WO2019112178A1 - Aptamère d'adn se liant spécifiquement à l'odam et utilisation associée - Google Patents

Aptamère d'adn se liant spécifiquement à l'odam et utilisation associée Download PDF

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WO2019112178A1
WO2019112178A1 PCT/KR2018/012958 KR2018012958W WO2019112178A1 WO 2019112178 A1 WO2019112178 A1 WO 2019112178A1 KR 2018012958 W KR2018012958 W KR 2018012958W WO 2019112178 A1 WO2019112178 A1 WO 2019112178A1
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odam
aptamer
nucleic acid
associated protein
dna
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구만복
이방현
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고려대학교 산학협력단
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/18—Dental and oral disorders

Definitions

  • the present invention relates to a nucleic acid aptamer specifically binding to odontogenic ameloblast-associated protein (ODAM) and a use thereof, and more particularly, to a nucleic acid aptamer which specifically binds to odontogenic ameloblast-associated protein (ODAM) And a composition, a sensor and a kit for detection.
  • ODAM odontogenic ameloblast-associated protein
  • Odontogenic ameloblast-associated protein is an essential protein in the connective epithelium of the gum (gingiva) to adhere to teeth. If periodontal disease occurs in the gum and adhesion between the tooth and epithelium is lost, odontogenic ameloblast-associated protein (ODAM) is released into the periodontal pouch and detected in the oral gingival crevicular fluid and saliva. Based on this, ODAM (Odontogenic AMeloblast-Associated protein) is used as an early periodontal disease-specific biomarker that can diagnose early periodontal disease (periodontal disease, implant periosteum) early (KR 10-2016-0060288).
  • Gingival inflammation is a chronic disease in which the patient's own subjective symptoms are delayed, and the inflammation progresses while the patient does not feel, resulting in severe destruction of the tissues surrounding the tooth (Lee et al., J Biol Chem. Vol. Pp. 14740-53, 2015). Therefore, it is very common to require dentures in a delayed state in a state requiring extraction, which causes great economic loss by requiring implant and denture treatment.
  • the currently used diagnostic method can not show the current state of the disease because it can only show the result of the disease, and it also has a limit showing high false positive. Therefore, it is necessary to develop a diagnostic tool that is easy to use in the dental or other medical field, and further, a self-diagnostic kit that can be easily self-diagnosed at home.
  • Aptamers are single-stranded DNA or RNA molecular structures that have high specificity and affinity for a particular target obtained from a random nucleic acid library of about 10 12 to 14 diversity.
  • aptamers are nucleic acid constructs, unlike antibodies, which are used in the field of sensors, and therefore have excellent thermal stability. Since they are synthesized in vitro, they do not require animals or cells, Economical and unlimited target material, it is possible to synthesize aptamers for various targets such as biomolecular substances such as proteins and amino acids, low molecular organic chemicals such as environmental hormones, antibiotics, residual drugs, bacteria and viruses.
  • aptamers for various target substances are being made, and due to the characteristics of aptamers that combine with target substances, specificity and strong affinity, a lot of studies recently applied aptamers to new drug development, drug delivery systems and biosensors .
  • aptamers are very suitable for the measurement of ODAM (Odontogenic Ameloblast-Associated protein) concentration in human body, and nanobiotechnology using this can be applied to the detection of specific proteins associated with periodontal disease-related diseases.
  • ODAM Oxdontogenic Ameloblast-Associated protein
  • aptamer The most important factor in the development of aptamer is to distinguish between DNA (or RNA) bound to a target substance and DNA that is not bound.
  • a method has been attempted in which a target is fixed or a DNA random library is fixed to distinguish DNA.
  • immobilization method has a low immobilization yield and requires much time and cost to analyze the immobilization yield itself.
  • the possibility of non-specific binding of DNA to the separating material (magnetic beads, columns, etc.) used for immobilization can not be totally excluded, and loss of DNA pool may occur in the process of separating DNA bound to a fixed target It is still a problem.
  • heavy metal ions are difficult to immobilize.
  • the fluorescence resonance energy transfer (FRET) diagnosis method is suitable for quick and simple checking of the operation of the aptamer due to its convenient preparation and simple operation.
  • Graphene oxide is a quencher that absorbs the fluorescent energy of FAM and the light energy of FAM that emits near 520 nm when it is within a certain distance. This phenomenon is called FRET, and this experiment consists of the following principle.
  • the 3 'end of single stranded DNA is labeled with FAM and reacted with graphene oxide.
  • Graphenol oxide nonspecifically binds to single-stranded DNA through a ⁇ - ⁇ bond, whereby the fluorescence of FAM is quenched by graphene oxide.
  • the affinity between the single strand DNA and the target is greater than the affinity with the graphene oxide, the DNA is separated from the graphene oxide and fluorescence of FAM appears again.
  • the affinity between the DNA and the target is lower, the target protein can not separate DNA from the graphene oxide and the fluorescence remains in the extinction state.
  • fluorescence can be measured to confirm the binding force between the single strand DNA and the target (Zhu Y et al., ACS Appl. Interfaces, Vol 7 -7496, 2015), and can also be used to determine the presence or absence of a target using an aptamer.
  • SPR diagnosis is a very sensitive diagnostic method and is useful for sensitively detecting the presence of a target.
  • the plasmon formed on the metal surface is fluctuated to cause a resonance wavelength change, resulting in a change in refractive index on the surface.
  • Immobilizing the aptamer using streptavidin-biotin binding on the SPR chip and flowing the target over it will increase the SPR signal in proportion to the amount of target associated with the aptamer.
  • target proteins can be quantitatively detected by SPR (Lee et al., Anal. Chem., Vol. 80 (8), pp. 2867-2873, 2008).
  • the sensitivity of the aptamer to the target can be improved by screening the aptamer duo capable of binding to different sites or several identical sites of one target protein and using it for target detection.
  • ODAM Oxdontogenic AMeloblast-Associated protein
  • ODAM Oxdontogenic AMeloblast-Associated protein
  • the present invention provides a nucleic acid aptamer which specifically binds to Odontogenic Ameloblast-Associated Protein (ODAM) represented by any one of SEQ ID NOS: 1 to 18.
  • ODAM Odontogenic Ameloblast-Associated Protein
  • the present invention also provides a method for detecting odor using the nucleic acid aptamer.
  • the present invention also provides a composition for diagnosing orphan-related diseases comprising the nucleic acid aptamer.
  • the present invention also provides a complex wherein the nucleic acid aptamer is immobilized on a solid support.
  • the present invention also provides compositions, sensors, and kits for detecting mutations that contain the nucleic acid aptamer or the complex.
  • Figure 1 shows a FRET assay using an aptamer.
  • FIG. 2 is a schematic diagram of a DNA aptamer preparation process capable of specifically binding ODAM (Odontogenic AMeloblast-Associated protein) using graphene oxide.
  • ODAM Oxdontogenic AMeloblast-Associated protein
  • FIG. 3 is a graph showing that the amount of ssDNA binding to odontogenic ameloblast-associated protein (ODAM) obtained from the selection round is increased
  • FIGS. 4 to 21 show the secondary structure of a DNA aptamer capable of specifically binding to ODAM (Odontogenic AMeloblast-Associated protein) of SEQ ID NO: 1 to SEQ ID NO: 18 according to the present invention.
  • ODAM Oxdontogenic AMeloblast-Associated protein
  • FIG. 22 shows the results of FRET analysis of the affinity for ODAM (Odontogenic AMeloblast-Associated protein) of DNA aptamer.
  • FIG. 23 is a graph showing the results of a comparison between ODAM (Odontogenic Ameloblast-Associated protein) using DNA aptamers OD 5R-35 and OD 5R-64 and alpha amylase, which is the most abundant protein in saliva, ) Of the aptamer was analyzed by SPR.
  • ODAM Oxdontogenic Ameloblast-Associated protein
  • FIG. 24 shows the result of SPR analysis after the OD 5R-35 aptamer duo was reacted in a sandwich manner with respect to the ODAM (Odontogenic Ameloblast-Associated protein) concentration using DNA aptamer OD 5R-64.
  • a DNA aptamer capable of specifically binding to odontogenic ameloblast-associated protein was selected using the GO-SELEX process.
  • ODAM odontogenic ameloblast-associated protein
  • " GO-SELEX process " according to the present invention is a method of selectively extracting DNA or RNA having a high binding capacity for a specific molecule from an arbitrarily synthesized DNA or RNA assembly to obtain a DNA binding sequence of the molecule Immobilization-free screening of aptamers assisted by graphene oxide, Chemical Communications, 48, 15, 2071-2073).
  • the present invention relates to a nucleic acid aptamer that specifically binds to Odontogenic Ameloblast-Associated Protein (ODAM) represented by any one of SEQ ID NOS: 1-18.
  • ODAM Odontogenic Ameloblast-Associated Protein
  • T is U in the nucleic acid sequence.
  • the nucleic acid aptamer of the present invention may be a nucleic acid aptamer of any base sequence that specifically binds to Odontogenic Ameloblast-Associated Protein (ODAM) selected by the GO-SELEX process.
  • ODAM Odontogenic Ameloblast-Associated Protein
  • nucleic acid aptamer capable of specifically binding to an ODAM (Odontogenic AMeloblast-Associated protein) of the present invention can be produced by a method comprising the steps of:
  • step e separating the single-stranded nucleic acid from the PCR product obtained in the step and adding the single-stranded nucleic acid to the mixed solution of step a) to repeat the graphene-based screening process.
  • step d) of the method for preparing a nucleic acid aptamer PCR is performed using a primer having fluorescein attached to one of the primer pairs, followed by separation of the unmodified single strand DNA through electrophoresis And further comprising
  • the present invention relates to a method for detecting Odontogenic Ameloblast-Associated Protein (ODAM) comprising contacting a nucleic acid aptamer of the present invention with a sample.
  • ODAM Odontogenic Ameloblast-Associated Protein
  • the sample may be selected from the group consisting of tissue, cell, blood, serum, plasma, saliva, sputum and urine, but is not limited thereto. It is obvious that the sample is not limited to the above, provided that it is a sample separated from a mammal, preferably a human body, a sample or secretory fluid which can be obtained by minimal invasion, a sample of an in vitro cell culture fluid component, and the like. More preferably, it may be saliva.
  • the detection may be performed by a method selected from the group consisting of Surface Plasmon Resonance (SPR), Fluorescence Resonance Energy Transfer (FRET) and gold nanoparticle-based chromaticity analysis , But is not limited thereto.
  • SPR Surface Plasmon Resonance
  • FRET Fluorescence Resonance Energy Transfer
  • gold nanoparticle-based chromaticity analysis but is not limited thereto.
  • the detection is performed by modifying the gold nanoparticle surface to fix the aptamer to the gold nanoparticle surface by covalent bonding or the like so that aggregation occurs due to the fact that two or more gold nanoparticle distances approach each other when the target substance is present.
  • the gold nanoparticle solution changes its color from red to blue, or when the aptamer is physically adsorbed on the surface of the undoped gold nanoparticles and when the target material is present, , It is possible to detect the defect by using the property that the color changes due to the phenomenon that the gold nanoparticles are separated from the surface.
  • the detection can also detect an impairment with magnetic resonance analysis SPR (Surface Plasmon Resonance) equipment using a gold chip. That is, it is possible to detect an impairment by using a method of measuring an increasing response unit value when a target material is present by fixing an aptamer to a gold chip by covalent bonding or the like.
  • SPR Surface Plasmon Resonance
  • the detection can also detect an impairment by the graphene oxide-based FRET method.
  • graphene oxide is a quencher that absorbs the light energy of FAM, which emits near 520 nm when it is within a certain distance from a fluorescent substance called FAM.
  • FRET fluorescent substance
  • the 3 'end of single stranded DNA is labeled with FAM and reacted with graphene oxide.
  • Graphene oxide binds nonspecifically with a single stranded DNA (aptamer of the present invention) through a pi-pi bond, whereby the fluorescence of FAM is quenched by graphene oxide.
  • the aptamers of the present invention may be chemically synthesized by methods well known in the art.
  • the nucleic acid aptamer of the present invention may be one in which a sugar residue (for example, ribose or deoxyribose) of each nucleotide is modified so as to improve binding, stability and the like to orgasm.
  • a sugar residue for example, ribose or deoxyribose
  • the moiety modified in the sugar residue include those obtained by substituting oxygen atoms at the 2'-, 3'-and / or 4'-positions of the sugar residue with other atoms.
  • Examples of the types of the formulas include fluorination, O-alkylation (e.g. O-methylation, O-ethylation), O-allylation, S-alkylation (e.g., S-methylation, S- , And amination (e.g., -NH).
  • Such modification of sugar residues can be carried out by a method known per se (Sproat et al., (1991) Nucle. Acid Res 19, 733-738; Cotton et al., (1991) Nucl. 19, 2629-2635; Hobbs et al., (1973) Biochemistry 12, 5138-5145).
  • the nucleic acid aptamer of the present invention may also be a nucleic acid base (for example, purine, pyrimidine) modified (for example, chemically substituted) so as to enhance the binding to the oram.
  • a nucleic acid base for example, purine, pyrimidine
  • modifications include, for example, a 5-site pyrimidine modification, a 6 and / or 8-site purine modification, a modification in an exocyclic amine, a substitution with 4-thiouridine, a 5-bromo or 5-iodo- And substitution with a silyl group.
  • the phosphate group contained in the nucleic acid aptamer of the present invention may be modified so as to have resistance to nuclease and hydrolysis.
  • each R or R ' is independently H, or substituted or unsubstituted alkyl (e.g., methyl, ethyl).
  • the linking group -O-, -N- or -S- are exemplified, and they can be bonded to adjacent nucleotides through these linking groups.
  • Variations in the present invention may also include 3 ' and 5 ' modifications such as capping.
  • Deformations may also be achieved by the addition of one or more excipients such as polyethylene glycols, amino acids, peptides, inverted dT, nucleic acids, nucleosides, Myristoyl, Lithocolic-oleyl, Docosanyl, Lauroyl, Stearoyl, Palmitoyl, Oleoyl, Linoleoyl, other lipids, steroids, cholesterol, caffeine, vitamins, pigments, fluorescent substances, anticancer agents, toxins, enzymes, Radioactive material, biotin or the like at the terminal thereof. See, for example, U.S. Patent No. 5,660,985, U.S. Patent No. 5,756,703 for such modifications.
  • OD 5R-35 which is the nucleic acid aptamer represented by SEQ ID NO: 4 showing the highest affinity of the nucleic acid aptamers of SEQ ID NOS: 1 to 18, and nucleic acid aptamer
  • SEQ ID NOS: 1 to 18 the nucleic acid aptamer represented by SEQ ID NOS: 1 to 18, and nucleic acid aptamer
  • the present invention relates, in another aspect, to a composition for diagnosing ovarian-related diseases comprising the nucleic acid aptamer.
  • othman-related diseases can be used as long as the disease occurs when the concentration of otham is out of the normal range, but it is preferably selected from the group consisting of periodontitis, gingivitis, epithelial periodontal cancer, intracranial aneurysm and implant periosteum But the present invention is not limited thereto.
  • the diagnosis of ovarian-related disease may include contacting the aptamer with a sample selected from tissues, cells, blood, serum, plasma, saliva, sputum and urine. It is obvious that the sample is not limited to the above, provided that the sample is separated from a mammal, preferably a human body, and may contain a sample such as a sample or secretory fluid capable of securing by minimal invasion, a sample of an in vitro cell culture medium component .
  • the present invention relates to a complex wherein the nucleic acid aptamer of the present invention is immobilized on a solid support.
  • the solid support may be selected from the group consisting of a substrate, a resin, a plate, a filter, a cartridge, a column, and a solid support in the form of a porous material.
  • the substrate may be selected from the group consisting of a nickel-PTFE (polytetrafluoroethylene) substrate, a glass substrate, an apatite substrate, a silicon substrate, a gold substrate, a silver substrate, a graphene oxide substrate and an alumina substrate
  • a nickel-PTFE polytetrafluoroethylene
  • the present invention is not limited thereto.
  • the substrate of the present invention can be a chip, a protein chip, or the like, and can be a substrate such as a nickel-PTFE (polytetrafluoroethylene) substrate, a glass substrate, an apatite substrate, a silicon substrate, gold, silver, An alumina substrate, and the like, and these substrates are coated with a polymer or the like.
  • a nickel-PTFE polytetrafluoroethylene
  • the resin may be selected from the group consisting of agarose particles, silica particles, copolymers of acrylamide and N, N'-methylenebisacrylamide, particles of polystyrene crosslinked divinylbenzene, particles of crosslinked dextran with epichlorohydrin, , A crosslinked polymer of allyldextran and N, N'-methylenebisacrylamide, a monodispersed synthetic polymer, a monodispersed hydrophilic polymer, Sepharose or Toyopearl, and the like.
  • a resin obtained by bonding various functional groups may be used.
  • the solid carrier may be useful for the purification of oram, the detection or quantification of opaque protein.
  • the nucleic acid aptamer of the present invention can be immobilized on a solid support by a known method. For example, a method of introducing an affinity substance or a predetermined functional group into an aptamer of the present invention and subsequently immobilizing the affinity substance or a predetermined functional group on the solid carrier using the affinity substance or a predetermined functional group.
  • the specific functional group may be a functional group capable of providing a coupling reaction, and examples thereof include an amino group, a thiol group, a hydroxyl group, and a carboxyl group.
  • the aptamer can be immobilized on the substrate surface.
  • a method of detecting an oram protein using an immobilized solid phase carrier can be performed by, for example, spotting a DNA aptamer of the present invention on a chip together with a fluorescent substance, a coloring substance or an antibody, By the reaction, it is possible to quickly measure the level of a trace amount of a test sample, for example, a trace amount of blood contained in the blood.
  • the bound DNA aptamer-otham complex can be separated by using a magnet. By separating the ocum again from the complex, As shown in FIG.
  • the present invention relates to a composition for detecting odor comprising a nucleic acid aptamer of the present invention or a complex wherein the nucleic acid aptamer is immobilized on a solid support.
  • the composition for detecting odalum of the present invention is for detecting an impairment and can be used in any form using the nucleic acid aptamer of the present invention for detecting odor.
  • the nucleic acid aptamer of the present invention is immobilized on a magnetic bead to bind an open cell, the bound DNA aptamer-adide complex can be separated using a magnet, and the open cell is separated again from the complex It is possible to selectively detect Odam.
  • a magnetic bead having the nucleic acid aptamer immobilized thereon may be filled in a column, and then the sample containing the impurity may be passed through to selectively remove odor.
  • the nucleic acid aptamer OD 5R-64 of SEQ ID NO: 6, which shows the highest affinity of the nucleic acid aptamers of SEQ ID NOS: 1 to 18, is coated with SPR chip and gold nanoparticle- As a result of conducting the sandwich analysis based on OD 5R-35, it was confirmed that sensitivity to oram was increased when the sandwich method was used.
  • the present invention provides a method for detecting a cancer, comprising the steps of: (a) contacting a sample to a kit or sensor containing a complex wherein the aptamer of SEQ ID NO: 6 is immobilized on a solid support or an aptamer of SEQ ID NO: 6; And (b) further contacting an aptamer represented by SEQ ID NO: 4; To an ODAM (Odontogenic Ameloblast-Associated protein) detection method.
  • ODAM Oxiontogenic Ameloblast-Associated protein
  • the aptamer represented by SEQ ID NO: 4 may be characterized by being labeled with gold nanoparticles, but the present invention is not limited thereto.
  • the present invention also relates to a detection sensor comprising an aptamer specifically binding to said sesame or a complex in which said nucleic acid aptamer is immobilized on a solid support.
  • a detection sensor system containing an aptamer that specifically binds to the hypothalamus or a complex in which the nucleic acid aptamer is immobilized on a solid support may be provided in the form of a kit.
  • the kit for detecting odds may take the form of a bottle, a tub, a sachet, an envelope, a tube, an ampoule, etc., which may be partly or wholly made of plastic, glass, paper, foil, wax And the like.
  • the container may be fitted with a cap which is initially part of the container or can be fully or partially detachable, which may be attached to the container by mechanical, adhesive, or other means.
  • the container may also be equipped with a stopper, which is accessible to the contents by the injection needle.
  • the kit may include an external package, and the external package may include instructions for use of the components.
  • the aptamer specifically binding to orophyllum according to the present invention can also provide a composition for detecting or eliminating oram as well as a specific detection of oedam, and it is an object of the present invention to a person skilled in the art It is.
  • the present invention relates to a method for eliminating odam using aptamer specifically binding to odam or a complex in which the nucleic acid aptamer is immobilized on a solid support.
  • the aptamer can fill the column with fixed beads and pass through a specimen containing odam to remove the impurities.
  • Example 1 DNA pool synthesis with arbitrary base sequence
  • a DNA pool with a primer region for PCR and an arbitrary base at the center was synthesized as follows.
  • the DNA pool used in the present invention was obtained from Genotech Inc. Korea for chemical synthesis.
  • SEQ ID NO: 19 CCATTCGTACGCAACAGG
  • SEQ ID NO: 20 GCATTCAGAGCCATCCAC
  • Example 2 Screening of ODAM (odontogenic ameloblast-associated protein) -specific DNA aptamers using GO-SELEX process
  • the random DNA pool synthesized in Example 1 was added to a buffer solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), mixed with a graphene oxide solution, reacted at room temperature for 30 minutes, DNA was removed by centrifugation and then the supernatant was removed.
  • the target mixture was mixed with the mixed solution for 30 minutes at 4 ° C. Then, the graphene oxide was removed by centrifugation, and the DNA isolated from the graphene oxide was obtained by the ethanol precipitation method.
  • the amount of DNA specifically binding to the odontogenic ameloblast-associated protein (ODAM) was measured. As a result, the ODAM obtained in each selection step as shown in FIG. 3, Odontogenic AMeloblast-Associated protein was increased by increasing the amount of DNA bound to the protein.
  • Example 3 Preparation of a DNA aptamer pool capable of binding ODAM (Odontogenic AMeloblast-Associated protein)
  • PCR was performed using a known primer region.
  • the PCR reaction product was purified using a purification kit, and then subjected to polyacrylamide gel electrophoresis to make double stranded DNA into a single strand.
  • the 10% polyacrylamide gel contains 6M urea and 20% formamide, resulting in two bands after electrophoresis, in which the two strands of DNA are denatured by electrophoresis, The DNA strands attached to the resein are located on the underside, while the DNA strands that are not attached are located below.
  • the DNA bands with fluororesin were cut out and subjected to gel extraction. DNA isolated by ethanol precipitation was obtained again.
  • the DNA pool is then mixed with the buffer solution containing the first target to initiate a new screening process.
  • the SELEX process is referred to as a selection.
  • four selection processes and two counter selection processes are used to determine ODAM, Odontogenic AMeloblast -Associated protein) was obtained.
  • the resulting pool of DNA was cloned using a Qiagen cloning kit. DNA was extracted from the obtained colonies and subjected to base analysis. As a result, a nucleic acid construct specifically binding to ODAM (Odontogenic AMeloblast-Associated protein) was obtained Respectively.
  • Table 1 shows the results of analyzing the nucleotide sequence of DNA that specifically binds to ODAM (Odontogenic AMeloblast-Associated protein) with high affinity. Further, the results of predicting the secondary structure of the odontogenic ameloblast-associated protein (ODAM) aptamer by using the m-fold program are shown in Figs. 4 to 21.
  • ODAM odontogenic AMeloblast-Associated protein
  • DNA aptamer has specificity for odontogenic ameloblast-associated protein (ODAM).
  • (F-F0) / F0 (F-F0) / F0 (F-F0) / F0 is defined as fluorescence when F0 is reacted with odontogenic ameloblast-associated protein (ODAM)
  • ODAM odontogenic ameloblast-associated protein
  • the SPR gold chip was treated with DTPA (dithiophosphoric acid), and the gold surface of the chip was immersed in 200 mM EDC / 50 mM NHS (Carbodiimide / N-Hydroxysuccinimide) for 1 hour, 100 ug / ml streptavidin 1 hour 30 minutes, 50 mM ethanol Amin for 30 minutes, biotin-labeled aptamer for 1 hour and 1 hour for 50ug / ml BSA for 1 hour at room temperature.
  • the process of washing the gold surface with the third-stage distilled water at each step and drying it with nitrogen gun was repeated.
  • an SPR chip fixed with an aptamer was inserted into an SPR machine and a buffer solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) for 10 minutes and ODAM, Odontogenic Ameloblast-Associated
  • the response unit (RU) value which was measured by flowing the buffer solution for 10 minutes and the buffer solution for 30 minutes at 30 ⁇ l / sec, is shown in FIG. 23, It was confirmed that the RU value was increased in proportion to the concentration, and it was confirmed that the RU value did not bind to the alpha amylase.
  • Example 6 Analysis of binding force as an aptamer duo with ODAM (Odontogenic AMeloblast-Associated protein)
  • OD 5R-35 and OD 5R-64 among the aptamers that specifically bind to 18 different odamorphous AMELoblast-Associated proteins are combined with odontogenic AMELoblast-Associated protein (ODAM) as an aptamer duo And whether they can do it.
  • ODAM odontogenic AMELoblast-Associated protein
  • the SPR chip in which the aptamer OD 5R-64 was immobilized was inserted into the SPR machine by the method of Example 5, and then 10 minutes of PBS, 10 minutes of the sample and 10 minutes of PBS were sequentially flowed.
  • the sample was ODNM, 1 nM, 2 nM, 4 nM, 8 nM, 16 nM, 32 nM and 64 nM odontogenic ameloblast-associated protein
  • the odontogenic ameloblast-associated protein (ODAM) was washed away, and then OD 5R-35 labeled with gold nanoparticles was flowed for 10 minutes.
  • OD 5R-35 labeled with gold nanoparticles unattached to odontogenic ameloblast-associated protein (ODAM) was washed away by re-flowing PBS for 10 minutes.
  • the SPR chip including OD 5R-64 and the OD 5R-35 labeled with gold nanoparticles were simultaneously used as a duo to measure the binding force.
  • the aptamer duo was sandwiched When used, it was confirmed that the sensitivity increased.
  • ODAM Oxdontogenic AMeloblast-Associated protein
  • ODAM odontogenic AMeloblast-Associated protein
  • the target can be selectively measured in a sample containing a very small amount of saturating agent by using the method of the present invention and the apparatus for detecting and detecting a DNA aptamer of the present invention. Therefore, it is useful for diagnosing ODAM (odontogenic ameloblast-associated protein) -related disease by measuring the odontogenic ameloblast-associated protein (ODAM) concentration in human blood easily and quickly.

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Abstract

La présente invention concerne un aptamère d'acide nucléique se liant spécifiquement à la protéine associée aux améloblastes odontogènes (ODAM) et une utilisation associée. Selon la présente invention, un aptamère d'acide nucléique pour la détection de l'ODAM, et un procédé, une composition, un capteur et un kit de détection de l'ODAM, comprenant chacun le même aptamère, sont utiles pour détecter l'ODAM présente dans un échantillon sur la base de la haute spécificité et de l'affinité de l'aptamère d'ODAM pour l'ODAM.
PCT/KR2018/012958 2017-12-06 2018-10-30 Aptamère d'adn se liant spécifiquement à l'odam et utilisation associée WO2019112178A1 (fr)

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KR1020170166589A KR102009016B1 (ko) 2017-12-06 2017-12-06 오담(ODAM, Odontogenic Ameloblast-Associated protein)에 특이적으로 결합하는 DNA 앱타머 및 그의 용도
KR10-2017-0166589 2017-12-06

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