WO2014010640A1

WO2014010640A1 - Method for producing aptamer

Info

Publication number: WO2014010640A1
Application number: PCT/JP2013/068901
Authority: WO
Inventors: 一典池袋; 吉田　亘
Original assignee: 国立大学法人東京農工大学
Priority date: 2012-07-10
Filing date: 2013-07-10
Publication date: 2014-01-16
Also published as: JP2018029601A; JPWO2014010640A1; JP6706417B2; JP6517017B2

Abstract

Disclosed is a method for producing an aptamer that binds specifically with a target substance without using SELEX. The method for producing an aptamer includes the chemical synthesis of an aptamer that has a gene sequence coding for a target protein or a base sequence identical to a region containing a G-quadruplex structure present in a control sequence or transcription product of the gene sequence, and that binds with the target protein or a related gene product of the gene sequence, or the chemical synthesis of an aptamer that has 90% or higher sequence identity with the base sequence of the aptamer, contains a G-quadruplex structure, and binds to the target protein to which the aptamer binds, or to the related gene product.

Description

Method for producing aptamer

The present invention relates to an aptamer production method and an aptamer produced thereby.

Aptamers that are nucleic acid molecules that specifically bind to arbitrary molecules are known. Since an aptamer specifically binds to a desired target molecule, it has been proposed to detect and quantify the target molecule using the aptamer. So far, aptamers that specifically bind to insulin, luciferase, thyroglobulin, C-reactive protein, vascular endothelial growth factor (Patent Document 1) and the like have been reported.

These aptamers that specifically bind to a desired target molecule are basically 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 10 times, aptamers having high binding power to the target molecule are 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.

JP 2011-92138 A WO 2005/049826 JP 2010-30999

SELEX is an excellent method for producing an aptamer that specifically binds to an arbitrary target substance, but it requires considerable labor to produce an aptamer. In addition, since it is a method utilizing chance, even if SELEX is applied for a considerable time, an aptamer that specifically binds to a target substance with satisfactory affinity may not be obtained.

An object of the present invention is to provide a method for producing an aptamer that specifically binds to a target substance without using SELEX.

It is known that a G-quadruplex structure exists in genomic DNA and takes a quadruplex structure, and promoters containing the G-quadruplex structure are also known. When the G-quadruplex structure is included in the promoter, the present inventors have found that the gene product of the structural gene controlled by the promoter or the gene product downstream of the cascade in which the gene product is included in the G-quadruplex structure. I thought that it might be combined to inhibit feedback. The new finding is that a single-stranded nucleic acid having the same base sequence as the G-quadruplex structure-containing region contained in the promoter can be used as it is as an aptamer that specifically binds to the substance to which the promoter binds. Thus, the first aspect of the present invention was completed. The present inventors further considered that when a G-quadruplex structure is present in mRNA, the mRNA and the gene product encoded by the mRNA may bind to each other. A new finding is that single-stranded nucleic acid having the same base sequence as the G-quadruplex structure-containing region contained in mRNA can be used as it is as an aptamer that specifically binds to the gene product encoded by the mRNA. To complete the second aspect of the present invention.

That is, the present invention has a base sequence identical to a region containing a G-quadruplex structure that is present in a gene sequence encoding a target protein or a regulatory sequence thereof or a transcription product thereof, and relates to the target protein or the gene sequence. An aptamer that binds to a gene product or an aptamer that has 90% or more sequence identity with the base sequence of the aptamer and includes a G-quadruplex structure, and binds to the target protein or the related gene product to which the aptamer binds There is provided a method for producing an aptamer, which comprises chemically synthesizing. Moreover, this invention provides the aptamer manufactured by the method of the said invention. Here, “control sequence” means a gene sequence that controls the expression of a gene encoding a target protein, such as a promoter, enhancer, silencer, insulator and the like. The “gene sequence encoding the target protein” means a gene sequence including not only a cDNA sequence but also an intron in the case of genomic DNA. In addition, “transcription product” includes not only mRNA but also mRNA precursor before splicing. As is well known, since thymine in DNA and uracil in RNA both specifically pair with adenine, in the present invention, thymine in DNA and uracil in RNA are “the same base”. I understand. Therefore, in the present invention, a certain DNA sequence and an RNA sequence in which thymine in this DNA sequence is replaced with uracil are understood to have “the same base sequence”.

According to the first aspect of the present invention, which is included in the scope of the present invention, the structural gene having the same base sequence as the region containing the G-quadruplex structure present in the promoter and controlled by the promoter An aptamer that binds to a gene product or a related gene product of the gene product, or that has a sequence identity of 90% or more with the base sequence of the aptamer and includes a G-quadruplex structure, and binds to the gene product to which the aptamer binds There is provided a method for producing an aptamer, which comprises chemically synthesizing an aptamer.

In addition, according to the second aspect of the present invention, which is included in the scope of the present invention, a gene having the same base sequence as a region containing a G-quadruplex structure present in mRNA and encoded by the mRNA An aptamer that binds to a product or a related gene product of the gene product, or has a nucleotide sequence of 90% or more with the base sequence of the aptamer, and includes a G-quadruplex structure, and binds to a gene product to which the aptamer binds A method for producing an aptamer is provided that includes chemically synthesizing an aptamer.

The present invention provides for the first time a method for producing an aptamer that binds to a desired target substance without using SELEX, which requires laborious operations. According to the method of the present invention, a nucleic acid having the same base sequence as the base sequence of the G-quadruplex structure-containing region in the promoter of the gene encoding the desired target protein can be chemically synthesized using a commercially available nucleic acid synthesizer without using SELEX. Since an aptamer that binds to a target substance can be produced by synthesis, an aptamer that binds to a desired target protein can be produced much more easily than conventional methods.

It is a figure which shows the result of the SPR measurement which shows the binding property of the aptamer produced in the following Example 1 and Comparative Example 1 and vascular endothelial growth factor (the left side is Example 1 and the right side is Comparative Example 1). It is a figure (the left side is Comparative Example 2 and the right side is Example 2) which shows the result of the SPR measurement which shows the binding property of the aptamer produced in the following Example 2 and Comparative Example 2 and platelet-derived growth factor. It is a figure which shows the result of the gel shift assay which shows the binding property of the aptamer produced in the following Example 3 and Comparative Example 3, and a retinoblastoma related protein. It is a figure which shows the result of having analyzed the coupling | bonding of HGF and HGF-PQS1, HGF-PQS7, and HGF-PQS8 by SPR performed in the following Examples 4-7. In any figure of FIG. 4, each curve shows the result of 5000 nM, 3000 nM, 1000 nM, 500 nM, 250 nM, 125 nM, and 62.5 nM from the top. It is a figure which shows the result of having analyzed the coupling | bonding of HBEGF, HBEGF-PQS8, and HBEGF-PQS9 by SPR performed in the following Examples 4-7. In any figure of FIG. 5, each curve shows the result of 5000 nM, 3000 nM, 1000 nM, 500 nM, 250 nM, 125 nM, and 62.5 nM from the top. It is a figure which shows the result of having analyzed in the following Examples 4-7 the analysis of the coupling | bonding of PDGF-BB and each oligonucleotide by a gel shift assay. It is a figure which shows the result of having analyzed in the following Examples 4-7 the analysis of the coupling | bonding of Annexin II and each oligonucleotide by gel shift assay. It is a figure which shows the CD spectrum of HGF-PQS1 to PQS9 obtained in the following Examples 4-7. It is a figure which shows the CD spectrum of HBEGF-PQS1 to PQS14 obtained in the following Examples 4-7. It is a figure which shows the result of having analyzed in SPR measurement the coupling | bonding to the VEGFA of the RNA sequence extracted from the VEGFA gene performed in the following Examples 8-10. In any figure of FIG. 10, each curve shows the results of 1000 nM, 500 nM, 100 nM, 50 nM, and 10 nM from the top. It is a figure which shows the result of having analyzed by the SPR measurement about the coupling | bonding to PDGFA of the RNA sequence extracted from the PDGFA gene performed in the following Examples 8-10. In the figure on the right side of FIG. 11, each curve shows the results of 1000 nM, 500 nM, 100 nM, 50 nM, and 10 nM from the top. It is a figure which shows the result of having analyzed by the SPR measurement about the coupling | bonding to PDGFB of the RNA sequence extracted from PDGFB gene performed in the following Examples 8-10. In any figure of FIG. 12, each curve shows the results of 1000 nM, 500 nM, 100 nM, 50 nM, and 10 nM from the top.

The G-quadruplex (also called G quartet, G4, guanine quadruplex, etc.) structure has two or three guanine bases in a nucleic acid that are close to each other and form a tetramer by hydrogen bonding. It is a structure and is known for taking a quadruplex structure. The G-quadruplex structure is known to exist in human telomeres and promoters.

The inventors of the present application use a single-stranded polynucleotide having the same base sequence as a region containing a G-quadruplex structure contained in a promoter (referred to as a “G-quadruplex structure-containing region”) as it functions as an aptamer. The aptamer was found to bind to the gene product of the structural gene controlled by its promoter or a related gene product of the gene product. The present invention is based on this new knowledge. Here, a “related gene product of a gene product” is, for example, a gene product upstream or downstream of a cascade in which the gene product is included, a protein that functions in cooperation with the gene product, and the like. A related gene product of a gene product can be examined, for example, by co-occurrence analysis in the literature. This co-occurrence analysis can be performed, for example, on the Jabion website published by the National Institute of Informatics, the National Institute of Genetics, and the like. According to the method provided on this website, the co-occurrence score is also known, so that a gene product strongly co-occurring with a certain gene product can be considered to be a highly related gene product. For example, a protein having high co-occurrence with vascular endothelial growth factor (VEGF) specifically tested in the following examples has high co-occurrence in the order of KDR, FLT1, and HIF1A, and with platelet-derived growth factor (PDGF), In the order of PDGFB, PDGFRA, and retinoblastoma-related protein (RB-1), E2F1, CDKN2A, and TP53 have the highest co-occurrence. G-quadruplex forming sequences in the promoters of these genes can be searched for by the method described later, and these sequences can be synthesized to confirm the binding ability.

The method of the present invention can be implemented as follows, for example. First, a promoter containing a G-quadruplex structure is selected. The human genome has already been decoded, and promoter sequences of various structural genes are also known. Among these various promoters, those containing a G-quadruplex structure can be used in the method of the present invention. Promoters known to contain a G-quadruplex structure can be used in the method of the present invention. Examples of promoters known to contain a G-quadruplex structure include c-Kit87up, which is the promoter of the c-Kit gene, c-Myb gene promoter, Rb gene promoter, c-myc gene promoter, c-myc- 23456, BCL-2 gene promoter, HIF1α gene promoter, PDGFRβ gene promoter, KRAS gene promoter, TERT gene promoter, MYB gene promoter, and the like. In the examples described below, the promoters were selected from among the vascular endothelial growth factor (VEGF) promoter, platelet-derived growth factor (PDGF-A) promoter, and retinoblastoma-related protein (RB-1) promoter. It is also known that a G-quadruplex structure is included in the promoter of.

Whether a promoter that is not known to contain a G-quadruplex structure contains a G-quadruplex structure can be examined as follows. In order for the promoter sequence to contain a G-quadruplex structure, it is necessary that four or more consecutive guanine sequences are included. Whether a promoter sequence that satisfies this requirement includes a G-quadruplex structure is analyzed by computer software (for example, Mapper of RAMAPOACOLLEGE published at http://bioinformatics.ramapo.edu/QGRS/analyze.php) (Nucleic Acids Research 2006 July; 34 (Web Server issue): W676-W682) or the actual G-quadruplex structure-containing region is chemically synthesized, for example, the known G-quadruplex structure described in Patent Document 3 It can be easily investigated by reacting with a detection reagent and examining whether or not it binds.

The inventors of the present application have also found that the method of the present invention is useful for creating an aptamer that binds to a protein having a heparin-binding domain. In the following examples, hepatocyte growth factor (HGF (hepatocyte growth factor)), heparin-binding EGF (HBEGF (Hparf-binding EGF-like growth factor))) and platelet derived are proteins having such heparin binding domains. Experiments were conducted on growth factors, annexin II (Annexin II) or apolipoprotein E4 (ApoE4), and aptamers that bind to each of them were successfully created by the method of the present invention for any of these.

Next, an aptamer having the same base sequence as the G-quadruplex structure-containing region of the selected promoter is chemically synthesized. Although the G-quadruplex structure exists in the promoter, the G-quadruplex structure-containing region may straddle parts other than the promoter. The size of the aptamer is not particularly limited, but is usually about 15 mer to 100 mer, preferably about 30 mer to 70 mer (mer represents the number of nucleotides). The aptamer may be DNA or RNA, but is preferably chemically stable DNA. In addition, chemical synthesis of DNA and RNA can be easily performed using a commercially available automatic synthesizer. In addition, since there are various contractors who undertake chemical synthesis of DNA or RNA having a predetermined base sequence, it is possible to request these contractors. In addition, synthesis of nucleic acid in a cell-free system such as RNA synthesis by in vitro transcription or DNA synthesis by a nucleic acid amplification method such as PCR is also included in “chemical synthesis” in the present invention. In the present specification and claims, the term “having a base sequence” means that bases are arranged in such a manner, for example, “an aptamer having a base sequence represented by SEQ ID NO: 1”. Means a 34-mer aptamer whose base sequence is arranged as shown in SEQ ID NO: 1.

Next, it is confirmed that the produced aptamer binds to the gene product of the structural gene controlled by the promoter. This can be performed by gel shift assay or SPR measurement specifically described in the following examples. That is, in the gel shift assay, a label such as a fluorescent label is bound to the end of the aptamer, mixed with the gene product, subjected to gel electrophoresis, the label is detected, and the protein is detected by silver staining. If positive, it can be confirmed that the aptamer and the gene product are bound. In SPR measurement, an aptamer and a gene product are immobilized by immobilizing the gene product on a chip for SPR measurement, bringing the gene product into contact with the aptamer, and measuring whether or not the aptamer binds to the gene product with a commercially available SPR measurement device. And can be confirmed.

When it is confirmed that the aptamer binds to the gene product, the aptamer is produced by chemical synthesis. In general, it is known that there are not a few cases where the binding property to the target substance can be maintained even if the base sequence of the aptamer that binds to the target substance is changed slightly. The aptamer base sequence confirmed to be may be modified while maintaining the binding property to the gene product. The base sequence of the aptamer after modification has 90% or more sequence identity with the original base sequence, and preferably the sequence identity is 95% or more. In this case, the modified aptamer also needs to have a G-quadruplex structure. Since it is thought that the G-quadruplex structure binds to the gene product, it is preferable to modify the part other than the G-quadruplex structure, and even if the part other than the G-quadruplex structure is modified, the binding to the gene product is maintained. Probability is high. Such modified aptamers can also be produced by chemical synthesis. Here, “sequence identity” refers to aligning two sequences so that the bases of the two base sequences to be compared match as much as possible (by inserting a gap if necessary), and By dividing the number by the total number of bases, it means a numerical value expressed as a percentage. If the number of bases is different, divide by the longer base number. Software for calculating sequence identity is well known and is also publicly available on the Internet. As described above, in the present invention, thymine in DNA and uracil in RNA are interpreted as “the same base”. Therefore, when comparing the sequence identity of a DNA sequence and an RNA sequence, thymine and uracil are the same. Calculate as the base of.

The aptamer having the same base sequence as the G-quadruplex structure-containing region is subjected to the known in-computer evolution (in silico maturation) method, and the base sequence is modified to further increase the affinity with the gene product. It is possible. The in-computer evolution method is a method invented by the joint inventor of the present application and is described in Non-Patent Document 2, Non-Patent Document 3, and Patent Document 2, and is also specifically described in Example 12 below. When this method is applied to an aptamer that has been confirmed to bind to a gene product, for example, a plurality of regions, for example, about 3 to 5 mer, which are not essential for maintaining a G-quadruplex structure, are obtained. Swap randomly between corresponding regions of the aptamer (shuffle), or perform crossover by genetic algorithm. Further, random single-base substitution is introduced into each region after shuffle or crossing. The introduction of these shuffles or crossings and single base substitutions is done by computer. Then, an aptamer having a new base sequence created by a computer is chemically synthesized into a nucleic acid library, which is subjected to a SELEX cycle. When preparing the second nucleic acid library after the end of the first cycle, the amount of aptamer having a region derived from the aptamer having the highest binding ability is maximized, and the ratio is decreased as the order is lowered. . As described above, the efficiency of evolution by SELEX can be enhanced by artificially introducing mutations by shuffling or crossing and single base substitution in a computer.

This in-computer evolution method uses computer support to perform SELEX, but since the original aptamer already has the ability to bind gene products, what is SELEX that starts from zero? Difficulty is different, and the ability to bind gene products can be further enhanced with high probability. In addition, since the in-computer evolution method is performed while confirming the binding ability with the gene product by experiment, the binding ability with the gene product is ensured even if the base sequence is considerably different from the original base sequence. Therefore, the sequence identity is not necessarily limited to 90% or more. However, since the in-computer evolution method introduces mutations in parts other than the G-quadruplex structure as described above, the G-quadruplex structure is maintained. Furthermore, as specifically described in Example 12 below, in the in-computer evolution method, by examining the aptamer sequence having high binding ability to the target gene product among aptamers obtained in each generation, A sequence motif that exhibits the ability to bind to a gene product may become apparent. For example, in Example 12 below, when the HGF-binding aptamer obtained in Example 4 below is used as a parent sequence and an in-computer evolution method is performed until the second generation, the first and second generation aptamers are used. Among them, when the base sequences of aptamers having high binding ability to HGF were examined, it was found that many sequences have a common sequence motif of ggtggagggg (SEQ ID NO: 57). Thus, when the third generation was carried out with this sequence motif portion fixed, an aptamer whose binding specificity for _HGF (Sp _(HGF) ) was about 240 times that of the parent sequence could be obtained. Thus, according to the in-computer evolution method, a sequence motif that exhibits high binding ability may be found in the middle stage. In this case, this sequence motif is fixed and subsequent generations are performed. This makes it possible to obtain an aptamer having a higher binding ability more efficiently.

As described above, according to the method of the present invention, an aptamer that binds to a desired target protein can be easily produced without performing SELEX using the promoter sequence of the structural gene of the desired target protein. Excellent effect is achieved.

Since aptamers have the property of binding specifically to a target protein, they can be used for quantification and detection of the target protein (for example, Patent Document 2). In this case, the aptamer can be labeled and used. Examples of the label include well-known labels such as a fluorescent label, a radiolabel, an enzyme label, and a chemiluminescent label. It is well known that the binding affinity to the target substance is maintained even if these labels are bound directly or via the spacer sequence to the end of the aptamer. In the examples below, the aptamer bound with FITC, which is a fluorescent label, is used. Used. Aptamers to which these labels are added can be used for measurement (detection or quantification) of gene products. The method for measuring a target substance using a labeled aptamer is well known in this field, and can be performed, for example, in the same manner as an immunoassay using a labeled antibody.

In addition, when the target protein has some physiological activity related to a disease and promoting its expression leads to treatment of the disease, the aptamer produced by the method of the present invention should be used as a nucleic acid drug. Can do. That is, when the aptamer is administered to a living body, a part of the gene product produced in the cell binds to the aptamer and cannot bind to the promoter, so that transcriptional repression by the gene product is reduced. In addition, if an aptamer can bind to a gene product and neutralize its activity, it can also inhibit the activity of the gene product, and if the inhibition of the gene product leads to treatment of the disease, the nucleic acid drug Can be used as When aptamers are used as nucleic acid drugs, for example, other structures such as polyethylene glycol (PEG) chains can be added to improve nuclease resistance. It is well known that the nuclease resistance of an aptamer is enhanced by attaching a PEG chain to the end of the aptamer and has already been put into practical use. The size of the PEG chain is not particularly limited, but usually has a molecular weight of about 10,000 to 30,000, preferably about 15,000 to 25,000, and the number of PEG chains may be one or two, but two are preferred. . Such a PEG chain can be bound via a known amino linker added to the end of the aptamer. When two PEG chains are bonded, an amino linker having a plurality of amino groups such as a lysine amino linker can be used, and the PEG chain can be bonded to each amino group.

Furthermore, when the target protein has some physiological activity related to a disease, and promoting its expression leads to the treatment of the disease, a new drug using the binding ability to the aptamer produced by the method of the present invention as an index Screening can also be performed. By administering a substance found by screening for new drugs, inhibition of the promoter by the gene product is reduced, so that the substance exerts a pharmacological effect.

As described above, the present inventors further considered that when a G-quadruplex structure is present in mRNA, the mRNA and the gene product encoded by the mRNA may be combined. A new finding is that single-stranded nucleic acid having the same base sequence as the G-quadruplex structure-containing region contained in mRNA can be used as it is as an aptamer that specifically binds to the gene product encoded by the mRNA. To complete the second aspect of the present invention. That is, the aptamer production method according to the second aspect of the present invention has the same base sequence as the region containing the G-quadruplex structure present in mRNA, and the gene product encoded by the mRNA or the relationship between the gene products Chemically synthesizing an aptamer that binds to a gene product, or an aptamer that has 90% or more sequence identity with the base sequence of the aptamer and includes a G-quadruplex structure and binds to the gene product to which the aptamer binds Including.

According to the second aspect of the present invention, mRNA itself can be used as an RNA aptamer, but the aptamer need not be RNA, and it is preferable to chemically synthesize aptamer DNA as described above. In this case, as a matter of course, uracil in mRNA becomes thymine in DNA, and as described above, uracil in mRNA is replaced with thymine in DNA, and it is understood that it has “the same base sequence”. .

As for the aptamer according to the second aspect of the present invention, the size is preferably 30 mer to 100 mer as in the first aspect of the present invention described above, and the affinity with the target substance can be further increased by the in-computer evolution method. preferable. Further, it can be used as a nucleic acid drug as in the first aspect, and in that case, it is preferable to perform nuclease resistance modification such as PEGylation. In the following examples, the second invention of the present application has succeeded in creating aptamers that bind to each of vascular endothelial growth factor and platelet-derived growth factor.

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 and Comparative Example 1
Analysis of binding ability of Gq structure formed in VEGF promoter region to VEGF

Experimental method VEGF promoter known to contain G-quadruplex structure (Sun, D., Guo, K., Rusche, JJ & Hurley, LH Facilitation of a structural transition in the polypurine / polypyrimidine tract within the proximal promoter region of The human VEGF gene by the presence of potassium and G-quadruplex-interactive agents. Nucleic Acids Res. 33, 6070-6080 (2005)) was selected as a promoter. G-quadruplex (hereinafter sometimes abbreviated as “Gq”) structure-forming sequence (VEGF promoter Gq (SEQ ID NO: 1)) formed within the VEGF promoter region (position on the genome: chr6: 43,737,633-43,739,852) Example 1) and evaluation of the binding ability of VEGF promoter Gq- (SEQ ID NO: 2), Comparative Example 1) to VEGF, which was designed to replace G base involved in Gq formation with T base and not form Gq Went. Each oligo DNA was synthesized by a commercially available DNA automatic synthesizer. All base numbers correspond to Human Feb. 2009 (GRCh37 / hg19) Assembly.

Evaluation was performed by gel shift assay and SPR measurement. The DNA sequences used for the evaluation are shown in Table 1 below. All aptamers were diluted to 1 μM with TBS buffer (10 mM Tris-HCl, 150 mM NaCl, 5 mM KCl, pH 7.4) and used after folding.

Gel Shift Assay VEGF ₁₆₅ (GenBank Accession No .: NP_001165097) and each aptamer (5′-FITC modification) were mixed to a final concentration of 1.3 μM or 500 nM, respectively, and shaken for 30 minutes at room temperature. Thereafter, each sample was applied to a 12% native polyacrylamide gel and electrophoresed at room temperature and 20 mA (constant current) for 25 minutes. After the electrophoresis, FITC fluorescence of each aptamer was detected by Typhoon 8600 (trade name, manufactured by GE Healthcare). Further, VEGF was stained by silver staining.

SPR measurement TBS buffer was used as the running buffer. Using the sensor chip CM5 (trade name, manufactured by GE Healthcare) to which VEGF _{165 of} about 4700 RU is immobilized by the amine coupling method, each aptamer prepared at 7.8 to 1000 nM is injected, and VEGF on the sensor chip is injected. The binding between ₁₆₅ and each aptamer was observed by SPR measurement.

Results and Discussion As a result of gel shift assay, a fluorescence band shift of VEap121 (SEQ ID NO: 3) was observed at the position of VEGF in the sample lane mixed with VEGF ₁₆₅ . A band shift was also observed at the position of VEGF in VEGF promoter Gq. This indicates that the Gq structure of the VEGF promoter region binds to VEGF. The results of SPR measurement of the VEGF promoter Gq sequence are shown in FIG.

When VEGF-promoter-Gq- was injected, no increase in SPR signal was observed, whereas when VEap121 or VEGF-promoter-Gq was used, an increase in aptamer concentration-dependent SPR signal was observed. As a result of calculating the dissociation constant of each aptamer by curve fitting, VEap121: 510 nM, VEGF promoter Gq: 240 nM.

Example 2 and Comparative Example 2
Analysis of binding ability of Gq structure formed in PDGF promoter region to PDGF by SPR

Experimental method PDGF-A promoter known to contain G-quadruplex structure (Qin, Y., Rezler, EM, Gokhale, V., Sun, D. & Hurley, LH Characterization of the G-quadruplexes in the duplex nuclease The hypersensitive element of the PDGF-A promoter and modulation of PDGF-A promoter activity by TMPyP4. Nucleic Acids Res. 35, 7698-7713 (2007)) was selected as the promoter. The Gq structure forming sequence (PDGF promoter Gq (SEQ ID NO: 4), Example 2) formed in the PDGF-A promoter region (position on the genome: chr7: 556,612-562,845) and the G base involved in Gq formation Evaluation of the binding ability to the PDGF-AA of the sequences (PDGF promoter Gq- (SEQ ID NO: 5), Comparative Example 2) designed so as to be substituted with T base and not form Gq was performed by SPR measurement. These sequences are shown in Table 2. All base numbers correspond to Human Feb. 2009 (GRCh37 / hg19) Assembly.

TBS バッファー buffer was used as the running buffer. Using the sensor chip CM5 on which PDGF-AA of about 9000 RU is immobilized by the amine coupling method, each aptamer prepared to 7.8-1000 nM is injected, and PDGF-AA (GenBank Accession No .: The binding between each aptamer and P04085) was observed by SPR measurement.

Results and Discussion FIG. 2 shows the results of SPR measurement of the PDGF promoter Gq sequence. When PDGF promoter Gq- was injected, no increase in SPR signal was observed, whereas when PDGF promoter Gq was used, an increase in aptamer concentration-dependent SPR signal was observed. As a result of calculating the dissociation constant of each aptamer by curve fitting, it was calculated as VEGF promoter Gq: 18 nM.

Example 3 and Comparative Example 3
Analysis of the binding ability of Gq structure in RB-1 promoter region to RB-1

Experimental method RB-1 promoter known to contain G-quadruplex structure (Xu, Y. & Sugiyama, H. Formation of the G-quadruplex and i-motif structures in retinoblastoma susceptibility genes (Rb) .Nucleic Acids Res. 34, 949-954 (2006)) were selected as promoters. Gq structure forming sequence (RB-1_Gq promoter Gq (SEQ ID NO: 6), Example 3) formed in the RB-1 promoter region (position on the genome: chr13: 48877460-48878501) and G involved in Gq formation The binding ability of the sequences (RB-1_Gq_Mut (SEQ ID NO: 7), Comparative Example 3) designed so as not to form Gq by replacing the base with T base was examined by gel shift assay. All base numbers correspond to Human Feb. 2009 (GRCh37 / hg19) Assembly. RB-1 (final concentration 0 nM or 470 nM) (GenBank Accession No .: P06400) and 1000 nM each DNA (5′-TAMRA modification, sequence shown in Table 3 below) were mixed and shaken at room temperature for 30 minutes. Thereafter, each sample was applied to 12% native polyacrylamide gel and electrophoresed at room temperature and 20 mA (constant current) for 20 minutes. After electrophoresis, TAMRA fluorescence of each DNA was detected with Typhoon 8600 (trade name). In addition, RB-1 was stained by silver staining.

Results and Discussion FIG. 3 shows the results of detecting fluorescence with Typhoon (trade name) and the results of detecting RB-1 protein with silver staining after electrophoresis. In the lane in which 470 nM RB-1 and 1000 nM RB-1_Gq were mixed, TAMRA fluorescence was observed at the position where the RB-1 band was observed. This band was not observed in other lanes. This indicates that the Gq structure formed in the RB-1 promoter binds to the RB-1 protein.

Examples 4-7
Creation of aptamers for proteins having heparin binding domains Method
(1) Sequences near transcription start sites of HGF, HBEGF, PDGFB, and Annexin II genes were obtained using USCS Genome Browser. For HGF, the sequence of transcription start point ± 1 kbp was obtained, and for HBEGF, PDGFB and Annexin II, sequences of CpG islands present in the vicinity of the transcription start point were obtained. As the sequence, both a plus strand and a minus strand were obtained.

(2) Among the sequences, sequences capable of forming a quadruplex structure were extracted under the following conditions.
HGF: Four or more consecutive Gs were included at four positions, the sequence between consecutive Gs and consecutive Gs was within 7 mer, and the total length was within 40 mer.
PDGFB, HBEGF, Annexin II: 3 or more consecutive Gs were included at 4 positions, the sequence between consecutive Gs and consecutive Gs was within 7 mer, and the total length was within 30 mer. If there is no sequence that meets the above conditions, extract 2 or more consecutive Gs in 4 or more locations, the sequence between consecutive Gs is within 7 mer, and the total length is within 30 mer did.

(3) The sequences extracted in (2) above were synthesized and analyzed by SPR for HGF and HBEGF, and by gel shift assay for PDGFB and Annexin II.

(4) SPR was performed by the following method.
HGF and HBEGF PBS buffer _{_{(Na 2 HPO 4 8.1 mM,}} KH 2 PO 4 1.47 mM, NaCl 137 mM, KCl 2.68 mM, pH7.4) was dissolved in was immobilized onto the sensor chip CM5 via amine coupling method . Thereafter, the synthesized oligonucleotide was folded in TBSK buffer (10 mM Tris-HCl, 150 mM NaCl, 100 mM KCl) (95 ° C. for 5 minutes and then cooled to 25 ° C. over 30 minutes). Each oligonucleotide was diluted to various concentrations and injected onto the sensor chip, and changes in the SPR signal were observed. The addition time was 120 seconds, the dissociation time was 120 seconds, and the flow rate was 30 μl / min. The dissociation constant (Kd) was calculated by Curve fitting analysis.

(5) The gel shift assay was performed by the following method.
Each oligonucleotide whose FI ′ end was modified with FITC was folded in TBSK buffer (10 mM Tris-HCl, 150 mM NaCl, 100 mM KCl, pH 7.4). Thereafter, 250 ng of protein and 5 pmol of oligonucleotide were mixed and shaken at room temperature for 30 minutes. Thereafter, each sample was applied to a 12% native polyacrylamide gel and electrophoresed at room temperature and 20 mA (constant current). After electrophoresis, the fluorescence of each aptamer was detected by Typhoon 8600.

(6) CD spectra were measured for sequences capable of forming a DNA quadruplex structure present in the HGF and HBEGF promoters. Prepare each oligonucleotide in TBSK buffer (10 mM Tris-HCl, 150 mM NaCl, 100 mM NaCl, KCl pH 7.4) or TBS buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4) to a final concentration of 2 μM. Folding (95 ° C for 5 minutes followed by cooling to 25 ° C over 30 minutes) was then performed. The prepared sample was put into a quartz cell having an optical path length of 10 mm, and a CD spectrum was measured using a J-820 type circular dichroism dispersometer. Sensitivity is 1000 mdeg, wavelength region is 220 320nm, data acquisition interval is 1 nm, scanning speed is 500 nm / min, response is 1 sec, bandwidth is 5.0 nm, integration number is 10 times. Measurements were made.

2. result
(1) When searching for a sequence capable of forming a DNA quadruplex structure from each promoter region, 9 sequences from the HGF promoter, 14 sequences from the HBEGF promoter, 8 sequences from the PDGFB promoter, 7 sequences from the Annexin II promoter Could be extracted (Table 4).

(2) When each oligonucleotide binds to HGF and analyzed by SPR, three sequences of HGF-PQS1 (SEQ ID NO: 8), HGF-PQS7 (SEQ ID NO: 9), and HGF-PQS8 (SEQ ID NO: 10) Increased DNA concentration-dependent SPR signals were observed in, indicating that these are aptamers of HGF (FIG. 4). The dissociation constants of HGF-PQS1, HGF-PQS7, and HGF-PQS8 for HGF were 73 nM, 45 nM, and 110 nM, respectively.

(3) SPR analysis was conducted to determine whether each oligonucleotide binds to HBEGF. DNA concentration-dependent increases in SPR signals were observed in the two sequences HBEGF-PQS8 and HBEGF-PQS9. (FIG. 5). The dissociation constants of HBEGF-PQS8 (SEQ ID NO: 11) and HBEGF-PQS9 (SEQ ID NO: 12) for HBEGF were 110 nM and 9 μM, respectively.

(4) When each oligonucleotide binds to PDGF-BB and analyzed by gel shift assay, all 8 oligonucleotides (SEQ ID NOs: 13 to 20 in order from the top in Table 4) bind to PDGF-BB. It was shown that these are aptamers that bind to PDGF-BB (FIG. 6). In FIG. 6, + indicates a lane in the presence of PDGF-BB, and-indicates a lane in the absence of PDGF-BB.

(5) Analysis of whether each oligonucleotide binds to Annexin II by gel shift assay shows that Annexin II-PQS6 (SEQ ID NO: 21) binds to Annexin II, which is an aptamer that binds Annexin II. It was shown (Figure 7). In FIG. 7, + indicates a lane in the presence of Annexin II, and-indicates a lane in the absence of Annexin II.

(6) When CD spectra of HGF-PQS1 to PQS9 were measured, only HGF-PQS1 and HGF-PQS7, in which binding to HGF was observed, were positive in the presence of potassium at around 260 nm, and negative at around 240 nm. (FIG. 8). This is a CD spectrum unique to the parallel DNA quadruplex structure, indicating that HGF-PQS1 and HGF-PQS7 form a parallel DNA quadruplex structure and bind to HGF. It was done.

When CD spectra of HBEGF-PQS1 to PQS14 were measured, only HBEGF-PQS8 and HBEGF-PQS9, in which binding to HBEGF was observed, showed a positive peak near 260 nm and a negative peak around 240 nm in the presence of potassium. (FIG. 9). That is, it was shown that HBEGF-PQS8 and HBEGF-PQS9 formed a parallel DNA quadruplex structure and bound to HBEGF.

Examples 8-10
1. Method
(1) Full length sequences of RNA transcribed from VEGFA, PDGFA, and PDGFB genes were obtained using USCS Genome Browser.

(2) Among the sequences, sequences capable of forming an RNA quadruplex structure were extracted under the following conditions.
Two or more consecutive Gs were included in four or more locations, the sequence between consecutive Gs was within 14 mer, and the sequence with a total length of 40 mer or less was extracted.

(3) PDGF-AA and PDGF-BB are dissolved in 10 mM HEPES buffer (pH 7.0) and VEGFA is dissolved in 10 mM sodium acetate buffer (pH 6.0) and immobilized on the sensor chip CM5 by the amine coupling method. did. Thereafter, the synthesized oligonucleotide was folded in PBS buffer (Na ₂ HPO ₄ 8.1 mM, KH ₂ PO ₄ 1.47 mM, NaCl 137 mM, KCl 2.68 mM, pH 7.4) (65 ° C. for 5 minutes and then 25 ° C. Cooling over 30 minutes). Each oligonucleotide was diluted to various concentrations and injected onto the sensor chip, and changes in the SPR signal were observed. The addition time was 120 seconds, the dissociation time was 120 seconds, and the flow rate was 30 μl / min. The dissociation constant (Kd) was calculated by Curve fitting analysis.

2. result
(1) From the transcribed RNA sequence of each gene, a sequence that can form a quadruplex RNA structure was searched. RNA of 159 sequences from the VEGFA gene, 247 sequences from the PDGFA gene, and 194 sequences from the PDGFB gene Could be extracted. From these, the following sequences were selected and synthesized (Table 5).

(2) When SPR analyzes whether each oligonucleotide binds to VEGFA, an increase in RNA concentration-dependent SPR signal was observed in all sequences, indicating that these are RNA aptamers that bind to VEGFA. It was done. VEGFA RNA1 (SEQ ID NO: 22) bound to VEGFA with a dissociation constant of about 140 nM, VEGFA RNA2 (SEQ ID NO: 23) was about 31 nM, and VEGFA RNA3 (SEQ ID NO: 24) was about 300 nM. (FIG. 10).

(3) When SPR analysis was performed to determine whether each oligonucleotide bound to PDGF-AA, an increase in RNA concentration-dependent SPR signal was observed in all sequences. These were RNA aptamers that bind to PDGF-AA. It was shown that there is. PDGFA RNA1 (SEQ ID NO: 25) bound to PDGFA with a dissociation constant of about 29 nM and PDGFA RNA2 (SEQ ID NO: 26) with a dissociation constant of about 30 nM. (FIG. 11).

(4) When SPR analysis was performed to determine whether each oligonucleotide bound to PDGF-BB, an increase in the SPR signal depending on the RNA concentration was observed in all sequences. These were RNA aptamers that bind to PDGF-BB. It was shown that there is. PDGFB RNA1 (SEQ ID NO: 27) is about 42 nM, PDGFB RNA2 (SEQ ID NO: 28) is about 30 nM, PDGFB RNA3 (SEQ ID NO: 29) is about 59 nM, PDGFB RNA4 (SEQ ID NO: 30) is about 35 nM, PDGFB RNA5 (SEQ ID NO: 31) bound to PDGFA with a dissociation constant of about 34 nM (FIG. 12).

Example 11
1. Search for the predicted G4 formation sequence in the promoter region of the target protein gene Genome Browser (http: //genome.ucsc. edu / cgi-bin / hgGateway). From the extracted sequences, a sequence that satisfies the following conditions was searched using QGRS Mapper (http://bioinformatics.ramapo.edu/QGRS/index.php).
(i) Total length within 30 mer (ii) The obtained sequence containing two or more consecutive Gs at intervals of 7 mer was designated as an aptamer candidate sequence for ApoE4.

2. Evaluation of binding between aptamer candidate sequence and target protein by surface plasmon resonance (SPR) measurement ApoE4 is diluted with 10 mM acetic acid buffer (pH 4.0) and immobilized on sensor chip CM4 by amine coupling method. did. After that, aptamer candidate sequences (Table.) Folded in TBS buffer (10 mM Tris-HCl, 150 mM NaCl, 100 mM KCl, pH 7.4) were diluted to various concentrations and added to the sensor chip. Changes in SPR signal were measured. After the measurement, the dissociation constant (K _d ) was calculated by curve fitting.

Results and discussion
1. From the promoter region of the gene encoding ApoE4, 8 sequences having the possibility of forming a G4 structure were obtained (named ApoE4_1 to 8). Among these, a DNA concentration-dependent increase in SPR signal was observed in ApoE4_1 (SEQ ID NO: 56). This suggests that ApoE4_1 is bound to ApoE4. The dissociation constant calculated by curve fitting was 60 nM.

Example 12
The three acquired HGF aptamers are referred to as HGFap1, HGFap2, HGFap3, and the two HBEGF aptamers are referred to as HBEGFap1, HBEGFap2. In in silico maturation of HGF aptamer binding specificity for HGF sequences were evaluated (Sp _(HGF)) includes a target / [HBEGF [association constant K _a in the case of the HGF targeted] Sp _(HGF) = The coupling constant K _a ] (Sp _(HGF) = _Ka _(HGF) / _{Ka (HBEGF)} ) was defined.

First generation sequence generation and specificity evaluation First, Sp _(HGF) was calculated for HGFap1, HGFap2, and HGFap3, which are the first generation parent sequences, and each sequence was replicated based on the ratio of Sp _(HGF) values, totaling 20 An array of books was made. Subsequently, 2 pairs were randomly formed in 20 sequences, and crossed at any one point. Thereafter, 10% mutations were randomly introduced into each of the 20 sequences, both in position and base type, to obtain first generation sequences (1R01 to 1R20). The base sequences of 1R01 to 1R20 are shown in SEQ ID NOs: 58 to 77 in this order.

HGF and HBEGF were immobilized on the sensor chip CM5 by PBS coupling (Na ₂ HPO ₄ 8.1 mM, KH ₂ PO ₄ 1.47 mM, NaCl 137 mM, KCl 2.68 mM, pH 7.4) by the amine coupling method. . As a protein dilution buffer, 10 mM HEPES buffer (pH 6.5) was used for HGF, and 10 mM acetic acid buffer (pH 5.0) was used for HBEGF. After that, each first-generation sequence folded in TBS buffer (10 mM Tris-HCl, 150 mM NaCl, 100 mM KCl) was used at various concentrations (fc 1000 nM, 500 nM, 100 nM) using TBS buffer. , 50 nM, 0 nM), and the change of the SPR signal when HGF or HBEGF was added to the immobilized sensor chip was measured. TBS buffer was used as a running buffer at the time of interaction measurement, and the measurement was performed at an addition time of 120 seconds, a dissociation time of 200 seconds, and a flow rate of 30 μL / min. After measurement, _Ka _(HGF) and _{Ka (HBEGF)} were obtained by curve fitting, and Sp _(HGF) was calculated.

Generation of second generation sequence and evaluation of specificity (i) Sequence lower than Sp _(HGF) (minimum value: 1.9) of the parent sequence from the first generation sequence, (ii) _{Ka (HGF)} is the parent sequence _Ka _{( HGF)} (minimum value: 9.1E + 06) 1/10 or less of the sequence was excluded, and the remaining sequence was used as a parent sequence for the generation of the second generation sequence. The obtained second generation parent sequence was replicated, crossed and mutagenized in the same manner as the first generation parent sequence to obtain second generation sequences (2R01 to 2R20). The nucleotide sequences of 2R01 to 2R20 are shown in SEQ ID NOs: 78 to 97 in this order. Evaluation of binding to HGF and HBEGF and calculation of Sp _(HGF) were performed in the same manner as the first generation sequence.

Generation of 3rd generation motif-fixed sequences and specificity evaluation The 1st and 2nd generation sequences do not bind to sequence groups with high Sp _(HGF) , sequence groups with low Sp _(HGF) , HGF and HBEGF As a result of comparing the sequences, the sequences having a high Sp _(HGF) have a common sequence motif of GGTGGAGGGG. Therefore, the first generation sequence and the second generation sequence that have a GGTGGAGGGG sequence motif with high Sp _(HGF) (1R01, 1R05, 1R08, 1R09, 2R07) For parent sequence. The obtained third generation motif-fixed parent sequence was replicated, crossed and introduced in the same manner as the first generation parent sequence and the second generation parent sequence to obtain third generation motif-fixed sequences (3R01mfix-3R20mfix). The nucleotide sequences of 3R01mfix to 3R20mfix are shown in SEQ ID NOs: 98 to 117 in this order. Evaluation of binding to HGF and HBEGF and calculation of Sp _(HGF) were performed in the same manner as the first generation sequence and the second generation sequence.

Results and Discussion In the first generation sequence, a plurality of sequences having higher Sp _(HGF) values than the parent sequence were obtained (Table 6). In the second generation sequence, a highly specific sequence showing Sp _(HGF) approximately 13 times Sp _(HGF) (minimum value: 1.9) of the parent sequence was obtained (Table 7, 2R07). In a further third generation motif fixed sequence, the parent sequence Sp _(HGF) approximately 50 times the Sp _(HGF) sequences showing the (3R02mfix), also approximately 240 times the Sp of the parent sequence Sp _{_(HGF) (HGF)} A sequence (3R14mfix) was obtained (Table 8). Thus, the specificity of the HGF aptamer obtained by the method of the present invention could be improved by the in-computer evolution method.

K _{d (HGF):} dissociation constant to HGF
K _{d (HBEGF)} : Dissociation constant for HBEGF
K _{a (HGF)} : Binding constant for HGF
K _{a (HBEGF)} : Binding constant for HBEGF
Sp _(HGF) : The value obtained by dividing the binding constant for HGF by the binding constant for HBEGF _(Ka _(HGF) / _{Ka (HBEGF)} )

K _{d (HGF)} : Dissociation constant for HGF
K _{d (HBEGF)} : Dissociation constant for HBEGF
K _{a (HGF):} binding constant for HGF
K _{a (HBEGF)} : Binding constant for HBEGF
Sp _(HGF) : The value obtained by dividing the binding constant for HGF by the binding constant for HBEGF _(Ka _(HGF) / _{Ka (HBEGF)} )

Claims

An aptamer that has the same base sequence as a region containing a G-quadruplex structure present in a gene sequence encoding the target protein or a regulatory sequence thereof or a transcription product thereof, and binds to the target protein or a related gene product of the gene sequence Or chemically synthesizing an aptamer having 90% or more sequence identity with the base sequence of the aptamer and containing a G-quadruplex structure and binding to the target protein or the related gene product to which the aptamer binds. A method for producing an aptamer.
An aptamer having the same base sequence as the region containing the G-quadruplex structure present in the promoter, and binding to the gene product of the structural gene controlled by the promoter or a related gene product of the gene product, or the base sequence of the aptamer The method according to claim 1, comprising chemically synthesizing an aptamer having a sequence identity of 90% or more and including a G-quadruplex structure and binding to a gene product to which the aptamer binds.
The method according to claim 2, wherein the aptamer produced binds to the gene product of the structural gene controlled by the promoter.
The method according to claim 2 or 3, wherein the size is 15mer to 100mer.
The method according to claim 3 or 4, wherein the promoter is a vascular endothelial growth factor promoter, a platelet-derived growth factor promoter, or a retinoblastoma-related protein promoter.
The method according to any one of claims 2 to 5, further comprising a step of further increasing the affinity of the produced aptamer with a target substance by an in-computer evolution method.
An aptamer produced by the method according to any one of claims 2 to 6.
The base sequence represented by SEQ ID NO: 1 or a base sequence having a sequence identity of 90% or more with the base sequence, comprising a G-quadruplex structure, and binding to vascular endothelial growth factor. Aptamer.
The base sequence represented by SEQ ID NO: 4 or a base sequence having a sequence identity of 90% or more with the base sequence, comprising a G-quadruplex structure, and binding to a platelet-derived growth factor. Aptamer.
A base sequence represented by SEQ ID NO: 6, or a base sequence having a sequence identity of 90% or more with the base sequence, comprising a G-quadruplex structure, and binding to a retinoblastoma-related protein. 7. The aptamer according to 7.
The method according to any one of claims 2 to 6, wherein the gene product has a heparin binding domain.
The method according to claim 11, wherein the gene product is hepatocyte growth factor, heparin-binding EGF, platelet-derived growth factor, or annexin II.
An aptamer produced by the method according to claim 11 or 12.
A nucleotide sequence represented by SEQ ID NO: 8, 9 or 10, or a nucleotide sequence having a sequence identity of 90% or more with each of these nucleotide sequences, comprising a G-quadruplex structure, 14. The aptamer according to claim 13, which binds.
A nucleotide sequence having the nucleotide sequence of SEQ ID NO: 11 or 12 or a nucleotide sequence having a sequence identity of 90% or more with each of these nucleotide sequences, comprising a G-quadruplex structure, and binding to heparin-binding EGF Item 14. An aptamer according to Item 13.
Platelet-derived growth factor having a base sequence represented by any of SEQ ID NOs: 13 to 20, or a base sequence having a sequence identity of 90% or more with each of these base sequences, including a G-quadruplex structure 14. The aptamer according to claim 13, which binds to.
The aptamer according to claim 13, which has the base sequence represented by SEQ ID NO: 21 or a base sequence having a sequence identity of 90% or more with the base sequence, includes a G-quadruplex structure, and binds to annexin II .
90% or more of the aptamer having the same base sequence as the region containing the G-quadruplex structure present in mRNA and binding to the gene product encoded by the mRNA or the related gene product of the gene product, or the base sequence of the aptamer The method according to claim 1, comprising chemically synthesizing an aptamer having a sequence identity of 2 and comprising a G-quadruplex structure and binding to a gene product to which the aptamer binds.
The method according to claim 18, wherein the aptamer produced binds to the gene product of the structural gene encoded by the mRNA.
The method according to claim 18 or 19, wherein the size is 15mer to 100mer.
The method according to any one of claims 18 to 20, wherein the mRNA is vascular endothelial growth factor or platelet-derived growth factor mRNA.
The method according to any one of claims 18 to 21, further comprising a step of further increasing the affinity of the produced aptamer with a target substance by an in-computer evolution method.
Platelet-derived growth factor having a base sequence represented by any of SEQ ID NOs: 22 to 24, or a base sequence having a sequence identity of 90% or more with each of these base sequences, including a G-quadruplex structure The aptamer according to claim 21, which binds to.
Platelet-derived growth factor having a base sequence represented by any one of SEQ ID NOs: 25 to 31 or a base sequence having a sequence identity of 90% or more with each of these base sequences and including a G-quadruplex structure The aptamer according to claim 21, which binds to.
The method according to claim 11, wherein the gene product is apolipoprotein E4.
An aptamer produced by the method according to claim 25.
The base sequence represented by SEQ ID NO: 56, or a base sequence having a sequence identity of 90% or more with the base sequence, comprising a G-quadruplex structure, and binding to apolipoprotein E4. Aptamer.
The aptamer according to claim 13, which comprises the base sequence represented by SEQ ID NO: 57 and binds to hepatocyte growth factor.
SEQ ID NOs: 58, 60 to 68, 70, 72, 77, 78, 84, 90, 99, 100, 102, 104, 107, 108, 111 or 115, or a sequence with each of these base sequences The aptamer according to claim 13, which has a nucleotide sequence having an identity of 90% or more, contains a G-quadruplex structure, and binds to a hepatocyte growth factor.
30. The aptamer according to claim 29, having a base sequence represented by SEQ ID NO: 111.