WO2014010640A1 - Procédé de production d'aptamère - Google Patents

Procédé de production d'aptamère Download PDF

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WO2014010640A1
WO2014010640A1 PCT/JP2013/068901 JP2013068901W WO2014010640A1 WO 2014010640 A1 WO2014010640 A1 WO 2014010640A1 JP 2013068901 W JP2013068901 W JP 2013068901W WO 2014010640 A1 WO2014010640 A1 WO 2014010640A1
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aptamer
sequence
base sequence
gene product
binds
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PCT/JP2013/068901
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Japanese (ja)
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一典 池袋
吉田　亘
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国立大学法人東京農工大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised

Definitions

  • 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.
  • 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).
  • SELEX® Systematic Evolution of Ligands by EXponential Enrichment
  • 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.
  • 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.
  • 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.
  • 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.
  • G-quadruplex structure exists in genomic DNA and takes a quadruplex structure
  • promoters containing the G-quadruplex structure are also known.
  • 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.
  • 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.
  • 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.
  • 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.
  • transcription product includes not only mRNA but also mRNA precursor before splicing.
  • 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”.
  • 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
  • a method for producing an aptamer which comprises chemically synthesizing an aptamer.
  • 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.
  • 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.
  • 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.
  • each curve shows the result of having analyzed the coupling
  • FIG. 4 shows the result of having analyzed the coupling
  • 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
  • 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
  • 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.
  • 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.
  • VEGF vascular endothelial growth factor
  • 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),
  • PDGF platelet-derived growth factor
  • PDGFB platelet-derived growth factor
  • PDGFRA platelet-derived growth factor
  • RB-1 retinoblastoma-related protein
  • 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.
  • 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, HIF1 ⁇ gene promoter, PDGFR ⁇ gene promoter, KRAS gene promoter, TERT gene promoter, MYB gene promoter, and the like.
  • 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.
  • VEGF vascular endothelial growth factor
  • PDGF-A platelet-derived growth factor
  • RB-1 retinoblastoma-related protein
  • 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.
  • 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.
  • hepatocyte growth factor hepatocyte growth factor
  • HGF hepatocyte growth factor
  • Hparin-binding EGF heparin-binding EGF-like growth factor
  • platelet derived proteins having such heparin binding domains.
  • growth factors annexin II (Annexin II) or apolipoprotein E4 (ApoE4)
  • aptamers that bind to each of them were successfully created by the method of the present invention for any of these.
  • an aptamer having the same base sequence as the G-quadruplex structure-containing region of the selected promoter is chemically synthesized.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the aptamer 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the in-computer evolution method introduces mutations in parts other than the G-quadruplex structure as described above, the G-quadruplex structure is maintained.
  • Example 12 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.
  • Example 12 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.
  • 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).
  • 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.
  • aptamers 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).
  • the aptamer can be labeled and used.
  • 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.
  • 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.
  • 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.
  • 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.
  • PEG polyethylene glycol
  • 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.
  • 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.
  • 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.
  • 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.
  • the aptamer production method 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.
  • 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.
  • 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”. .
  • 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.
  • 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.
  • Example 1 and Comparative Example 1 Analysis of binding ability of Gq structure formed in VEGF promoter region to VEGF
  • 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.
  • Gq 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.
  • Gq G-quadruplex
  • VEGF 165 (GenBank Accession No .: NP_001165097) and each aptamer (5′-FITC modification) were mixed to a final concentration of 1.3 ⁇ M 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.
  • CM5 trade name, manufactured by GE Healthcare
  • 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.
  • VEGF-promoter-Gq- 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.
  • 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
  • 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 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 ⁇ ⁇ ⁇ ⁇ ⁇ buffer was used as the running buffer.
  • PDGF-AA GenBank Accession No .: The binding between each aptamer and P04085) was observed by SPR measurement.
  • FIG. 2 shows the results of SPR measurement of the PDGF promoter Gq sequence.
  • PDGF promoter Gq- 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.
  • VEGF promoter Gq 18 nM.
  • 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.
  • 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.
  • Typhoon trade name
  • 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.
  • 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 .
  • 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
  • the flow rate was 30 ⁇ l / min.
  • the dissociation constant (Kd) was calculated by Curve fitting analysis.
  • 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.
  • CD spectra were measured for sequences capable of forming a DNA quadruplex structure present in the HGF and HBEGF promoters.
  • 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.
  • HGF-PQS1 SEQ ID NO: 8
  • HGF-PQS7 SEQ ID NO: 9
  • 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.
  • Annexin II-PQS6 SEQ ID NO: 21
  • Annexin II-PQS6 SEQ ID NO: 21
  • HGF-PQS1 to PQS9 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.
  • HBEGF-PQS8 and HBEGF-PQS9 formed a parallel DNA quadruplex structure and bound to HBEGF.
  • 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.
  • 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).
  • 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
  • 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
  • the flow rate was 30 ⁇ l / min.
  • the dissociation constant (Kd) was calculated by Curve fitting analysis.
  • 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
  • VEGFA RNA3 (SEQ ID NO: 24) was about 300 nM. (FIG. 10).
  • 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 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.
  • HGFap1, HGFap2, HGFap3, and the two HBEGF aptamers are referred to as HBEGFap1, HBEGFap2.
  • Sp (HGF) includes a target / [HBEGF [association constant K a in the case of the HGF targeted]
  • HGF 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.
  • 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
  • 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.
  • 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 ⁇ L / min. After measurement, Ka (HGF) and Ka (HBEGF) were obtained by curve fitting, and Sp (HGF) was calculated.
  • 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.
  • HGF dissociation constant to HGF K d
  • HGF Dissociation constant for HBEGF K a
  • HGF Binding constant for HGF K a
  • HBEGF Sp HGF
  • HGF Dissociation constant for HGF K d
  • HGF 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) )
  • HGF dissociation constant to HGF K d
  • HGF Dissociation constant for HBEGF K a
  • HGF Binding constant for HGF K a
  • HBEGF Sp HGF

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Abstract

La présente invention concerne un procédé de production d'un aptamère, qui se lie spécifiquement à une substance cible, sans utiliser SELEX. Le procédé de production d'un aptamère comprend la synthèse chimique d'un aptamère, qui présente une séquence de gène codant pour une protéine cible ou une séquence de base identique à une région contenant une structure G-quadruplex, présente dans une séquence de contrôle ou un produit de transcription de la séquence de gène et qui se lie à la protéine cible ou à un produit de gène associé de la séquence de gène ou la synthèse chimique d'un aptamère qui présente une identité de séquence de 90 % ou plus avec la séquence de base de l'aptamère, contient une structure G-quadruplex et se lie à la protéine cible, à laquelle se lit l'aptamère ou au produit de gène associé.
PCT/JP2013/068901 2012-07-10 2013-07-10 Procédé de production d'aptamère WO2014010640A1 (fr)

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WO2019131727A1 (fr) * 2017-12-27 2019-07-04 コニカミノルタ株式会社 Procédé pour évaluer un médicament
JP2020000051A (ja) * 2018-06-26 2020-01-09 国立大学法人東京農工大学 アプタマーの創製が可能か否かの判定方法及びそれを利用したアプタマーの創製方法

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019131727A1 (fr) * 2017-12-27 2019-07-04 コニカミノルタ株式会社 Procédé pour évaluer un médicament
JPWO2019131727A1 (ja) * 2017-12-27 2021-02-12 コニカミノルタ株式会社 医薬の評価方法
JP2020000051A (ja) * 2018-06-26 2020-01-09 国立大学法人東京農工大学 アプタマーの創製が可能か否かの判定方法及びそれを利用したアプタマーの創製方法
JP7224011B2 (ja) 2018-06-26 2023-02-17 国立大学法人東京農工大学 アプタマーの創製が可能か否かの判定方法及びそれを利用したアプタマーの創製方法

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