JP2010158237A - Thyroglobulin-binding aptamer and method for producing aptamer polymer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a thyroglobulin-binding aptamer useful as a means for quickly and simply detecting thyroglobulin, and to provide a means capable of improving the binding ability of the aptamer. <P>SOLUTION: It was succeeded to obtain the thyroglobulin-binding aptamer by SELEX method comprising a gel shift method for binding a target molecule of thyroglobulin to ssDNA library in a liquid phase and then separating and recovering the product by electrophoresis, and an aptamer blotting method for binding the thyroglobulin in the coexistence of a competing protein on a solid phase and then recovering ssDNA bound to the thyroglobulin, and a base sequence thereof was determined. Also, a method for polymerizing the aptamer through a connected molecule such as avidin-biotin was developed. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to aptamers that bind to thyroglobulin and methods for multimerizing aptamers.

Thyroglobulin is an iodoprotein that exists in large amounts in the thyroid gland and is used in the clinical and diagnostic fields as a marker for thyroid diseases. For measurement of thyroglobulin, an antibody is generally used, and is mainly performed by an immunoradiometric assay (IRMA). IRMA is a detection system that uses two antibodies that recognize different epitopes on the thyroglobulin molecule. In the detection system, an antibody Ab ^* labeled with a radioisotope and an immobilized antibody Ab are used to form an Ab ^* -Tg-Ab complex, and its radioactivity is measured. Determine the concentration. As other methods, an enzyme that is not a radioisotope is modified with an antibody to detect the activity of the enzyme, an chemiluminometric assay (ICML) that is detected by chemiluminescence, or the like is also used.

  Since an antibody does not emit a signal when bound to a target, two types of antibodies that bind to different portions of the target molecule are required when used for immunoassay. It is difficult to produce two types of antibodies that bind to different parts of one target molecule, and in the first place, it takes time and labor to produce antibodies, and the price is expensive.

Further, as described above, in the detection method using an antibody, thyroglobulin binds to an immobilized capture antibody Ab, and further, the detection antibody Ab ^* binds to the thyroglobulin, and Ab ^* -Tg- When the Ab complex is formed, the presence of thyroglobulin is detected by the radioactivity of Ab ^* . Therefore, an operation of B / F separation for removing the unlabeled free labeled antibody Ab ^* is essential, and it cannot be said to be a simple system.

  On the other hand, aptamers that are oligonucleotides that specifically bind to arbitrary molecules are known. Since aptamers can be chemically synthesized using a commercially available nucleic acid synthesizer, they are much cheaper than specific antibodies and can be easily modified. Therefore, aptamers are expected to be applied as sensing elements. Aptamers that specifically bind to a desired target molecule can be produced by a method called SELEX (Systematic Evolution of Ligands by EXponential Enrichment) (Non-patent Document 1). In this method, a target molecule is immobilized on a carrier, a nucleic acid library consisting of nucleic acids having a large number of random base sequences is added to the target molecule, nucleic acids that bind to the target molecule are recovered, and this is amplified by PCR. Again, the target molecule is added to the immobilized carrier. By repeating this step about 5 to 10 times, the aptamer having high binding power to the target molecule is concentrated, the base sequence thereof is determined, and the aptamer that recognizes the target molecule is obtained. The nucleic acid library can be easily prepared by binding nucleotides at random using an automatic nucleic acid synthesizer. Thus, an aptamer that specifically binds to an arbitrary target substance can be produced by a method that actively uses chance by using a nucleic acid library having a random base sequence.

  As a disease marker detection technique that does not require a B / F separation operation, the inventors of the present application link a recognition aptamer that recognizes a target molecule and an enzyme-controlled aptamer that binds to an enzyme and changes the activity of the enzyme. Thus, AES (Aptameric Enzyme Subunit), which is a sensing technique using aptamers as enzyme subunits, is constructed (Patent Documents 1 and 2). The detection principle is that when the molecule to be measured is present, the molecule binds to the recognition aptamer, which causes a change in the structure of the enzyme-controlled aptamer linked to it, resulting in the activity of the enzyme contained in AES. Therefore, the target molecule is detected by measuring the change in its activity. The advantage of this detection method is that, unlike the detection by ELISA, the binding of the target molecule is detected directly as a signal of the enzyme activity, so that rapid and simple detection without requiring B / F separation is possible. It is done. In addition, once an aptamer that inhibits enzyme activity is acquired, an aptamer that binds to the target molecule to be detected can be arbitrarily selected to detect various target molecules. Various aptamers that inhibit such enzyme activity are already known. Furthermore, aptamers are easier to make and less expensive than antibodies.

  If an aptamer that binds to thyroglobulin can be obtained, the above-described detection system using AES can be constructed, which can contribute to the diagnosis of thyroid diseases. However, aptamers that bind to thyroglobulin have not yet been obtained. Since the aptamer creation method is a method that actively uses chance as described above, whether or not an aptamer having a high binding ability to a target substance can be obtained must actually be subjected to enormous experiments. do not know. Once created and its base sequence is clarified, it can be easily prepared by conventional methods. However, enormous experimentation and trial and error are required for creation.

  In addition, when an aptamer having a certain binding ability is obtained, if there is a simple means that can further enhance the binding ability of the aptamer to the target molecule, it is far more than creating a new aptamer from the beginning. It is useful to save time and cost. As a means for enhancing aptamer binding ability, a method is known in which aptamer molecules are appropriately linked via a linker, and a plurality of aptamer molecules are prepared and used as a single molecule of polynucleotide (Non-patent Document 2). No other useful means are known.

International Publication WO / 2005/049826 International Publication WO / 2007/032359 Publication

Tuerk, C. and Gold L. (1990), Science, 249, 505-510 Sensors, 8, p.1090-1098 (2008)

  Accordingly, an object of the present invention is to provide an aptamer that binds to thyroglobulin, which is useful as a rapid and simple means for detecting thyroglobulin. A further object of the present invention is to provide a useful means for increasing the affinity of aptamers.

  The inventors of the present invention have made a gel shift method in which thyroglobulin as a target molecule and an ssDNA library are bound in a liquid phase and then separated and recovered by electrophoresis, and thyroglobulin and thyroglobulin in the presence of a competing protein on a solid phase. We succeeded in obtaining aptamers that bind to thyroglobulin by the SELEX method combined with the aptamer blotting method that recovers ssDNA bound to thyroglobulin after binding. Furthermore, by using a biotin-avidin system and linking aptamers modified with biotin via avidin, the aptamer can be easily multimerized, and the aptamer thus multimerized has improved affinity for the target molecule. As a result, the present invention has been completed.

  That is, the present invention provides a thyroglobulin-binding aptamer comprising any of the following polynucleotides (a) to (d): (a) any one of SEQ ID NOs: 8 to 12, 26 and 31 in the sequence listing (B) a polynucleotide having the ability to substitute, delete and / or insert one or several bases and to bind to thyroglobulin in the polynucleotide of (a) (C) a polynucleotide comprising a structural region containing at least one stem-loop structural unit present in the polynucleotide of (a) or (b) and having the ability to bind to thyroglobulin; (d) (a) A polynucleotide comprising the polynucleotide of any one of (c) as a partial region and having the ability to bind to thyroglobulin. The present invention also provides an aptamer multimer having a structure in which a plurality of aptamers are linked via a linking molecule. Furthermore, the present invention provides an aptamer multimer comprising contacting an aptamer to which one of a set of linking molecules is bound with the other of the set of linking molecules and linking the aptamer by a bond between the linking molecules. A manufacturing method is provided. Furthermore, the present invention provides an aptamer multimer preparation kit containing one or more aptamers and a linking molecule.

  The present invention provides for the first time an aptamer that binds to thyroglobulin. Since the aptamer of the present invention has high affinity and specificity for thyroglobulin, it contributes to rapid and simple measurement of thyroglobulin. The present invention also provides for the first time an aptamer multimerization method using a linking molecule such as biotin-avidin. In this field, the use of biotin-avidin for the purpose of binding an aptamer to some other molecule is a common technique, but it is not known to be used for aptamer multimerization. According to this multimerization method, multimerization is possible only by mixing an aptamer to which one of the linking molecule sets is bound and the other of the linking molecule set, and this is a very simple and highly versatile technique. For example, when the aptamer target molecule is a molecule in the form of a homomultimer, the aptamer can be multimerized using the same aptamer molecule to enhance the binding ability. Alternatively, it is also easy to multimerize multiple types of aptamers that bind to different sites on one target molecule, so that aptamer binding is possible regardless of the target molecule form (whether it is a homomultimer or not). It becomes possible to improve performance.

In Example 3, the result of having evaluated the binding ability by the aptamer blotting method about 37 aptamers by which stable secondary structure is estimated is shown. In Example 4, the result of having evaluated the binding ability of the aptamer by the gel shift assay is shown. The arrows in the figure are aptamers bound to thyroglobulin (a phenomenon in which the molecular weight increases by forming a complex with thyroglobulin and the migration position shifts from the original position. This is the fluorescence of FITC modified to DNA. .) In Example 5, the result of having evaluated specificity by the aptamer blotting method in the presence of various competitive proteins is shown. C: C-reactive protein, V: VEGF ₁₆₅ , G: PQQGDH, T: thyroglobulin. The secondary structure prediction figure by m-fold (brand name) of TgA7 (sequence number 8) is shown. The secondary structure prediction figure by m-fold (brand name) of TgA8 (sequence number 9) is shown. The secondary structure prediction figure by m-fold (brand name) of TgA9 (sequence number 10) is shown. The secondary structure prediction figure by m-fold (brand name) of TgA10 (sequence number 11) is shown. The secondary structure prediction figure by m-fold (brand name) of TgA11 (sequence number 12) is shown. The secondary structure prediction figure by m-fold (brand name) of TgA25 (sequence number 26) is shown. The secondary structure prediction figure by m-fold (brand name) of TgA30 (sequence number 31) is shown. In Example 6, the result of having evaluated the binding ability of the aptamer by SPR is shown. (A) SPR sensorgram of each aptamer, (B) Dissociation constant of each aptamer. The structure of the truncated mutant constructed in Example 7 (region extracted from the original aptamer structure) is shown. The result of having evaluated the binding ability of the truncated mutant constructed in Example 7 by the aptamer blotting method is shown. The result of having evaluated the competitive relationship of the aptamer in Example 8 by the aptamer blotting method is shown. The secondary structure prediction figure by m-fold (brand name) of the dimer aptamer TgA25-7TL (sequence number 43) constructed | assembled in Example 9 is shown. In Example 9, the dimer aptamer TgA25-7TL and the monomeric TgA25 were each evaluated for their ability to bind to thyroglobulin. (A) Results for monomer TgA25, (B) Results for dimer TgA25-7TL. It is a schematic diagram explaining the multimerization of aptamer using a biotin-avidin system. In Example 10, the result of having examined the multimerization of aptamer TgA25 using the biotin-avidin system is shown. (A) shows the results of evaluating the binding ability of multimerized TgA25 by the aptamer blotting method. (B) shows the results of examining the efficiency of multimerization by electrophoresis of multimerized TgA25.

The thyroglobulin-binding aptamer of the present invention consists of any one of the following polynucleotides (a) to (d).
(a) A polynucleotide comprising the base sequence represented by any of SEQ ID NOs: 8 to 12, 26 and 31 in the sequence listing.
(b) A polynucleotide in which one or several bases are substituted, deleted and / or inserted in the polynucleotide of (a) and capable of binding to thyroglobulin.
(c) A polynucleotide comprising a structural region containing at least one stem-loop structural unit present in the polynucleotide of (a) or (b) and capable of binding to thyroglobulin.
(d) A polynucleotide comprising the polynucleotide of any one of (a) to (c) as a partial region and capable of binding to thyroglobulin.

  The polynucleotide may be DNA or RNA, or an artificial nucleic acid such as PNA, but is preferably DNA that is chemically stable and easy to perform automatic chemical synthesis.

  The nucleotide sequences shown in SEQ ID NOs: 2 to 38 in the sequence listing are obtained by screening a single-stranded DNA (ssDNA) library (SEQ ID NO: 1) containing a random region of 30 mer (“mer” indicates the number of nucleotide residues). Among the obtained ssDNA, it is the base sequence of ssDNA that is considered to have a stable structure from the prediction of secondary structure. All of the ssDNAs composed of these base sequences showed binding to thyroglobulin in the evaluation of binding ability by the aptamer blotting method (see Examples below). Among them, the ssDNA consisting of the nucleotide sequences shown in SEQ ID NOs: 8 to 12, 26 and 31 is considered to have high specificity to thyroglobulin with almost no binding to competitive proteins in the evaluation by the aptamer blotting method. Therefore, a preferred example of the aptamer of the present invention includes a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 8 to 12, 26 and 31 ((a) above). Among these, aptamers consisting of the base sequences shown in SEQ ID NOs: 8, 9, 11, 12, and 26, which have been confirmed to have high specificity even in evaluation by SPR, are more preferred examples.

  The secondary structure formed by the aptamer under a predetermined condition (folding condition) can be easily determined by a conventional method using a computer. Various programs used for predicting the secondary structure of nucleic acids are known. For example, m-fold (trade name, Nucleic Acids Res. 31 (13 ), 3406-15, (2003), which can be downloaded from The Bioinformatics Center at Rensselaer and Wadsworth website), but is not limited thereto. The secondary structure diagrams shown in FIGS. 4 to 10 are secondary structure prediction diagrams based on the aptamer m-fold (trade name) consisting of the base sequences shown in SEQ ID NOs: 8, 9, 10, 11, 12, 26 and 31. is there.

The “folding condition” is a condition in which a part of complementary regions of one molecule of aptamer forms a stem part consisting of a double strand by base pairing within the molecule. It is also a usage condition. Usually, it is in an aqueous buffer solution having a predetermined salt concentration at room temperature and optionally containing a surfactant. For example, in addition to TBS (10-20 mM Tris-HCl, 100-150 mM NaCl, 0-5 mM KCl, pH 7.0) and TBST (TBS containing about 0.05 v / v% Tween 20), 10 mM MOPS and 1 mM CaCl ₂ A buffer solution such as an aqueous solution containing can be used. After heat denaturation by heating to about 95 ° C in these buffers, aptamer molecules can be folded by gradually cooling to room temperature (over about 30 minutes if the amount is about 100 μL). . In the present specification, the term “folding” includes not only the formation of a base pair within a molecule of an aptamer but also the formation of a base pair between molecules of a plurality of polynucleotide molecules. Used to mean For example, as will be described later, when preparing a thyroglobulin measurement reagent using a known AES method, the polynucleotide portion of the reagent can be composed of two molecules of the polynucleotide. In this case, the reagent is subjected to the folding conditions described above. In addition, two polynucleotides form a unique three-dimensional structure by forming base pairs within and / or between molecules.

  If the aptamer has the same positional relationship and size of the stem part and the loop part in its three-dimensional structure, it can usually exhibit the binding ability to the target molecule as well. For example, even if a small number of bases are deleted from the terminal, the same binding ability as the original aptamer can be maintained. The base forming the stem part may have a base sequence in which the positions of the bases to be paired with each other are replaced with each other, or the base pair to be paired may be replaced with, for example, an at-t pair to a g-c pair. As for the base forming the loop part, other base sequences may be adopted as long as a loop of the same size is formed at that position. In addition, if the region is not important for aptamer binding ability, even if a small number of bases are inserted, the same binding ability as that of the original aptamer can usually be maintained. Accordingly, in the polynucleotide (a), one or several (preferably 1 or 2) bases are substituted, deleted and / or inserted as exemplified above (the above (b) As long as it has the ability to bind to thyroglobulin, it is also included in the scope of the present invention. The secondary structure of the polynucleotide (b) may be confirmed anew using a known nucleic acid structure prediction program.

  Whether or not a polynucleotide has the ability to bind to thyroglobulin can be easily evaluated by, for example, the aptamer blotting method described in the Examples below. Specifically, for example, thyroglobulin and any other protein (competitive protein) are immobilized on a support such as a nitrocellulose membrane by a conventional method, and the protein-immobilized support and aptamer are TBS or the like. The ability of the aptamer to bind to thyroglobulin can be evaluated by examining the extent of aptamer molecules bound to each of thyroglobulin and the competitor protein. A polynucleotide having a greater amount of binding to thyroglobulin than the amount of binding to a competitor protein can be evaluated as having a preferred binding ability to thyroglobulin. In particular, a polynucleotide that hardly binds to a competitive protein has high specificity for thyroglobulin and is preferable as the aptamer of the present invention. The competitive protein is not particularly limited, and for example, PQQGDH (pyrroloquinoline quinone glucose dehydrogenase) used in the following examples can be used. The amount of aptamer molecule bound can be determined, for example, by labeling the aptamer molecule with biotin, FITC or the like in advance, and performing an immunoassay by a conventional method using an antibody against the labeled substance after reaction with the protein-immobilized support. The amount of aptamer binding can be examined.

  In aptamers, usually, three-dimensional structural parts such as stem loops and guanine quartets play an important role in aptamer activity. All of the aptamers of the present invention have one or several stem loop structures. Therefore, even if the single-stranded structure region of the molecular end is deleted and only the stem loop structure portion is taken out, the original aptamer The ability to bind to a target molecule can be exerted in the same manner as described above. As long as such a polynucleotide comprising a structural region containing at least one stem-loop structural unit present in the polynucleotide (a) or (b) has the ability to bind to thyroglobulin, Included in the range (above (c)).

  The “stem loop structural unit” as used herein refers to a group of stem loop structures existing at one place on the single-stranded structure serving as the main chain. For example, in the example of SEQ ID NO: 8 (FIG. 4), the stem loop structure formed by the 3 nt to 28 nt region and the stem loop structure formed by the 32 nt to 40 nt region are each one unit stem loop structure (note that "X nt" represents the Xth base from the 5 'end). “Structural region” refers to a series of partial regions on a molecule of a polynucleotide containing at least one stem-loop structural unit. The polypeptide comprising the structural region may contain 1 to several bases of the single-stranded structural region in the original polynucleotide in the terminal region.

  The polynucleotide (c) will be described below using SEQ ID NO: 8 (FIG. 4) as an example. The polynucleotide consisting of the base sequence of SEQ ID NO: 8 has a 2-unit stem loop structure (3 nt to 28 nt, 32 nt to 40 nt). Accordingly, examples of one containing a stem loop structure include a polynucleotide consisting of 3 nt to 28 nt, a polynucleotide consisting of 32 nt to 40 nt, and the like. Since one to several bases of the single-stranded structure region in the original polynucleotide (SEQ ID NO: 8) may be included, for example, a polynucleotide comprising 1 nt to 31 nt is also included. Examples of all of the stem loop structural units include a polynucleotide consisting of 3 nt to 40 nt, a polynucleotide consisting of 1 nt to 49 nt, and the like.

  The polynucleotide (c) is preferably composed of a structural region containing at least one stem-loop structural unit present in a polynucleotide comprising the base sequence shown in SEQ ID NO: 8 or 11. In particular, the nucleotide sequence shown in SEQ ID NO: 39 or 40 is preferred.

  In addition, even if an arbitrary base sequence is added to one or both ends of the aptamer, as long as the positional relationship and size of the stem part and the loop part are equal in the aptamer region, the aptamer has the ability to bind to the target molecule in the same manner as the original aptamer. Can demonstrate. For example, in the known AES constructed by the inventors of the present application (Patent Document 1), even when aptamers that bind to different target molecules are linked to each other, the respective aptamer activities (binding ability to the target molecule and target If the molecule is an enzyme, it exhibits the ability to increase or decrease the activity of the enzyme. This indicates that the original binding ability can be maintained even if an arbitrary sequence is added to the end of the aptamer. Therefore, even if an arbitrary sequence is added to one or both ends of the above-mentioned polynucleotides (a) to (c), binding to thyroglobulin is the same as the original polynucleotides (a) to (c). The ability can be demonstrated. Such a polynucleotide containing the above-mentioned polynucleotides (a) to (c) as a partial region is also included in the scope of the present invention as long as it has an ability to bind to thyroglobulin (the above (d)).

  The arbitrary sequence to be added may be any nucleotide sequence as long as it does not adversely affect the ability to bind thyroglobulin, such as completely losing the ability of the aptamer to bind to thyroglobulin, It is not limited. However, if the total length of the aptamer is too long, it takes time and cost for synthesis. Therefore, the size of the additional sequence is generally about 40 bases or less in total, preferably about 10 bases or less, more preferably about 1 to 2 bases. However, this is not the case when the additional sequence is an aptamer sequence, and the size is determined according to the chain length of the added aptamer sequence. The size of the total aptamer molecule is usually about 200 mer or less, preferably about 150 mer or less. The lower limit of the aptamer size is not particularly limited, but is usually about 15 mer or more.

  Among the polynucleotides of (d) above, examples of the polynucleotide to which an arbitrary aptamer sequence is added include, for example, a polynucleotide having a structure in which several polynucleotides (a) to (c) are linked (hereinafter, for convenience). And a polynucleotide obtained by adding an aptamer sequence targeting a substance other than thyroglobulin to the polynucleotides (a) to (c).

  Techniques for using aptamers in multimerization are known (Sensors, 8, p. 1090-1098 (2008)). Also in the present invention, several aptamers (a) to (c) (hereinafter sometimes referred to as “monomer” for convenience) can be linked and used. In particular, linking aptamers that bind to different sites on thyroglobulin can increase the ability to bind to thyroglobulin than when individual aptamers (hereinafter sometimes referred to as “monomers”) are used alone. it can. Whether or not the aptamer competes for binding to thyroglobulin can be determined by, for example, a competition experiment described in Example 8 below. Specifically, for example, different aptamers with different labels (for example, biotin label and FITC label) are mixed at various concentration ratios, and this is reacted with thyroglobulin immobilized on a solid phase, and one label ( For example, FITC) is detected and the binding amount of one aptamer is measured. The binding amount of the other aptamer is measured in the same manner under the condition where the label is replaced. If the detected FITC signal does not decrease even under a high concentration ratio of biotin-labeled aptamer, it is highly likely that the aptamers are bound to different sites on thyroglobulin. Therefore, when aptamers are linked in such a combination, the binding ability can be improved as compared with the case of using them as monomers. For example, in the present invention, since the combination of SEQ ID NO: 9 (TgA8) and SEQ ID NO: 26 (TgA25) is considered not to compete with each other (see Example 8 below), it is preferable as a combination for producing a multimer. In addition, as described in Example 9 below, the ability to bind to thyroglobulin can be improved by linking SEQ ID NO: 26 (TgA25) and SEQ ID NO: 43 (TgA7T). Since thyroglobulin is a protein in the form of a homodimer, there are two sites to which the same aptamer binds. Therefore, even if the same aptamer is linked, the binding ability can be improved.

  When the aptamer is multimerized, it may have a structure in which monomers are directly linked, but can be linked via a linker if necessary. For example, if a stem loop structure exists in the 3 ′ terminal region of the aptamer monomer on the 5 ′ upstream side and / or the 5 ′ terminal region of the aptamer monomer on the downstream side of 3 ′, the stem loop can be directly linked. In such a case, it is preferable that the structure is connected via a linker because the structure may not be formed desirably. In addition, when the site recognized by the monomer is at a distant position on the thyroglobulin, a linker with a sufficient chain length is inserted so that each multimerized aptamer can bind to each recognition site. Then, the binding ability of the aptamer can be preferably improved (Sensors, 8, p. 1090-1098 (2008), supra). The chain length of the linker can be appropriately determined and is not particularly limited, but is usually about 1 mer to 20 mer, preferably about 5 mer to 15 mer. Although it does not specifically limit, As a linker, what consists only of adenine or thymine is preferable.

  The number of monomers to be linked is about several, preferably two.

  Among the polynucleotides in (d), examples of the polynucleotide to which an aptamer sequence targeting a substance other than thyroglobulin is added include an enzyme-controlled polynucleotide using AES described later.

  The aptamer of the present invention is folded under known folding conditions by a method known per se, and can be used for measurement (detection, quantification, semi-quantification) of the target molecule thyroglobulin. As a test sample, a biopsy tissue of thyroid gland, a body fluid such as serum or plasma, or a diluted product thereof can be used. The measurement of thyroglobulin in a test sample using the aptamer of the present invention can be performed by a well-known ordinary method using an aptamer. For example, an immunoassay method (immunochromatography or the like) using the aptamer of the present invention instead of an antibody. ELISA (Enzyme linked Immunosorbent Assay)). In particular, the combination of the aptamer monomer TgA8 consisting of the base sequence shown in SEQ ID NO: 9 and the aptamer monomer TgA25 consisting of the base sequence shown in SEQ ID NO: 26 has little influence on the binding to the target molecule thyroglobulin. However (see Example 8), if such a combination of aptamers with little adverse effect on each other, a detection system similar to the sandwich method using two types of antibodies that bind to two different epitopes can be constructed. The measurement of thyroglobulin can also be performed by known methods such as aptamer blotting and surface plasmon resonance (SPR) specifically described in the following examples.

  Moreover, if the thyroglobulin binding aptamer of this invention is used, the thyroglobulin measuring reagent using the aptamer enzyme subunit (AES) (patent document 1) which the present inventors developed can be prepared. The polynucleotide portion in the reagent binds to a thyroglobulin recognition aptamer site (hereinafter sometimes simply referred to as “recognition aptamer site”) including the thyroglobulin-binding aptamer of the present invention, and the activity of the enzyme is reduced. A polynucleotide comprising an enzyme-controlled aptamer site having the ability to change, wherein the ability of the enzyme-controlled aptamer site to change the activity of the enzyme is altered by binding of thyroglobulin to the thyroglobulin recognition aptamer site (Hereinafter, this polynucleotide is referred to as “enzyme-controlled polynucleotide”).

  Note that “changing the activity of the enzyme” means increasing or decreasing the activity of the enzyme as compared with the enzyme in a state where it is not bound to the enzyme-controlled aptamer site. Hereinafter, in this specification, the ability to change such enzyme activity is referred to as “enzyme control ability”, and an aptamer having enzyme control ability is referred to as “enzyme control aptamer”.

  Enzyme-controlled polynucleotides consist of one or two molecules of a polynucleotide chain (hereinafter referred to as “single-molecule enzyme-controlled polynucleotides” consisting of one molecule and “bi-molecular enzyme-controlled polynucleotides consisting of two molecules”). ), The ability of the enzyme-controlled aptamer site to change the enzyme activity is changed by binding of the target substance thyroglobulin to the recognition aptamer site. Changes in the ability of the enzyme-regulated aptamer site to change the enzyme activity can be determined by examining changes in the activity of the enzyme bound to the site. The enzyme bound to the enzyme-controlled aptamer site is in a state where the enzyme activity is increased or decreased due to the action of the site, but in this state, when thyroglobulin binds to the recognition aptamer site, the enzyme activity further changes. (That is, the “ability to change enzyme activity” of the enzyme-regulated aptamer site changes). Usually, the enzyme activity of the enzyme bound to the enzyme-regulated aptamer site returns to the original activity from the state where the activity is increased or decreased (that is, the “ability to change enzyme activity” of the enzyme-regulated aptamer site). Decreases). The details of the principle of reducing the ability to change enzyme activity are unknown, but the following may be considered. That is, it is considered that the steric structure of the enzyme control polynucleotide is changed by the binding of thyroglobulin to the recognition aptamer site, and the enzyme is detached from the enzyme control aptamer site.

  Specifically, for example, when the enzyme-controlled aptamer site has the ability to increase the activity of the enzyme, the ability to increase the enzyme activity usually decreases due to the binding of thyroglobulin to the recognition aptamer site, and the enzyme-controlled aptamer The activity of the enzyme bound to the site will be reduced by the binding of thyroglobulin to the recognition aptamer site. In addition, when the enzyme-regulated aptamer site has the ability to reduce the activity of the enzyme, thyroglobulin usually binds to the recognition aptamer site, thereby reducing the ability to reduce the enzyme activity and binding to the enzyme-controlled aptamer site. The activity of the enzyme is increased by the binding of thyroglobulin to the recognition aptamer site. Therefore, if an enzyme-polynucleotide complex prepared by binding an enzyme corresponding to the enzyme-controlled aptamer site of the enzyme-controlled polynucleotide is used, thyroglobulin in a sample can be measured using the change in enzyme activity as an index. Can do.

  The enzyme-controlled aptamer site is configured based on the three-dimensional structure (including a planar secondary structure) of the enzyme-controlled aptamer molecule. The enzyme-regulated aptamer molecule to be employed is not particularly limited, and any aptamer having the enzyme-controlling ability described above may be used. As such an enzyme-controlled aptamer, for example, as described in Patent Document 1, a thrombin-inhibiting aptamer is known.

  Here, “configured based on the three-dimensional structure of the aptamer molecule” means that the three-dimensional structure taken by the enzyme-controlled aptamer moiety in the enzyme-controlled polynucleotide is taken by the enzyme-controlled aptamer molecule consisting of one molecule. It is configured to approximate. Therefore, the region constituting the enzyme-regulated aptamer site does not necessarily have to be present as one continuous region in the polynucleotide chain constituting the enzyme-regulated polynucleotide, but in one molecule or two molecules of the polynucleotide chain. It may be divided and present. Even if they are disrupted, the regions are combined by hybridizing the polynucleotide strands under folding conditions and forming base pairs at appropriate sites within and / or between the molecules. As long as it is possible to form a three-dimensional structure similar to the three-dimensional structure of the aptamer molecule, it is allowed as a configuration aspect of the enzyme-controlled aptamer site.

  As the position at which the base sequence of the enzyme-controlled aptamer molecule is divided, any site in the loop structure is preferable. By dividing within the loop structure, even if two molecules of the polynucleotide chain each containing the fragment after the fragmentation are prepared, these polynucleotide chains are subjected to base pairing under folding conditions, so that the guanine quartet structure and the stem part are formed. It is highly possible that the three-dimensional structure of the original aptamer molecule can be reproduced (forms a three-dimensional structure similar to the three-dimensional structure of the original aptamer molecule). The ability to maintain the binding ability and enzyme control ability of the aptamer molecule of this protein is increased (hereinafter, it consists of two polynucleotide chains that can take a three-dimensional structure similar to the original aptamer molecule). Polynucleotides are sometimes referred to as “split aptamers”). When such a split aptamer is applied to an enzyme-controlled aptamer site, the effect of the binding of thyroglobulin to the recognition aptamer site (change in the three-dimensional structure) is efficiently transmitted to the enzyme-controlled aptamer site, and changes in enzyme activity as an index are measured. Since it becomes easy to do, it is preferable.

  In the enzyme-controlled polynucleotide molecule, the position at which the recognition aptamer site is provided is not particularly limited, and may be the terminal part of the enzyme-controlled aptamer structure, or provided so as to be added to the loop structure of the enzyme-controlled aptamer site. Also good. For example, a thyroglobulin-recognizing aptamer site was set in the loop structure of an enzyme-controlled aptamer by linking a thyroglobulin-binding aptamer to the split site side end of one polynucleotide chain of a split aptamer fragmented in the loop structure. A bimolecular enzyme-controlled polynucleotide can be obtained. At this time, when a fragment consisting of a sequence complementary to at least a partial region in the thyroglobulin-binding aptamer sequence is linked to the fragment site side end of the other fragment, the thyroglobulin-binding aptamer and the complementary sequence fragment Hybridize to form a base pair, which is preferable because the three-dimensional structure of the enzyme-controlled aptamer site can be stabilized. The size of the complementary sequence fragment can be selected from any size not more than the same as the full length of the thyroglobulin-binding aptamer, and is not particularly limited. The following) degree. Thus, the bimolecular enzyme-controlled polynucleotide prepared using the enzyme-controlled aptamer fragmented in the loop structure efficiently transmits the structural change of the thyroglobulin recognition aptamer site to the enzyme-controlled aptamer as described later. Therefore, it is preferable to a single molecule enzyme-controlled polynucleotide prepared by linking a recognition aptamer to the end of one molecule of an enzyme-controlled aptamer.

  The enzyme-controlled aptamer site and the thyroglobulin recognition aptamer site may be linked directly or via a linker. For example, when a monomolecular enzyme-controlled polynucleotide is prepared by ligating an enzyme-controlled aptamer and a thyroglobulin-binding aptamer at each terminal portion, it is appropriate from the viewpoint of maintaining the three-dimensional structure in the vicinity of the linking portion. They may be linked via a linker having a chain length. In addition, when preparing a bimolecular enzyme-controlled polynucleotide using a split aptamer split at the loop portion, the thyroglobulin-binding aptamer can be linked to the end of the split portion via a linker. When a linker is interposed, the complementary sequence fragment may include a region complementary to the linker. The complementary sequence fragments may be linked via a linker. The linker used here is not particularly limited, as is the case with the linker that can be used in the multimeric aptamer, but it is preferably composed of only adenine or only thymine. The chain length of the linker is such that when an enzyme-controlled aptamer and a thyroglobulin-binding aptamer are linked at their respective terminal portions, a sufficient amount of space can be secured for each aptamer to take the desired three-dimensional structure. The length is not particularly limited and is usually about 1 mer to 20 mer, preferably about 5 mer to 15 mer. Moreover, when providing a thyroglobulin recognition aptamer site | part in a loop part using a split aptamer, although it does not specifically limit, Usually, it is about 1mer-10mer, Preferably it is about 1mer-5mer.

  The thyroglobulin recognition aptamer site may consist of only a thyroglobulin-binding aptamer sequence, but may contain a small number of bases (about 1 to several) at the end. For example, when the enzyme-regulated polynucleotide is bimolecular, even if one to several irrelevant bases are added to the free terminal portion that is not linked to the split aptamer among the thyroglobulin-binding aptamers, There is no hindrance to the function of the enzyme-controlled polynucleotide.

  A thyroglobulin measurement reagent using AES contains the enzyme-controlled polynucleotide described above and an enzyme bound to an enzyme-controlled aptamer site in the polynucleotide. As described above, the enzyme activity of the enzyme changes due to the binding of thyroglobulin to the recognition aptamer site in the reagent. Specifically, when the enzyme-controlled aptamer employed in the enzyme-controlled polynucleotide has an action to increase the activity of the enzyme, the thyroglobulin measurement reagent has the enzyme activity due to the binding of thyroglobulin to the recognition aptamer site. descend. On the other hand, when the enzyme-controlled aptamer employed has an action of reducing the activity of the enzyme, the activity of the enzyme increases in the thyroglobulin measuring reagent due to the binding of thyroglobulin to the recognition aptamer site. Therefore, thyroglobulin in a specimen can be measured by bringing a specimen that can contain thyroglobulin into contact with the reagent of the present invention and examining the change in enzyme activity of the reagent. That is, a step of contacting the thyroglobulin measuring reagent with a sample that may contain thyroglobulin, a step of measuring a change in enzyme activity of the enzyme bound to the enzyme-controlled aptamer site, and using the change as an index in the sample A method for measuring thyroglobulin, comprising the step of measuring thyroglobulin.

  The change in enzyme activity can be measured, for example, by separately preparing a reagent that is not brought into contact with the specimen and a reagent that is brought into contact with the specimen, and comparing the enzymatic activities of the two. The enzyme activity may be measured before and after contact with the sample to compare the two, or the enzyme activity from before contact to the sample until after contact may be continuously measured to measure the change. Also good. If the enzyme activity is examined using a sample with a known thyroglobulin concentration using the thyroglobulin measuring reagent and a calibration curve is prepared, thyroglobulin in the sample can be quantified.

  The enzyme activity can be easily measured by a conventional method based on the common general technical knowledge in this field depending on the type of enzyme employed. For example, when thrombin is used as an enzyme, thrombin activity can be measured by measuring the absorbance (410 nm) of released p-nitroaniline using N-benzoyl-Phe-Val-Arg-p-nitroanilide as a substrate. . Alternatively, fibrinogen can be used to measure the coagulation time of a fibrinogen solution initiated by fibrinogen cleavage by thrombin, and to measure thrombin activity using the coagulation time as an index. The solidification of the solution can be measured by a well-known conventional method, for example, a method of measuring a change in refractive index by a spectroscopic method, or adding a metal sphere to the solution to stop the movement accompanying the solidification of the solution. Although the method of observing etc. is mentioned, it is not limited to these. Alternatively, since a measuring means such as a glucose sensor for measuring a target substance by electrochemically measuring a change in enzyme activity is known, the enzyme-controlled aptamer and enzyme employed in the thyroglobulin measuring reagent described above are appropriately used. By selection, such electrochemical measurement means can be applied.

  The aptamer of the present invention and the enzyme-controlled polynucleotide described above can be easily prepared by a conventional method using a commercially available nucleic acid synthesizer. The complex of enzyme and polynucleotide (enzyme-polynucleotide complex) contained in the thyroglobulin assay reagent is, for example, an enzyme to be bound to the enzyme control site of the polynucleotide after folding the enzyme control polynucleotide. And can be easily prepared by incubating at room temperature for about 5 to 30 minutes. In addition, when the enzyme to be bound is one that requires a coenzyme or a metal in order to exhibit activity, it is not particularly limited. Mix with control polynucleotide.

  The thyroglobulin assay reagent may consist only of the enzyme-polynucleotide complex described above, or may contain other components useful for stabilizing the complex. For example, it may be in the form of a solution in which only the complex is dissolved in an appropriate buffer, or a component useful for stabilizing the complex may be further included in the solution. Alternatively, the thyroglobulin measurement reagent can be provided in the form of a set of reagents separately containing the enzyme control polynucleotide and the enzyme to be bound thereto. In this case, the user can prepare a thyroglobulin measuring reagent by mixing each reagent at the time of use.

  In addition, the inventors of the present application have invented a novel method different from the above-described method for preparing aptamer as a single molecule polynucleotide by directly connecting aptamer via a linker as appropriate. In the method for producing a linked aptamer of the present invention (hereinafter sometimes referred to as “multimerization method”), aptamers are linked via a linking molecule to form a multimer. Here, the “linking molecule” is a molecule that can exist separately from the aptamer to be linked, and is a molecule capable of directly or indirectly binding a plurality of aptamers to be linked to the molecule itself.

  The linking molecule is preferably a set of two types of molecules that specifically bind to each other. The specific binding here has the same meaning as the specific binding described above for the antibody of the present invention. When using such a set of two types of molecules as a linking molecule, one of the set of linking molecules is bound to the aptamer to be linked. Then, just by bringing the aptamer that binds one side of the linking molecule set and the other side of the linking molecule set into contact, a plurality of aptamers are linked onto the linking molecule by a specific bond between the linking molecules, resulting in a multimer. . A person skilled in the art can easily determine which of the linking molecule sets is bound to the aptamer according to the type of the linking molecule set. Specific examples of the set of linking molecules include a set of biotin and avidin or streptavidin, a set of a zinc finger protein and a region consisting of a base sequence that recognizes and binds the zinc finger, and the like. Any combination of structures that can specifically bind can be used, but is not limited thereto. These specific examples and sets of linking molecules other than these are known in this field, and some of them are commercially available, so they are easily available.

  The multimerization method is not limited to the above-described thyroglobulin-binding aptamer of the present invention, and can be applied to various aptamers. Up to now, aptamers for various target molecules (for example, various proteins, saccharides, lipids, nucleic acids, low molecular compounds, etc.) have been prepared and publicly known, and aptamer preparation methods such as SELEX are also known. Therefore, a new aptamer for a desired target molecule can be produced. Any of them can be linked using the method. Furthermore, any aptamer multimer prepared as a single molecule polynucleotide by the method described above can be further multimerized by a method of multimerization via a linking molecule.

  Hereinafter, the aptamer multimer production method of the present invention will be described by taking as an example the case where a set of biotin and avidin is used as a linking molecule. However, as described above, the method is not limited only to the case where biotin-avidin is used.

  Biotin, one of the linking molecules, is bound to the aptamer to be linked. Methods for modifying biotin on nucleic acids are well known, and binding of biotin to aptamers can be performed as usual. Biotin may be bound to either end of the aptamer molecule. For example, a labeling substance can be bound to the other end from the viewpoint of use as a detection reagent. As a labeling substance, a labeling substance such as a fluorescent label, a chemiluminescent label, an enzyme label or the like usually used for detecting binding in a known immunoassay can be used. It is also possible to prepare aptamers labeled with radiolabeled or DIG labeled nucleotides.

  When an aptamer to which biotin is bound and avidin are brought into contact with each other, biotin binds to avidin, so that the biotin-modified aptamer is linked via avidin. Since avidin has four binding sites for biotin, a maximum of four aptamers can be linked according to the method using a biotin-avidin set. As shown in the examples below, the population of aptamer multimers obtained by this method is a mixture of multimers having different aptamer linkage numbers. The ratio of the biotin-modified aptamer and avidin in contact with each other is not particularly limited and can be appropriately determined by those skilled in the art. However, from the viewpoint of efficient ligation, the biotin-modified aptamer: avidin molar ratio is usually 1: It is selected within the range of 1 to 4: 1. The avidin solution and the biotin-modified aptamer solution may be mixed and placed at 4 ° C. to room temperature for about 15 minutes to 30 minutes. The concentration at the time of mixing avidin and biotin-modified aptamer is not particularly limited, and generally the concentration of avidin in the mixed system may be about 10 nM to 100 μM. For example, when a biotin-modified aptamer of about 80 nM is linked, the concentration of avidin in the system after mixing may be about 10 nM to 100 nM.

  When aptamer multimers are used for target molecule measurement, the aptamer is usually folded under the above-mentioned known folding conditions. In the folding step, the aptamer is heated at a high temperature of about 95 ° C. Since it is not preferable to expose the protein avidin to high temperature, the folding step is preferably performed before contacting the avidin and the biotin-modified aptamer. That is, the biotin-modified aptamer is preferably folded before contact with avidin.

  The aptamers linked by the method of the present invention bind to the same target molecule, but may be only aptamers having the same sequence or two or more aptamers having different sequences. When a plurality of aptamers that bind to different sites with respect to a certain target molecule are obtained, one of the linking molecules may be bound to all of them and brought into contact with the other of the linking molecules for multimerization. When a plurality of aptamers are multimerized, the composition of the aptamer contained in the obtained aptamer multimer can be different for each multimer molecule, but such aptamer multimer population is also within the scope of the present invention. Is included. If the aptamer target molecule is a homomultimeric protein such as thyroglobulin or PQQGDH, there will be multiple identical binding sites, so only aptamers of the same sequence that bind to the same site will be multimerized. Even so, the affinity of the aptamer can be preferably improved. Although it does not specifically limit, The kind of aptamer couple | bonded through a connecting molecule is about 1 type-several types normally.

  For example, when the thyroglobulin-binding aptamer of the present invention is linked by this method, since thyroglobulin is a homodimer, only one of the aptamers of the present invention described above may be linked, or different sites. Two or more types of aptamers that bind to can be linked, and in any case, the affinity of aptamers can be improved. In the following examples, only aptamer TgA25 consisting of the base sequence shown in SEQ ID NO: 26 is linked by biotin modification, but the affinity of the multimer is improved compared to the TgA25 monomer by aptamer blotting. Has been confirmed specifically. Examples of combinations of different aptamers include the combination of SEQ ID NO: 9 (TgA8) and SEQ ID NO: 26 (TgA25), and the combination of SEQ ID NO: 26 (TgA25) and SEQ ID NO: 43 (TgA7T) as described above. However, the present invention is not limited to this.

  Aptamer multimers via linking molecules can also be used for measurement of target molecules by a well-known ordinary method using aptamers, similarly to aptamer multimers and monomers having a single molecular structure. For example, it can be performed by a method according to an immunoassay using the aptamer of the present invention instead of an antibody. For example, a multimer of the thyroglobulin-binding aptamer of the present invention can be used for measurement of thyroglobulin in a test sample such as a biopsy tissue of a thyroid gland, a body fluid such as serum or plasma, or a dilution thereof. . The folding process is performed in an appropriate order depending on the heat resistance of the linking molecule. When using a linking molecule such as a protein that is not desirable to be exposed to high temperatures, or when binding to the linking molecule is thermally unstable, contact the aptamer with one of the linking molecules and the other of the linking molecules. It is preferable to carry out folding before making it happen. When the bond between the linking molecules is stable to heat, folding can be performed after multimerization. Other conditions are the same as those described above for the aptamer having a single molecular structure.

  Aptamers and linking molecules can be provided as kits for producing aptamer multimers. Only one type of aptamer may be used, or two or more types of aptamers that bind to the same target molecule may be included. One or more aptamers that bind to different target molecules may further be included. The linking molecule is preferably a set of two types of molecules that specifically bind to each other. Specific examples of the linking molecule are the same as described above. Further, one of the sets of linking molecules may be in a form bound to an aptamer. Optionally, it may further contain other reagents such as a folding buffer.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Example 1 Search for DNA aptamer targeting thyroglobulin In this example, SELEX was performed 5 rounds on thyroglobulin. A single-stranded DNA random library having a total length of 66mer having an 18mer primer region and a 30mer random region (SEQ ID NO: 1) was used. PCR amplification from the library was carried out by a conventional method using an 18-mer primer set consisting of the same nucleotide sequence as the primer region or its complementary strand (reaction conditions: 95 ° C. 60 seconds, 52 ° C. 60 seconds, 72 ° C. 60 35 cycles of seconds).

  Specific methods are shown below. FITC-modified single-stranded DNA library was prepared to 1 nmol / 100 μL with TBS buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mM KCl), heated at 95 ° C. for 3 minutes, and then room temperature (25 ° C. for 30 minutes). It was made to fold by cooling gradually. The thyroglobulin and the library after folding were mixed using TBS buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mM KCl) so that the final concentrations were 1 μM and 500 μM, respectively, and incubated at room temperature for 1 hour. Then, Native-PAGE was performed with TBE buffer (89 mM Tris-HCl, 89 mM boric acid, 2 mM EDTA) using 6% polyacrylamide gel. The fluorescence of FITC modified to DNA was detected, and the shift of DNA band due to binding with thyroglobulin was confirmed. After CBB staining, the gel at the position where the thyroglobulin band appeared was cut out, DNA was extracted, and PCR amplified to obtain a library for the next round. This was done for 2 rounds.

From the third round, a method called aptamer blotting in which thyroglobulin was immobilized on a nitrocellulose membrane was used. In the aptamer blotting method, PQQGDH having a high pI was used as a competitive protein in order to reduce nonspecific binding to thyroglobulin. Immobilization of thyroglobulin and PQQGDH at 1.5 μg each (thyroglobulin was equivalent to 2.3 pmol and PQQGDH was equivalent to 30 pmol) on a nitrocellulose membrane, followed by blocking with 10% serum at room temperature for 1 hour. After washing with TBST (20 mM Tris-HCl, 150 mM NaCl, 15 mM KCl, 0.05% Tween), the mixture was incubated with 100 μM of the library solution extracted from the third round for 1 hour at room temperature, and washed with TBST. A part of the membrane on which thyroglobulin was immobilized was cut out and treated with phenol chloroform to extract and collect ssDNA bound to thyroglobulin. Using this as a template, PCR amplification was performed, and single-stranded preparation was performed to obtain a library for the next round. Specifically, single strand preparation was performed as follows. That is, 1/10 times the amount of x50 TE buffer and 1/5 times the amount of 5 M NaCl were added to the PCR product, and this solution was added to avidin-immobilized agarose and incubated for 30 minutes. Thereafter, the supernatant was removed and washed twice with Column buffer (30 mM HEPES, 500 mM NaCl, 5 mM EDTA, pH 7.0). After removing the supernatant, 0.15 M NaOH was added and stirred for 10 minutes, and the operation of recovering the supernatant was repeated twice to elute ssDNA. The supernatant containing ssDNA was neutralized with 2M HCl, and ssDNA was recovered by ethanol precipitation.
The above operation was set as 1 round, and a total of 3 rounds (3rd to 5th rounds) were performed. In addition, in each round, in order to confirm the amount of DNA bound to each protein, a membrane for detection was prepared in the same manner as described above, and after incubation with DNA, an anti-FITC antibody modified with HRP was used as a secondary antibody. The amount of DNA bound to each protein was confirmed by chemiluminescence.

  Spots indicating the amount of DNA bound to the thyroglobulin-immobilized portion became darker as the round progressed, and in the fifth round, became equal to or higher than PQQGDH. From this, it is considered that the concentration of single-stranded DNA consisting of a base sequence that binds to thyroglobulin has occurred by the fifth round. In the fifth round, DNA binding was also observed against the competitive protein PQQGDH, but the immobilization amount (pmol) of thyroglobulin and PQQGDH was more than 10 times different. This is not a problem if

Example 2 Sequence Analysis of Single-Stranded DNA that Binds to Thyroglobulin Obtained In this example, the sequence of DNA collected in the fourth round was analyzed.

  A specific method is shown below. The single-stranded DNA collected in the fourth round was PCR amplified and the DNA was purified. Thereafter, TA cloning, transformation and culture were performed, and 100 white colonies obtained were cultured overnight in LB medium, and then the plasmid was extracted. The extracted plasmid was subjected to PCR amplification of the target fragment using primers, and the target fragment was confirmed by colony PCR. The plasmid in which the target fragment was confirmed to be inserted was determined for the base sequence by the dideoxy method.

  As a result of sequence analysis, 60 sequences were obtained. For the obtained 60 sequences, the secondary structure was predicted by m-fold (trade name), and 37 considered to have a stable structure were designated TgA1-37. These base sequences are shown in Table 1. In Table 1, the primer region sequences are omitted.

Example 3 Evaluation of Aptamer Binding Ability by Aptamer Blotting Method In this example, aptamer blotting method (see Example 1) was used for 37 aptamers considered to have the stable structure shown in Example 2. Thus, the binding ability to thyroglobulin was evaluated.

  A specific method is shown below. Thyroglobulin and PQQGDH were immobilized on a nitrocellulose membrane and blocked with 10% serum for 1 hour at room temperature. After washing with TBST, the solution was incubated with a FITC-modified single-stranded DNA solution and further incubated with an anti-FITC antibody, and DNA bound to thyroglobulin was detected by chemiluminescence.

  The detection results of each aptamer for thyroglobulin are shown in FIG. All aptamers bound to thyroglobulin. Some aptamers bound only to thyroglobulin and no binding to competing PQQGDH.

Example 4 Evaluation of Aptamer Binding Ability by Gel Shift Assay In this example, several aptamers that showed strong binding in systems where thyroglobulin was immobilized by the aptamer blotting method, TgA2, 3, 7, 8, 10 , 11, 12, 16, 17, 20 and TgA 22-24, 26-37, the ability to bind to thyroglobulin in a solution system was evaluated using a gel shift assay.

  A specific method is shown below. Thyroglobulin and FITC-modified single-stranded DNA (folded, see Example 1) were mixed using TBS buffer so that the final concentrations were 1 μM and 500 μM, respectively, and incubated for 1 hour. Then, Native-PAGE was performed with TBE buffer using 6% polyacrylamide gel. The fluorescence of FITC modified to DNA was detected, and the shift of DNA band due to binding with thyroglobulin was confirmed.

  The detection results are shown in FIG. In any aptamer, binding to thyroglobulin could be confirmed. Aptamers have also been shown to bind to thyroglobulin in solution systems.

Example 5 Evaluation of Aptamer Specificity In this example, each aptamer TgA1-37 was evaluated for specificity using an aptamer blotting method. Thyroglobulin and PQQGDH, vascular endothelial growth factor 165 (VEGF ₁₆₅ ), and C-reactive protein as competitor proteins were each immobilized on a nitrocellulose membrane in an amount of 4.5 pmol. The membrane was blocked with 10% serum using TBST buffer, and incubated with 100 nM (70 pmol / L) of each aptamer modified with FITC at room temperature for 1 hour. Thereafter, it was incubated with an HRP-modified anti-FITC antibody, and the aptamer bound to the protein was detected by chemiluminescence.

  All aptamers bound to thyroglobulin, but TgA7, 8, 9, 10, 11, 25, 30 hardly bound to the competitor protein (FIG. 3). These aptamers are considered to have some specificity for thyroglobulin. The predicted secondary structures of these aptamers are shown in FIGS. Other aptamers also bound to competitive proteins and were less specific.

Example 6 Evaluation of Aptamer Binding Ability by SPR In this example, TgA7 was found to have high binding ability and specificity for thyroglobulin in Examples 3 and 4 using SPR (surface plasmon resonance method). , 8, 9, 10, 11, 25, 30 were calculated.

  The detailed method is shown below. Each aptamer modified with biotin at the 5 ′ end was folded, and then immobilized on flow cell 1 of Sensor chip SA, and control DNA (5′-ACTAGTTCAGAACTAGTTGCCAGAAAGCTACCTTGACGTCAGGGCCTACTGACC-3 ′: SEQ ID NO: 44) was immobilized on flow cell 2. Thereafter, 100 μl of thyroglobulin prepared at various concentrations was injected, and the interaction between the aptamer and thyroglobulin was observed. Measurement of SPR was performed at room temperature at a flow rate of 10 μl / min or 20 μl / min. Preparation of aptamer and thyroglobulin and SPR immobilization buffer, running buffer was TBS buffer, and regeneration buffer was 0.5% SDS. .

  A value obtained by subtracting the amount of thyroglobulin bound to the control DNA immobilized on the flow cell 2 from the amount of thyroglobulin bound to the aptamer immobilized on the flow cell 1 is defined as ΔRU. When thyroglobulin was injected into each aptamer and the binding at each concentration was observed, an increase in concentration-dependent signal was observed at TgA7, 8, 10, 11, 25 (FIG. 11 (A)). TgA9, 30 gave almost no signal indicating binding to thyroglobulin. Therefore, in TgA7, 8, 10, 11, 25, global fitting was performed using BIAevaluation 3.1 (Biacore) and the respective dissociation constants were calculated (FIG. 11 (B)).

Example 7 Evaluation of Aptamer Truncated Mutant In this example, an aptamer truncated mutant was prepared and the aptamer was minimized.

  Each aptamer is a 66-mer one containing a primer sequence, but it may be minimized by removing a part not involved in binding. A truncated mutant was prepared from the predicted secondary structure of TgA7, 10, 25, and its ability to bind to thyroglobulin was evaluated by the aptamer blotting method. Each truncated mutant is designated as TgA7T, TgA10T, TgA25T1, TgA25T2 (each SEQ ID NO: 39 to 42) (FIG. 12). Immobilizing 1.5 μg of thyroglobulin and PQQGDH on a nitrocellulose membrane, blocking the membrane with 10% serum using TBST buffer, and incubating each aptamer modified with FITC with 100 nM for 1 hour at room temperature . Thereafter, the mixture was incubated with an HRP-modified anti-FITC antibody, and ssDNA bound to the protein was detected by chemiluminescence.

  The detection results of each aptamer for thyroglobulin are shown in FIG. TgA7T and TgA10T were shown to have the ability to bind to thyroglobulin. However, TgA25T1 and TgA25T2 are considered to have low binding ability to thyroglobulin.

Example 8 Aptamer Competition Experiment In this example, aptamers TgA7, 8, 10, 11, 25 having high specificity and affinity and TgA7T, which is a truncated mutant of TgA7, bind aptamers that bind to different regions of thyroglobulin. Combinations were explored by competition studies using aptamer blotting. 4.5 pmol of thyroglobulin was immobilized on a nitrocellulose membrane, and aptamer blotting was performed using a mixed solution of FITC-modified aptamer (final concentration 80 nM) and biotin-modified aptamer (final concentration 0 nM, 80 nM, 800 nM). went. Thyroglobulin and PQQGDH were immobilized on a nitrocellulose membrane at 1.5 μg each, and the membrane was blocked with 10% serum using TBST buffer. Furthermore, the two aptamers were incubated with an aptamer solution mixed at a concentration ratio of 1: 0, 1: 1, 1:10. Thereafter, the cells were incubated with HRP-modified avidin, and ssDNA bound to the protein was detected by chemiluminescence.

  The results of detecting aptamer binding by chemiluminescence are shown in FIG. The concentration ratio of the two aptamers incubated with each membrane is also shown on the left. When the spot showing aptamer binding becomes thinner as the concentration ratio of biotin-modified aptamer increases, the two aptamers are considered to be in competition. However, such a decrease in spot intensity was confirmed in all combinations.

  Although it is possible that each aptamer recognizes the same site, the binding of one aptamer causes a conformational change in the target molecule thyroglobulin, which indirectly inhibits the binding of other aptamers. It is possible that this is happening. Accordingly, it cannot be said that all aptamers recognize the same site. However, the combination of TgA25 and TgA8 does not diminish the spot even when the concentration ratio of FITC-labeled TgA and biotin-labeled TgA is 1:10 (10: 1), and it has an effect on binding to the target molecule. Are considered to be few.

Example 9 Aptamer Dimerization In this example, dimerization of TgA25 and TgA7T was performed. By linking two different aptamers, avidity may improve affinity. Thus, TgA25-7TL (FIG. 15) in which TgA25, which has been known to have high affinity and specificity so far, and TgA7T, which is a TgA7 truncated mutant, are linked by a 5-mer thymine, was prepared and bound to thyroglobulin. Noh was evaluated.

  TgA25-7TL (final concentration 400 nM) and thyroglobulin (final concentration 0 nM to 800 nM) were mixed using TBS buffer and incubated at room temperature for 1 hour. Then, Native-PAGE was performed with TBE buffer (89 mM Tris-HCl, 89 mM boric acid, 2 mM EDTA) using 6% polyacrylamide gel. The separated thyroglobulin and TgA25-7TL were transferred to a nitrocellulose membrane at a constant current of 400 mA for 1 hour. The membrane was blocked with 10% serum, incubated with HRP-modified avidin and detected by chemiluminescence. The same operation was performed for TgA25 as a control.

  The result of detecting the binding of each aptamer to thyroglobulin is shown in FIG. Aptamers of both TgA25 (FIG. 16A) and TgA25-7TL (FIG. 16B) were confirmed to bind to thyroglobulin, but TgA25-7TL had stronger band intensity than TgA25. Therefore, it is considered that TgA25-7TL has a higher affinity than TgA25, which is the original aptamer. From the result of the competition experiment of Example 8, it was considered that the binding of TgA25 to thyroglobulin had an adverse effect on the binding of TgA7T in the combination of TgA25 and TgA7T. It was confirmed that the property was improved.

Example 10 Aptamer multimerization using biotin-avidin system
Aptamer TgA25 modified with FITC at the 5 ′ end and biotin at the 3 ′ end was multimerized via streptavidin, and the binding ability to Thyroglobulin was evaluated using Aptamer Blotting. Streptavidin and modified TgA25 are mixed at a molar ratio of 1: 2, 1: 3, 1: 4, 0: 1, and TgA dimer, trimer, tetramer and non-rich Formed TgA (monomer) was prepared. TgA25 was folded in the same manner as in Example 1 before mixing with streptavidin. The concentration of the biotin-modified TgA25 solution used for mixing was 80 nM, and the concentration of the streptavidin solution was 20 to 80 nM. A schematic diagram illustrating the multimerization process is shown in FIG.

  Thyroglobulin was immobilized on a nitrocellulose membrane at 0.5 μg, 1 μg, and 1.5 μg each. The membrane was blocked with 10% serum using TBST buffer, and the membrane after blocking with TgA dimer, trimer, tetramer and monomer DNA solutions (TgA25 concentration 80 nM) prepared above. Was incubated. Then, it was incubated with an HRP-modified anti-FITC antibody, and TgA25 bound to Thyroglobulin was detected by chemiluminescence.

  The detection result of chemiluminescence is shown in FIG. All TgA25 multimerized via streptavidin bound to Thyroglobulin more strongly than monomeric TgA25. It is thought that the affinity was improved by multimerizing the aptamer. Aptamer blotting was performed under three conditions of 1: 2, 1: 3, and 1: 4, respectively, with the concentration ratio to streptavidin, but no difference in affinity for Thyroglobulin was observed among the three. However, when electrophoresis was performed with agarose, the aptamers were most efficiently multimerized at a concentration ratio of 1: 3 (FIG. 18 (B)).

Claims (23)

A thyroglobulin-binding aptamer comprising the polynucleotide according to any one of the following (a) to (d).
(a) A polynucleotide comprising the base sequence represented by any of SEQ ID NOs: 8 to 12, 26 and 31 in the sequence listing.
(b) A polynucleotide in which one or several bases are substituted, deleted and / or inserted in the polynucleotide of (a) and capable of binding to thyroglobulin.
(c) A polynucleotide comprising a structural region containing at least one stem-loop structural unit present in the polynucleotide of (a) or (b) and capable of binding to thyroglobulin.
(d) A polynucleotide comprising the polynucleotide of any one of (a) to (c) as a partial region and capable of binding to thyroglobulin.   The aptamer according to claim 1, wherein the polynucleotide (b) is a polynucleotide in which one or two bases in the polynucleotide (a) are substituted, deleted and / or inserted.   The aptamer according to claim 1, comprising a polynucleotide comprising the nucleotide sequence shown in any one of SEQ ID NOs: 8 to 12, 26 and 31, or a polynucleotide comprising the polynucleotide as a partial region and capable of binding to thyroglobulin. .   The aptamer according to any one of claims 1 to 3, wherein the polynucleotide has a size of 150 mer or less.   The aptamer according to claim 3, which comprises a polynucleotide comprising a base sequence represented by any of SEQ ID NOs: 8 to 12, 26 and 31 in the sequence listing.   The aptamer according to claim 5, which comprises a polynucleotide comprising a base sequence represented by any one of SEQ ID NOs: 8, 9, 11, 12, and 26 in the sequence listing.   2. A polynucleotide comprising a structural region comprising at least one stem-loop structural unit present in a polynucleotide comprising the base sequence shown in SEQ ID NO: 8 or 11 in the sequence listing, and having the ability to bind to thyroglobulin. The aptamer described.   The aptamer according to claim 7, which consists of the base sequence represented by SEQ ID NO: 39 or 40 in the sequence listing.   A number of polynucleotides selected from polynucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 8-12, 26, 31, 39, and 40 have a linked structure and an ability to bind to thyroglobulin. The aptamer according to 1.   The aptamer according to claim 9, which has a structure in which two polynucleotides are linked.   40. It has a structure in which two polynucleotides consisting of the base sequences shown in SEQ ID NO: 9 and SEQ ID NO: 26, respectively, or two polynucleotides consisting of the base sequences shown in SEQ ID NO: 26 and SEQ ID NO: 39, are linked. 10. The aptamer according to 10.   The aptamer according to any one of claims 9 to 11, wherein the several polynucleotides are linked via a linker composed of 1mer to 20mer bases.   The aptamer according to claim 12, comprising a polynucleotide comprising a base sequence represented by SEQ ID NO: 43 in the sequence listing.   An aptamer multimer having a structure in which a plurality of aptamers are linked via a linking molecule.   The aptamer multimer according to claim 14, wherein the linking molecule is a set of two molecules that bind to each other.   The aptamer multimer according to claim 15, wherein the set of linking molecules is a set of biotin and avidin or streptavidin.   The aptamer multimer according to any one of claims 14 to 16, wherein the aptamer is at least one selected from thyroglobulin-binding aptamers according to any one of claims 1 to 13.   The aptamer multimer according to claim 17, wherein the aptamer has a base sequence represented by SEQ ID NO: 26 in the Sequence Listing.   A method for producing an aptamer multimer, comprising bringing an aptamer to which one of a set of linking molecules is bound into contact with the other of the set of linking molecules and linking the aptamer by a bond between the linking molecules.   The method according to claim 19, wherein the linking molecule linked to the aptamer is biotin, and the other linking molecule is avidin or streptavidin.   A kit for producing an aptamer multimer comprising one or more aptamers and a linking molecule.   The kit according to claim 21, wherein the linking molecule is a set of biotin and avidin or streptavidin.   The kit according to claim 22, wherein the biotin is in a form bound to the aptamer.