JP6517017B2 - Method of producing aptamer - Google Patents

Description

  The present invention relates to a method for producing an aptamer and an aptamer produced thereby.

  Aptamers that are nucleic acid molecules that specifically bind to any molecule are known. Since the 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 cell 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 variety of random base sequences is added to this, nucleic acid binding to the target molecule is recovered, and this is amplified by PCR. Again, the target molecule is added to the immobilized carrier. By repeating this process about 10 times, an aptamer with high avidity to the target molecule is concentrated, and its base sequence is determined to obtain an aptamer that recognizes the target molecule. The above nucleic acid library can be easily prepared by binding nucleotides at random by an automatic chemical synthesizer for nucleic acid. Thus, an aptamer that specifically binds to any target substance can be created by a method that actively utilizes chance, using a nucleic acid library having a random base sequence.

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

Tuerk, C. and Gold L. (1990), Science, 249, 505-510 Kazunori Ikebukuro et al., Nucleic Acids Research, 33 (12), e108 Nasa Savory et al., Biosensors and Bioelectronics, Volume 26, Issue 4, 15 December 2010, Pages 1386-1391

  Although SELEX is an excellent method that can create an aptamer that specifically binds to any target substance, it requires considerable labor to create an aptamer. Also, as it is a method utilizing chance, applying SELEX for a considerable amount of time may not result in an aptamer that specifically binds to a target substance with satisfactory affinity.

  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 G-quadruplex structure exists in genomic DNA and takes a quadruplex structure, and a promoter including G-quadruplex structure is also known. The present inventors indicate that, when the G-quadruplex structure is contained in the promoter, the gene product of a structural gene controlled by the promoter or the gene product downstream of the cascade in which the gene product is contained is in the G-quadruplex structure. I thought that binding would cause feedback inhibition. And, as it is, single-stranded nucleic acid having the same base sequence as the G-quadruplex structure-containing region contained in the promoter can be used as an aptamer specifically binding to a substance to which the promoter binds. I got it . The present inventors further considered that, when a G-quadruplex structure is present in the mRNA, the mRNA may be bound to the gene product encoded by the mRNA. And, a new finding that a single-stranded nucleic acid having the same base sequence as the G-quadruplex structure-containing region contained in mRNA can be used as an aptamer that specifically binds to the gene product encoded by the mRNA as it is It was completed the present onset Akira obtained.

That is, the present invention is identical to the region containing the G-quadruplex structure present in the mRNA has a nucleotide sequence, the aptamer or the aptamer to bind to related gene product of a gene product or the gene product said mRNA encodes It has nucleotide sequence and 95% or more sequence identity, and includes the G-quadruplex structure comprises chemically synthesizing an aptamer that binds to the gene product or the related gene product wherein the aptamer binds, aptamers Provide a manufacturing method of Further, the present invention is that provides an aptamer produced by the method of the present invention. As circumferential intelligence, uracil thymine in RNA in DNA, since it is intended to both adenine specifically pairing, in the present invention, uracil thymine in RNA in DNA "Identity of 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 by uracil are understood as “having the same base sequence”.

According to the present invention, an aptamer having the same base sequence as a region containing a G-quadruplex structure present in mRNA and binding to a gene product encoded by the mRNA or a related gene product of the gene product have nucleotide sequence and 95% or more sequence identity, and includes the G-quadruplex structure comprises chemically synthesizing an aptamer that binds to a gene product the aptamer binds, method for producing aptamers are provided Ru.

  The present invention provides, for the first time, a method for producing an aptamer that binds to a desired target substance without using SELEX requiring time-consuming manipulation. 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 is chemically analyzed using a commercially available nucleic acid synthesizer without using SELEX. Since the synthesis | combination can manufacture the aptamer which couple | bonds with a target substance, the aptamer couple | bonded with desired target protein can be manufactured much more easily compared with the conventional method.

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

  The G-quadruplex (also called G-quartet, G4, guanine quadruple chain, etc.) structure has two to three faces where four guanine bases in the nucleic acid are close to form a tetramer by hydrogen bonding. It is a structure and is known to have a quadruple helical structure. G-quadruplex structures are known to exist in human telomeres and promoters.

The present inventors function as an aptamer as they are, as a single-stranded polynucleotide having the same base sequence as a region containing a G-quadruplex structure (referred to as "G-quadruplex structure-containing region") contained in a promoter. The aptamer was found to bind to a gene product of a structural gene controlled by the promoter or a related gene product of the gene product . In here, there "related gene product of a gene product", for example, upstream or downstream of the gene product of the cascade of the gene product is included, a protein or the like that function in cooperation with its gene product. Related gene products 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 website of Jabion published by the National Institute of Informatics, the National Institute of Genetics, and the like. According to the method provided on this website, since the co-occurrence score is also known, it is possible to consider that a gene product strongly co-occurring with a certain gene product is a highly relevant gene product. For example, proteins highly co-occurring with vascular endothelial growth factor (VEGF) specifically tested in the following examples have high co-occurrence in the order of KDR, FLT1, HIF1A, and platelet-derived growth factor (PDGF) PDGFB, PDGFRA, and retinoblastoma-related protein (RB-1) have high co-occurrence in the order of E2F1, CDKN2A, and TP53. The G-quadruplex-forming sequence in the promoter of these genes can be searched by the method described later, and that sequence can be synthesized to confirm the binding ability.

The method according to the reference example of the present invention can be implemented, for example, as follows. 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. Of these various promoters, those containing the G-quadruplex structure are available in the methods of the present invention. Promoters known to contain the G-quadruplex structure are available in the methods of the invention. Examples of promoters known to contain the G-quadruplex structure include c-Kit 87up, which is a promoter of 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. Also, in the examples described later, the promoters for vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF-A) and retinoblastoma related protein (RB-1) were selected as promoters. It is also known that the G-quadruplex structure is contained in the promoter of

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

The present inventors also found that the method of the reference example of the present invention is also useful for creating an aptamer that binds to a protein having a heparin binding domain. In the following reference example , hepatocyte growth factor (HGF) which is a protein having such a heparin-binding domain, heparin-binding EGF (HBEGF (Heparin-binding EGF-like growth factor)), and platelet-derived Experiments were conducted on growth factors, annexin II (Anexin II) or apolipoprotein E4 (ApoE4), and any of these was successfully created by the method of the reference example of the present invention to create an aptamer that binds to each of them. There is.

  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 is present in the promoter, the G-quadruplex structure-containing region may span 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 indicates the number of nucleotides). The aptamer may be DNA or RNA, but is preferably chemically stable DNA. Chemical synthesis of DNA and RNA can be easily performed using a commercially available automatic synthesizer. In addition, there are various vendors undertaking chemical synthesis of DNA and RNA having a predetermined base sequence, and these vendors can also be requested. In addition, synthesizing a nucleic acid in a cell-free system such as RNA synthesis by in vitro transcription, DNA synthesis by a nucleic acid amplification method such as PCR, etc. is also included in the “chemical synthesis” in the present invention. In the present specification and claims, the term "having a base sequence" means that the bases are aligned as such, for example, "an aptamer having the base sequence represented by SEQ ID NO: 1". Means a 34-mer aptamer whose base sequence is arranged as SEQ ID NO: 1.

  Next, it is confirmed that the produced aptamer binds to the gene product of a 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, and the label is detected and the protein is detected by silver staining. If it is positive, it can be confirmed that the aptamer and the gene product are bound. In SPR measurement, a gene product is immobilized on a chip for SPR measurement, the gene product and aptamer are brought into contact, and the aptamer and gene product are measured by measuring whether or not the aptamer binds to the gene product with a commercially available SPR measurement device It can be checked whether or not to be combined.

  If it is confirmed that the aptamer binds to the gene product, the aptamer is produced by chemical synthesis. Generally, it is known that even if the base sequence of the aptamer binding to the target substance is slightly changed, the binding property to the target substance can be maintained in many cases, so that it binds to the gene product. The nucleotide sequence of the aptamer in which the is confirmed may be modified while maintaining the binding 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 95% or more sequence identity. In this case, the modified aptamer also needs to have a G-quadruplex structure. Since the G-quadruplex structure is considered to bind to the gene product, it is preferable to modify the portion other than the G-quadruplex structure, and even if the portion 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 (inserting gaps as necessary) so that the bases of the two base sequences whose sequences are to be compared match as much as possible. The number is divided by the total number of bases, meaning the numerical value expressed as a percentage. When the numbers of bases of the two are different, they are divided by the number of longer bases. Software for calculating sequence identity is well known and published free of charge on the Internet. As described above, in the present invention, thymine in DNA and uracil in RNA are understood to be "identical bases", so thymine and uracil are identical when comparing the sequence identity of DNA sequences and RNA sequences. Calculate as the base of

  Aptamers having the same base sequence as the G-quadruplex structure-containing region are subjected to a known in-situ computer evolution method to further enhance the affinity to the gene product by modifying the base sequence. It is possible. The in-computer evolution method is a method invented by the co-inventor of the present application and 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 confirmed to bind to a gene product, for example, a plurality of regions not necessary for maintaining the G-quadruplex structure, for example, about 3 to 5 mers, are obtained. Random exchange (shuffle) between corresponding regions of the aptamer or crossover by genetic algorithm is performed. Then, random single base substitutions are introduced into the above regions after shuffle or intersection. The introduction of these shuffles or crossovers and single base substitutions is done by computer. Then, an aptamer having a new base sequence generated by a computer is chemically synthesized to form a nucleic acid library, which is subjected to the cycle of SELEX. When producing the second nucleic acid library after completion of the first cycle, the amount of aptamers having a region derived from the aptamer having the highest binding ability order is maximized, and the ratio is decreased as the order is lowered. . As described above, the efficiency of SELEX evolution can be enhanced by artificially introducing mutations by shuffle or crossover and single base substitution in a computer.

Although this in-house evolution method performs SELEX with the assistance of a computer, since the original aptamer already has the ability to bind to the gene product, with SELEX starting from an entirely zero, The level of difficulty is different, and the ability to bind to a gene product 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 secured even if the base sequence becomes considerably different from the original base sequence. Therefore, the sequence identity is not necessarily limited to 90% or more. However, as described above, since the in-computer evolution method is to introduce mutations in parts other than the G-quadruplex structure, the G-quadruplex structure is maintained. Furthermore, as specifically described in Example 12 below, in the in-house evolution method, among the aptamers obtained in each generation, the target sequence can be determined by examining the sequence of an aptamer having high binding ability with the target gene product. Sequence motifs that exert the ability to bind to gene products 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, the in-computer evolution method is performed, and when the second generation is performed, aptamers of the first generation and the second generation Among them, when the base sequences of aptamers having high binding ability to HGF were examined, it was found that many sequences share the sequence motif of ggtggagggg (SEQ ID NO: 57). Then, when the portion of this sequence motif was fixed and the third generation was performed, it was possible to obtain an aptamer with about 240 times the binding specificity (Sp _(HGF) ) for _{HGF as the} parent sequence. Thus, according to the in-house evolution method, a sequence motif that exerts a high binding ability may be found in the middle, and in this case, this sequence motif is fixed and subsequent generations are performed. This makes it possible to obtain an aptamer having high binding ability more efficiently.

As described above, according to the method of the reference example of the present invention, easily using the promoter sequence of the structural gene of the desired target protein to easily produce an aptamer that binds to the desired target protein without performing SELEX. The excellent effect of being able to

  Aptamers have the property of binding specifically to a target protein, and can therefore be used for quantification and detection of the target protein (for example, Patent Document 2). In this case, an aptamer can be labeled and used. Examples of labels include well-known labels such as fluorescent labels, radio labels, enzyme labels, chemiluminescent labels and the like. It is well known that the binding affinity to the target substance is maintained even if these labels are bound to the end of the aptamer directly or through a spacer sequence, and in the following example, the fluorescent label FITC conjugated aptamer is used. It is used. Aptamers added with these labels can be used for measurement (detection or quantification) of gene products. Methods of measuring target substances using labeled aptamers are well known in the art, and can be performed, for example, in the same manner as immunoassays using a labeled antibody.

  In addition, when the target protein has some physiological activity related to a disease, and promoting its expression leads to the treatment of the disease, the aptamer produced by the method of the present invention is used as a nucleic acid drug. Can. That is, when the aptamer is administered to a living body, part of the gene product produced in the cell binds to the aptamer and can not bind to the promoter, so that the transcriptional repression by the gene product is reduced. In addition, when the aptamer can bind to the gene product and neutralize its activity, it is also possible to inhibit the activity of the gene product, and also when the inhibition of the gene product leads to the treatment of the disease It can be used as When an aptamer is used as a nucleic acid drug, other structures such as polyethylene glycol (PEG) chain can be added, for example, to improve nuclease resistance. It is well known to increase the nuclease resistance of aptamers by attaching a PEG chain to the end of the aptamer, and has already been put to practical use. The size of the PEG chain is not particularly limited, but usually, the molecular weight is about 10,000 to 30,000, preferably the molecular weight is about 15,000 to 25,000, and the number of PEG chains may be one or two, but two is preferable. . Such PEG chains can be attached via the addition of a well-known amino linker at the end of the aptamer. When two PEG chains are linked, an aminolinker having a plurality of amino groups such as a lysineated aminolinker can be used to link the PEG chain 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 based on the binding to the aptamer produced by the method of the present invention It is also possible to screen. By administering the substance found by the drug screening, the inhibition of the promoter is reduced by the gene product, 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 the mRNA, the mRNA may be bound to the gene product encoded by the mRNA. And, a new finding that a single-stranded nucleic acid having the same base sequence as the G-quadruplex structure-containing region contained in mRNA can be used as an aptamer that specifically binds to the gene product encoded by the mRNA as it is It was completed the present onset Akira obtained. That is, the production method of aptamer by application onset Ming has the same base sequence region containing the G-quadruplex structure present in the mRNA, and related gene product of a gene product or the gene product said mRNA encodes The chemical synthesis of the aptamer to be bound or the aptamer having a sequence identity of 90% or more with the nucleotide sequence of the aptamer and comprising a G-quadruplex structure and binding to the gene product to which the aptamer binds.

According to the present onset bright, although the mRNA itself may be used as the RNA aptamer, the aptamer need not be RNA, as described above, it is preferred to chemically synthesize the aptamer DNA. In this case, of course, uracil in mRNA is thymine in DNA, and as described above, those in which uracil in mRNA is replaced with thymine in DNA are understood to have “the same base sequence”. .

For even A aptamer of the present invention, similarly to the reference example of the present invention described above, preferably size is 30Mer～100mer, also preferably further enhance the affinity for a target substance by a computer evolution method. Also, as in the reference example, it can be used as a nucleic acid drug, and in that case, it is preferable to carry out nuclease resistant modification such as PEGylation. In the following examples, the present gun onset bright, have succeeded in creation of aptamers that bind to each of the vascular endothelial growth factor and platelet-derived growth factor.

Hereinafter, the present invention will be more specifically described based on examples. However, the present invention is not limited to the following examples. In the following examples, Examples 1 to 7 are respectively read as Reference Examples 1 to 7.

Example 1, Comparative Example 1
Analysis of the ability of Gq structures formed in the VEGF promoter region to bind to VEGF

experimental method
VEGF promoter known to contain G-quadruplex structure (Sun, D., Guo, Rusche, JJ & Hurley, LH Facilitation of a structural transition in the polypurine / polypyrimidine tract within the proximal promoter region of the human Nucleic acid Res. 33, 6070-6080 (2005)) was selected as a promoter for VEGF gene by the presence of potassium and G-quadruplex-interactive agents. G-quadruplex (hereinafter sometimes abbreviated as “Gq”) structure-forming sequence (VEGF promoter Gq (SEQ ID NO: 1), formed in the VEGF promoter region (location on the genome: chr6: 43, 737, 633-43, 739, 852) Example 1) Evaluation of the binding ability of an oligo DNA (VEGF promoter Gq- (SEQ ID NO: 2), Comparative Example 1) designed to substitute G bases involved in Gq formation with T bases and not form Gq. Did. Each oligo DNA was synthesized by a commercially available automatic DNA synthesizer. All nucleotide 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 modified) were mixed to a final concentration of 1.3 μM or 500 nM, respectively, and shaken at room temperature for 30 minutes. Thereafter, each sample was applied to a 12% non-denaturing polyacrylamide gel, and electrophoresis was performed at room temperature and 20 mA (constant current) for 25 minutes. After electrophoresis, FITC fluorescence of each aptamer was detected by Typhoon 8600 (trade name, manufactured by GE Healthcare). In addition, VEGF was stained by silver staining.

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

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

  While an increase in SPR signal was not observed when VEGF promoter Gq- was injected, an aptamer concentration-dependent increase in SPR signal was observed when VEap121 or VEGF promoter Gq was used. As a result of calculating the dissociation constant of each aptamer by curve fitting, it was calculated with VEap121: 510 nM and VEGF promoter Gq: 240 nM.

Example 2, Comparative Example 2
Analysis of the ability of Gq structures formed in the PDGF promoter region to bind to PDGF by SPR

experimental method
PDGF-A promoter which is 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 hypersensitive element Nucleic acid Res. 35, 7698-7713 (2007)) was selected as a promoter of the PDGF-A promoter and the modulation of the PDGF-A promoter activity by TMPyP4. The Gq structure-forming sequence (PDGF promoter Gq (SEQ ID NO: 4), Example 2) formed in the PDGF-A promoter region (location on the genome: chr7: 556, 612-562, 845) and the G bases involved in Gq formation Evaluation of the binding ability to PDGF-AA of a sequence (PDGF promoter Gq- (SEQ ID NO: 5), Comparative Example 2) designed to substitute T bases and not form Gq was performed by SPR measurement. These sequences are shown in Table 2. All nucleotide numbers correspond to Human Feb. 2009 (GRCh37 / hg19) Assembly.

  TBS buffer was used as a running buffer. Each aptamer prepared to 7.8 to 1000 nM is injected using a sensor chip CM5 on which approximately 9000 RU of PDGF-AA is immobilized by an amine coupling method, and PDGF-AA (GenBank Accession No .: The binding of each aptamer with P04085 was observed by SPR measurement.

Result and consideration
The results of SPR measurement of the PDGF promoter Gq sequence are shown in FIG. While an increase in SPR signal was not observed when PDGF promoter Gq- was injected, an increase in aptamer concentration-dependent SPR signal was observed when PDGF promoter Gq was used. 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, Comparative Example 3
Analysis of the ability of Gq structures present in the promoter region of RB-1 to bind to RB-1

experimental method
The RB-1 promoter is known to contain the 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)) was selected as a promoter. Gq structure-forming sequence (RB-1_Gq promoter Gq (SEQ ID NO: 6), Example 3) formed in the RB-1 promoter region (location on the genome: chr13: 48877460-48878501), and G involved in Gq formation The binding ability to RB-1 of the sequence (RB-1_Gq_Mut (SEQ ID NO: 7), Comparative Example 3) designed to substitute T bases for bases and not form Gq was examined by gel shift assay. All nucleotide 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, the sequence is shown in Table 3 below) was mixed and shaken at room temperature for 30 minutes. Thereafter, each sample was applied to a 12% non-denaturing polyacrylamide gel, and electrophoresis was performed for 20 minutes at room temperature and 20 mA (constant current). After electrophoresis, the fluorescence of TAMRA of each DNA was detected by Typhoon 8600 (trade name). In addition, RB-1 was stained by silver staining.

Results and Discussion After electrophoresis, the results of fluorescence detection by Typhoon (trade name) and the results of detection of RB-1 protein by silver staining are shown in FIG. 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 band of RB-1 was observed. This band was not observed in the other lanes. This indicates that the Gq structure formed in the RB-1 promoter binds to the RB-1 protein.

Examples 4 to 7
Creation of an aptamer for a protein having a heparin binding domain Method
(1) The sequences near the transcription start point of the HGF, HBEGF, PDGFB, and Annexin II genes were obtained using the USCS Genome Browser. The sequence of transcription start point ± 1 kbp was obtained for HGF, and the sequence of CpG island present near the transcription start point was obtained for HBEGF, PDGFB and Annexin II. The sequence acquired both plus strand and minus strand sequences.

(2) Among the sequences, sequences capable of forming a quadruplex structure were extracted under the following conditions.
HGF: A sequence containing G at two or more consecutive positions at 4 or more positions, a sequence between consecutive Gs and consecutive Gs was within 7 mer, and a sequence having a total length of 40 mer or less was extracted.
PDGFB, HBEGF, Annexin II: A sequence containing at least 3 consecutive Gs, and a sequence between consecutive Gs and a continuous G was within 7 mer, and a sequence having a total length of 30 mer was extracted. When there is no sequence that falls under the above conditions, it contains 4 or more of 2 or more consecutive G, and the sequence between continuous G and continuous G 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 gel shift assay for PDGFB and Annexin II using SPR for HGF and HBEGF.

(4) SPR was performed by the following method.
HGF and HBEGF were dissolved 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) and immobilized on sensor chip CM5 by amine coupling method . Thereafter, the synthesized oligonucleotides were 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, injected onto a sensor chip, and changes in 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.
The 5'-end FITC-modified oligonucleotides were folded in TBSK buffer (10 mM Tris-HCl, 150 mM NaCl, 100 mM KCl, pH 7.4). Then, 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% non-denaturing polyacrylamide gel, and electrophoresis was performed 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 quadruple helix structure present in the HGF and HBEGF promoters. Prepare each oligonucleotide in TBSK buffer (10 mM Tris-HCl, 150 mM NaCl, 100 mM KCl pH 7.4) or TBS buffer (10 mM Tris-HCl, 150 mM NaCl pH 7.4) to a final concentration of 2 μM And folding (95 ° C. for 5 minutes and then cooling to 25 ° C. over 30 minutes). The prepared sample was placed in a quartz cell with an optical path length of 10 mm, and a CD spectrum was measured using a Model J-820 circular dichroism spectrometer. The sensitivity is 1000 mdeg, the wavelength range is 220 nm to 320 nm, the data acquisition interval is 1 nm, the scanning speed is 500 nm / min, the response is 1 sec, the bandwidth is 5.0 nm, and the integration number is 10 times. It measured.

2. result
(1) Sequences capable of forming a quadruple DNA structure were searched from each promoter region, 9 sequences from the HGF promoter, 14 sequences from the HBEGF promoter, 8 sequences from the PDGFB promoter and 7 sequences from the Annexin II promoter , Could be extracted (Table 4).

(2) Three oligonucleotides of HGF-PQS1 (SEQ ID NO: 8), HGF-PQS7 (SEQ ID NO: 9) and HGF-PQS8 (SEQ ID NO: 10) were analyzed by SPR to analyze whether each oligonucleotide binds to HGF An increase in SPR signal dependent on DNA concentration was observed in A, 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) When each oligonucleotide binds to HBEGF or analyzed by SPR, an increase in DNA concentration-dependent SPR signal is observed in two sequences of HBEGF-PQS8 and HBEGF-PQS9, and these are aptamers of HBEGF Was shown (Figure 5). The dissociation constants for 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 or analyzed by gel shift assay, all oligonucleotides bind to PDGF-BB in 8 sequences (SEQ ID NOS: 13 to 20 in order from the top in Table 4) It was shown that these are aptamers that bind to PDGF-BB (FIG. 6). In FIG. 6, + indicates the presence of PDGF-BB, and-indicates the lane in the absence of PDGF-BB.

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

(6) HGF-PQS1 to PQS9 CD spectra measured HGF-PQS1 and HGF-PQS7 only show binding to HGF, positive peak near 260 nm and negative peak near 240 nm in the presence of potassium (Figure 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 HBEGF-PQS1 to PQS14 CD spectra were measured, only HBEGF-PQS8 and HBEGF-PQS9 in which binding was observed to HBEGF showed a positive peak near 260 nm and a negative peak near 240 nm in the presence of potassium (Figure 9). That is, it was shown that HBEGF-PQS8 and HBEGF-PQS9 form parallel DNA quadruplexes and bind to HBEGF.

Examples 8 to 10
1. Method
(1) The full-length sequence of RNA transcribed from the VEGFA, PDGFA and PDGFB genes was obtained using the USCS Genome Browser.

(2) Among the sequences, sequences capable of forming an RNA quadruple helix structure were extracted under the following conditions.
Sequences containing two or more consecutive Gs at four positions, and between the continuous Gs and the consecutive Gs were within 14 mers, and a sequence having 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), VEGFA is dissolved in 10 mM sodium acetate buffer (pH 6.0), and immobilized on sensor chip CM5 by amine coupling method did. The synthesized oligonucleotides were then 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, then 25 ° C. Cool down to 30 minutes). Each oligonucleotide was diluted to various concentrations, injected onto a sensor chip, and changes in 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) When sequences capable of forming a quadruple-helix RNA structure were searched among the transcribed RNA sequences of each gene, RNAs of 159 sequences from the VEGFA gene, 247 sequences from the PDGFA gene, and 194 sequences from the PDGFB gene Could be extracted. The following sequences were respectively selected and synthesized from these (Table 5).

(2) Analysis of each oligonucleotide binding to VEGFA or analysis by SPR shows that an RNA concentration-dependent increase in SPR signal is observed in all sequences, indicating that they are RNA aptamers binding 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) about 31 nM, and VEGFA RNA3 (SEQ ID NO: 24) with a dissociation constant of about 300 nM. (Figure 10).

(3) When each oligonucleotide binds to PDGF-AA or analyzed by SPR, an RNA concentration-dependent increase in SPR signal is observed in all sequences, and these are RNA aptamers that bind to PDGF-AA. It was shown to be. PDGFA RNA1 (SEQ ID NO: 25) and PDGFA RNA2 (SEQ ID NO: 26) bound PDGFA with a dissociation constant of about 30 nM, respectively. (Figure 11).

(4) When each oligonucleotide binds to PDGF-BB or analyzed by SPR, an increase in SPR signal dependent on RNA concentration is observed in all sequences, and these are RNA aptamers that bind to PDGF-BB. It was shown to be. 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, respectively, with a dissociation constant of about 34 nM (FIG. 12).

Example 11
1. Search for a G4 formation prediction sequence in the promoter region of the target protein gene The sequence of ± 1 kbp from the transcription start point of the gene encoding ApoE4, which is a protein having a heparin binding domain, is referred to as Genome Browser (http: //genome.ucsc. It extracted using edu / cgi-bin / hgGateway). From the extracted sequences, sequences satisfying the following conditions were searched using QGRS Mapper (http://bioinformatics.ramapo.edu/QGRS/index.php).
(i) Total length of 30 mer or less (ii) The obtained sequence containing G of two or more consecutive lines at an interval of 7 mer or less was defined 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 was diluted with 10 mM acetic acid buffer (pH 4.0), and approximately 900 RU was immobilized on the sensor chip CM4 by amine coupling method. After that, the aptamer candidate sequence (Table.) Folded in TBS buffer (10 mM Tris-HCl, 150 mM NaCl, 100 mM KCl, pH 7.4) is diluted to various concentrations and added to the sensor chip Changes in SPR signal were measured. After measurement, the dissociation constant (K _d ) was calculated by curve fitting.

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

Example 12
The three obtained HGF aptamers are referred to as HGFap1, HGFap2 and HGFap3, and the two HBEGF aptamers are referred to as HBEGFap1 and HBEGFap2. Moreover, in the in silico maturation of the HGF aptamer, the binding specificity (Sp _(HGF) ) with respect to HGF of the evaluated sequence is Sp _(HGF) = [the binding constant K _a when targeting HGF] / [HBEGF as the target The binding constant K _a ] (Sp _(HGF) = K _{a (HGF)} / K _{a (HBEGF)} ) in the case of the _reaction was defined.

Preparation of first generation sequences and evaluation of specificity First, Sp _(HGF) is determined for first generation parent sequences HGFap1, HGFap2 and HGFap3, and each sequence is replicated based on the ratio of Sp _(HGF) values, for a total of 20 An array of books was made. Subsequently, a pair was randomly made out of 20 sequences, and crossover was made at any one point. Thereafter, 10% of mutations were randomly introduced into each of the 20 sequences for the position and the type of the base, to obtain first generation sequences (1R01 to 1R20). The nucleotide sequences of 1R01 to 1R20 are shown in this order in SEQ ID NOs: 58 to 77.

HGF and HBEGF were immobilized on sensor chip CM5 by amine coupling method using 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) . As a dilution buffer for proteins, 10 mM HEPES buffer (pH 6.5) was used for HGF, and 10 mM acetic acid buffer (pH 5.0) was used for HBEGF. Thereafter, each first-generation sequence folded in TBS buffer (10 mM Tris-HCl, 150 mM NaCl, 100 mM KCl) was subjected to various concentrations (fc 1000 nM, 500 nM, 100 nM) using TBS buffer. , 50 nM, 0 nM), and the change in SPR signal was measured when HGF or HBEGF was added to the immobilized sensor chip. The 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 determined by curve fitting, and Sp _(HGF) was calculated.

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

Generation and specificity evaluation of third generation motif fixed sequences First generation sequence and second generation sequence, sequence group with high Sp _(HGF) , sequence group with low Sp _(HGF) , not bound to HGF and HBEGF When classified into sequence groups and compared the sequences, many sequences of the sequence group having high Sp _(HGF) share a sequence motif of GGTGGAGGGG in common. Therefore, among the first-generation sequences and second-generation sequences, sequences having a GGTGGAGGGGG sequence motif and having high Sp _(HGF) (1R01, 1R05, 1R08, 1R09, 2R07) are used to prepare a third-generation motif-fixed sequence The parent array for The obtained third generation motif fixed parent sequence was replicated, crossed and mutagenized in the same manner as the first generation parent sequence and the second generation parent sequence to obtain a third generation motif fixed sequence (3R01mfix to 3R20mfix). The nucleotide sequences of 3R01mfix to 3R20mfix are shown in this order in SEQ ID NOs: 98 to 117. Evaluation of the 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 high Sp _(HGF) values compared to the parent sequence were obtained (Table 6). In the second generation sequence, a highly specific sequence showing Sp _(HGF) approximately 13 times that of the parent sequence Sp _(HGF) (minimum value: 1.9) 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)} The sequence (3R14mfix) showing is obtained (Table 8). From this, it was possible to improve the specificity of the HGF aptamer obtained by the method of the present invention by in-house evolution.

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

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

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

Claims (2)

Nucleotide sequence, the nucleotide sequence represented by any of SEQ ID NO: 22-24, or sequence identity between the nucleotide sequences of these is the nucleotide sequence is 95% or more, and includes a G-quadruplex structure, vessel Aptamers that bind to endothelial growth factor. Nucleotide sequence, the nucleotide sequence represented by any of SEQ ID NO: 25 to 31, or sequence identity between the nucleotide sequences of these is the nucleotide sequence is 95% or more, and includes a G-quadruplex structure, platelets Aptamers that bind to derived growth factors.