocrine disrupting substance according to claim 6, which is obtained by dissociating the complex into the single-stranded nucleic acid and the endocrine disrupting substance by supplying an eluate for dissociating the endocrine disrupting substance and the single-stranded nucleic acid. Single-stranded nucleic acid. 8. The single-stranded nucleic acid that binds to an endocrine disrupting substance according to claim 6, wherein the single-stranded nucleic acid is a single-stranded DNA. 9. The single-stranded nucleic acid that binds to an endocrine disrupting substance according to any one of claims 6 to 8, wherein the endocrine disrupting substance is an endocrine disrupting substance having a benzene ring structure. 10. The single-stranded nucleic acid binding to an endocrine disrupting substance according to claim 9, wherein the endocrine disrupting substance having a benzene ring structure is a dioxin.