WO2023177242A1 - Dna aptamer specifically binding to apolipoprotein b100 and use of dna aptamer - Google Patents
Dna aptamer specifically binding to apolipoprotein b100 and use of dna aptamer Download PDFInfo
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- WO2023177242A1 WO2023177242A1 PCT/KR2023/003549 KR2023003549W WO2023177242A1 WO 2023177242 A1 WO2023177242 A1 WO 2023177242A1 KR 2023003549 W KR2023003549 W KR 2023003549W WO 2023177242 A1 WO2023177242 A1 WO 2023177242A1
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- dna aptamer
- dyslipidemia
- vascular disease
- apob100
- aptamer
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- A—HUMAN NECESSITIES
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- A61P9/00—Drugs for disorders of the cardiovascular system
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2200/00—Function of food ingredients
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- A23V2200/326—Foods, ingredients or supplements having a functional effect on health having effect on cardiovascular health
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/3262—Foods, ingredients or supplements having a functional effect on health having an effect on blood cholesterol
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
- C12Q2525/10—Modifications characterised by
- C12Q2525/205—Aptamer
Definitions
- the present invention relates to a DNA aptamer that specifically binds to apolipoprotein B100 (ApoB100) and the use of the DNA aptamer.
- ApoB100 apolipoprotein B100
- Dyslipidemia refers to a state in which total cholesterol, low density lipoprotein (LDL) cholesterol, and triglycerides in the blood are increased, or the level of HDL (high density lipoprotein) cholesterol is decreased. Most cases of dyslipidemia are caused by causes such as obesity, diabetes, and drinking, but in some cases, it is caused by an increase in specific lipids in the blood due to genetic factors.
- LDL low density lipoprotein
- HDL high density lipoprotein
- dyslipidemia does not show any special symptoms, but if left uncontrolled for a long period of time, cholesterol can build up on the walls of blood vessels, narrowing or blocking them, causing cardiovascular and cerebrovascular diseases such as angina, myocardial infarction, and stroke (cerebral infarction).
- Dyslipidemia can be broadly divided into primary and secondary causes.
- the primary cause is primary dyslipidemia caused by a fat-centered diet, lack of exercise, and genetic factors
- the secondary cause is underlying diseases such as hypothyroidism, chronic liver disease, nephrotic syndrome, pregnancy, or drug use. It refers to dyslipidemia caused by factors such as: In cases of genetic predisposition such as familial hypercholesterolemia or familial hypertriglyceridemia, xanthomas, xanthomatoma, hepatomegaly, and renal enlargement may appear, but they are usually asymptomatic and are mostly discovered through blood tests.
- the purpose of the present invention is to provide a DNA aptamer that specifically binds to apolipoprotein B100 (ApoB100) composed of a specific base sequence.
- ApoB100 apolipoprotein B100
- Another object of the present invention is to provide a composition and kit for detecting ApoB100 containing the DNA aptamer, and a method for detecting ApoB100 using the DNA aptamer.
- Another object of the present invention is to provide a composition and kit for diagnosing dyslipidemia or vascular disease containing the DNA aptamer, and a method of providing information for diagnosing dyslipidemia or vascular disease using the DNA aptamer. It is done.
- Another object of the present invention is to provide a use of the DNA aptamer for treating dyslipidemia or vascular disease.
- the present invention provides a DNA aptamer that specifically binds to apolipoprotein B100 (ApoB100) consisting of a base sequence selected from the group consisting of the base sequences shown in SEQ ID NOs: 1 to 16, respectively. .
- ApoB100 apolipoprotein B100
- the present invention provides a composition or kit for detecting ApoB100 containing the DNA aptamer.
- the present invention provides a method for detecting ApoB100 including the step of reacting the DNA aptamer with a sample.
- the present invention provides a composition or kit for diagnosing dyslipidemia or vascular disease containing the DNA aptamer.
- the present invention provides a method for providing information on diagnosing dyslipidemia or vascular disease, including the step of reacting the DNA aptamer with a sample.
- the present invention provides a pharmaceutical composition for preventing or treating dyslipidemia or vascular disease containing the DNA aptamer as an active ingredient.
- the present invention provides a health functional food for preventing or improving dyslipidemia or vascular disease containing the DNA aptamer as an active ingredient.
- the present invention provides a method for preventing, improving, or treating dyslipidemia or vascular disease, comprising administering the DNA aptamer to an individual.
- the present invention provides the use of the DNA aptamer for use in a method of preventing, improving, or treating dyslipidemia or vascular disease.
- the DNA aptamer according to the present invention specifically binds to ApoB100 with high affinity and can be usefully used to detect ApoB100 by reducing cholesterol, or to diagnose or treat diseases such as dyslipidemia and vascular disease.
- Figure 1 is a graph showing the results of confirming the binding affinity to ApoB100 according to the number of SELEX times in one embodiment of the present invention.
- Figure 2 is a graph showing the results of confirming the cytotoxicity of the DNA aptamer in an example of the present invention.
- Figure 3 is a graph showing the results of confirming the cholesterol-reducing effect of DNA aptamers in one embodiment of the present invention.
- Figure 4 is a graph showing the results of confirming the cholesterol reduction effect by combined treatment of DNA aptamer and lecithin in one embodiment of the present invention.
- the present invention provides a DNA aptamer that specifically binds to apolipoprotein B100 (ApoB100) consisting of a base sequence selected from the group consisting of the base sequences shown in SEQ ID NOs: 1 to 16, respectively.
- ApoB100 apolipoprotein B100
- apolipoprotein B100 is one of the lipoproteins that transport liver or intestinal cholesterol to tissues, and is recognized by LDL receptors in various types of cells.
- ApoB100 is a protein included in the largest ApoB group, consisting of 4,563 amino acids, and exists in two isoforms in plasma: ApoB100 and ApoB48.
- the level of ApoB100 is related to vascular disease caused by cholesterol and is known to be a better predictor than LDL-C.
- the ApoB100 may include all polypeptide sequences known as ApoB100 in the art.
- aptamer refers to a nucleic acid molecule that can bind to a specific substance with high affinity and specificity.
- the aptamer can be prepared by a method well known in the art, and the method can be appropriately modified as needed.
- the DNA aptamer according to the present invention may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16, respectively. As long as the DNA aptamer maintains the characteristic of specifically binding to ApoB100, it is at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% homologous to the base sequences shown in SEQ ID NOs: 1 to 16, respectively. You can have
- the DNA aptamer may be modified with the sugar residue of each nucleotide, specifically ribose or deoxyribose, to increase binding and stability to the target substance.
- the above formula may be one in which one or more oxygen atoms selected from the group consisting of the 2', 3', and 4' positions of the sugar residue are replaced with another atom. Examples of modifications may include fluorination, O-alkylation, O-allylation, S-alkylation, S-allylation, amination, etc. Modification of the sugar residue as described above can be performed by conventional methods.
- nucleotide base of the DNA aptamer may be modified to increase binding to the target substance.
- the modification of the nucleotide base includes pyrimidine modification at position 5, purine modification at position 6, purine modification at position 8, modification at extracyclic amine, substitution with 4-thiouridine, and substitution with 5-bromo. Or it may include substitution with 5-iodine-uricyl, etc.
- the phosphate group of the DNA aptamer may be modified to be resistant to nucleases, hydrolases, etc.
- the phosphate group may be substituted with thioate, dithioate, amidate, formacetal, 3'-amine, etc.
- the DNA aptamer may include modifications at the 3' end or 5' end, and the modifications include capping, biotinyl, polyethyl glycol, amino acids, peptides, nucleic acids, nucleosides, and myristoyl.
- lithocolic-oleyl docosanyl, lauroyl, stearoyl, palmitoyl, oleoyl, linoleoyl , lipids, steroids, cholesterol, caffeine, vitamins, pigments, fluorescent substances, anticancer drugs, toxins, enzymes, radioactive substances, biotin, etc. may be added.
- the present invention provides a composition or kit for detecting ApoB100 containing the DNA aptamer.
- the DNA aptamer included in the composition and kit for ApoB100 detection according to the present invention may have the characteristics described above.
- the DNA aptamer may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16.
- the DNA aptamer included in the composition for detecting ApoB100 according to the present invention may be a conjugate labeled with a detection agent such as a chromogenic enzyme, a fluorescent substance, a radioactive isotope, or a colloid.
- a detection agent such as a chromogenic enzyme, a fluorescent substance, a radioactive isotope, or a colloid.
- the chromogenic enzyme may be peroxidase, alkaline phosphatase, or acid phosphatase
- the fluorescent substance may be thiourea (FTH), 7-acetoxycoumarin-3-yl, fluorescein-5-yl, or fluorescein-6-yl.
- the DNA aptamer may include a ligand that can specifically bind to the DNA aptamer.
- the ligand may be a conjugate labeled with a detector such as a chromogenic enzyme, a fluorescent substance, a radioactive isotope, or a colloid, and a ligand treated with streptavidin or avidin.
- the detection composition of the present invention may further include distilled water or a buffer solution to keep their structures stable.
- a kit containing the detection composition according to the present invention can be bound to a solid substrate to facilitate subsequent steps, such as washing the DNA aptamer contained therein or separating the complex.
- synthetic resin nitrocellulose, glass substrate, metal substrate, glass fiber, microspheres, or microbeads may be used as the solid substrate.
- polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF, or nylon may be used as the synthetic resin.
- the kit may be manufactured by conventional manufacturing methods known to those skilled in the art, and may further include buffers, stabilizers, inactive proteins, etc.
- the kit is used for ultra-high throughput screening through a fluorescence method performed by detecting the fluorescence of a fluorescent substance attached as a detector to detect the amount of reagent, or a radiography method performed by detecting the radiation of a radioisotope attached as a detector.
- screening (HTS) system the SPR (surface plasmon resonance) method that measures plasmon resonance changes on the surface in real time without labeling the detector, or the SPRI (surface plasmon resonance imaging) method that confirms the SPR system by imaging it.
- the present invention provides a method for detecting ApoB100 including the step of reacting the DNA aptamer with a sample.
- the DNA aptamer used in the ApoB100 detection method according to the present invention may have the characteristics described above.
- the DNA aptamer may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16.
- the sample may include any sample for which ApoB100 is to be detected. Additionally, the sample may be pretreated using methods such as anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, continuous extraction, centrifugation, or gel electrophoresis.
- the present invention provides a composition or kit for diagnosing dyslipidemia or vascular disease containing the DNA aptamer.
- the DNA aptamer included in the composition for diagnosing dyslipidemia or vascular disease according to the present invention may have the characteristics described above.
- the DNA aptamer may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16.
- the compositions and kits may also have the characteristics described above.
- dyslipidemia refers to a state in which total cholesterol, LDL cholesterol, and triglycerides in the blood are increased or HDL cholesterol is decreased, and is a risk factor for developing cardiovascular disease.
- the dyslipidemia may include all types of dyslipidemia known in the art.
- the dyslipidemia may include hyperlipidemia, hypercholesterolemia, or hypertriglyceridemia.
- vascular disease refers to a disease that occurs when blood vessels become narrowed due to accumulation of fat or blood clots such as blood clots.
- the vascular disease may include all types of vascular diseases known in the art.
- the vascular disease may include not only diseases caused by narrowing of blood vessels, but also diseases caused by rupture of blood vessels.
- the vascular disease may include arteriosclerosis, atherosclerosis, atherosclerosis, cerebral infarction, angina pectoris, peripheral vascular occlusive disease, myocardial infarction, diabetic retinopathy, or hypertensive retinopathy.
- the present invention provides a method for providing information on diagnosing dyslipidemia or vascular disease, including the step of reacting the DNA aptamer with a sample.
- the DNA aptamer used in the method for providing information for diagnosing dyslipidemia or vascular disease according to the present invention may have the characteristics described above.
- the DNA aptamer may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16.
- the dyslipidemia or vascular disease may have the characteristics described above.
- the sample may include any sample that is intended to diagnose dyslipidemia or vascular disease.
- the sample may be a mammal-derived sample, and specifically, it may be a human-derived sample.
- the sample may be any one or more selected from the group consisting of blood, saliva, tear fluid, urine, synovial fluid, mucus, cells, and tissues. Additionally, the sample may be pretreated in the manner described above before being used in the method according to the present invention.
- the present invention provides a pharmaceutical composition for preventing or treating dyslipidemia or vascular disease containing the DNA aptamer as an active ingredient.
- the DNA aptamer included in the pharmaceutical composition for preventing or treating dyslipidemia or vascular disease according to the present invention may have the characteristics described above.
- the DNA aptamer may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16.
- the dyslipidemia or vascular disease may have the characteristics described above.
- the pharmaceutical composition according to the present invention may contain 10 to 95% by weight of the DNA aptamer according to the present invention, which is an active ingredient, based on the total weight of the composition.
- the pharmaceutical composition of the present invention may further include one or more active ingredients that exhibit the same or similar functions in addition to the above-mentioned effective ingredients.
- the pharmaceutical composition of the present invention may include carriers, diluents, excipients, or mixtures thereof commonly used in biological products.
- Any pharmaceutically acceptable carrier can be used as long as it is suitable for delivering the composition in vivo.
- the carrier is Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or mixtures thereof.
- conventional additives such as antioxidants, buffers, bacteriostatic agents, etc. can be added as needed.
- diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be added.
- the composition of the present invention may be formulated as an oral preparation or a parenteral preparation.
- Oral preparations may include solid preparations and liquid preparations.
- the solid preparation may be a tablet, pill, powder, granule, capsule, or troche, and such solid preparation may be prepared by adding at least one excipient to the composition.
- the excipient may be starch, calcium carbonate, sucrose, lactose, gelatin, or mixtures thereof.
- the solid preparation may contain a lubricant, examples of which include magnesium styrate and talc.
- the liquid preparation may be a suspension, oral solution, emulsion, or syrup.
- the liquid formulation may contain excipients such as wetting agents, sweeteners, fragrances, preservatives, etc.
- the parenteral preparations may include injections, suppositories, powders for respiratory inhalation, aerosols for sprays, powders and creams.
- the injection may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, etc.
- the non-aqueous solvent or suspension may be propylene glycol, polyethylene glycol, vegetable oil such as olive oil, or injectable ester such as ethyl oleate.
- the present invention provides a health functional food for preventing or improving dyslipidemia or vascular disease containing the DNA aptamer as an active ingredient.
- the DNA aptamer included in the health functional food for preventing or improving dyslipidemia or vascular disease according to the present invention may have the characteristics described above.
- the DNA aptamer may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16.
- the dyslipidemia or vascular disease may have the characteristics described above.
- the DNA aptamer of the present invention can be added as is to food or used together with other foods or food ingredients.
- the content of the active ingredient added may be determined depending on the purpose, and may generally be 0.01 to 90 parts by weight of the total weight of the food.
- the form and type of health functional food are not particularly limited.
- the health functional food may be in the form of tablets, capsules, powders, granules, liquids, and pills.
- the health functional food may contain various flavors, sweeteners, or natural carbohydrates as additional ingredients.
- the sweetener may be a natural or synthetic sweetener, and examples of natural sweeteners include thaumatin and stevia extract. Meanwhile, examples of synthetic sweeteners include saccharin and aspartame.
- the natural carbohydrate may be monosaccharide, disaccharide, polysaccharide, oligosaccharide, sugar alcohol, etc.
- the health functional food of the present invention contains nutrients, vitamins, electrolytes, flavors, colorants, pexane and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, and preservatives. , glycerin, alcohol, etc. may be further included. These ingredients can be used independently or in combination.
- the ratio of the additive may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.
- the present invention provides a method for preventing, improving, or treating dyslipidemia or vascular disease, comprising administering the DNA aptamer to an individual.
- the DNA aptamer used in the method for preventing, improving, or treating dyslipidemia or vascular disease according to the present invention may have the characteristics described above.
- the DNA aptamer may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16.
- the dyslipidemia or vascular disease may have the characteristics described above.
- the subject may be a mammal, and may specifically be a human.
- the above administration may be oral administration or parenteral administration depending on the desired method.
- Parenteral administration may be selected from the following injection methods: external dermal application, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. It can be.
- the administration may be administered in a pharmaceutically effective amount. This may vary depending on the type and severity of the disease, drug activity, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, drugs used simultaneously, etc.
- the administration may be single administration or combined administration with other therapeutic agents. When administered in combination, administration may be sequential or simultaneous.
- the amount to be administered may be 0.001 to 10,000 mg/kg, specifically 0.1 g to 5 g/kg. This may be once a day, or it may be divided into several sessions.
- the present invention provides the use of the DNA aptamer for use in a method of preventing, improving, or treating dyslipidemia or vascular disease.
- the DNA aptamer for use in the method for preventing, improving, or treating dyslipidemia or vascular disease according to the present invention may have the characteristics described above.
- the DNA aptamer may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16.
- the dyslipidemia or vascular disease may have the characteristics described above.
- DNA aptamer that specifically binds to ApoB100 (apolipoprotein B100)
- ApoB100 apolipoprotein B100
- a 76 bp template DNA containing 40 random base sequences (SEQ ID NO: 17: 5'-ATACCAGCTTATTCAATT-N 40 -AGATAGTAAGTGCAATCT-3') and a forward primer for it (SEQ ID NO: 18: 5'-ATACCAGCTTATTCAATT-3) ') and reverse primer (SEQ ID NO: 19: 5'-AGATTGCACTTACTATCT-3') were custom-made by Cosmogenetech (Korea).
- the obtained PCR product was confirmed through agarose gel electrophoresis, and the PCR product was obtained using a PCR purification kit (Qiagen, USA) using 50 ⁇ l of distilled water. The obtained product was confirmed by agarose gel electrophoresis.
- Asymmetric PCR was performed using the PCR product obtained in Example 1-1 as a template to amplify dsDNA into ssDNA.
- PCR reaction was prepared by mixing ExTaq polymerase (Takara, Japan) (1 unit/ ⁇ l) and 76.5 ⁇ l of distilled water. At this time, a biotin-conjugated primer was used as the reverse primer. PCR was performed under the conditions described in Table 1, except that the denaturation, annealing, and elongation steps for the prepared reaction were repeated 15 times instead of 5 times.
- the obtained PCR product was confirmed through agarose gel electrophoresis. Afterwards, 3 times the volume of 100% ethanol and 1/1000 times the volume of tRNA were added to the PCR product and reacted at -70°C for 1 hour. This was centrifuged at 13,000 rpm for 20 minutes at 4°C to obtain only ssDNA.
- ssDNA aptamer 50 ⁇ l of 2 ⁇ TBS buffer was added to 50 ⁇ l of ssDNA aptamer, denatured by boiling at 85°C for 5 minutes, and then slowly cooled at room temperature for over 1 hour to form a stable three-dimensional structure of the ssDNA aptamer.
- ssDNA aptamers that specifically bind to ApoB100 were selected using the SELEX method.
- the reaction product was centrifuged at 13,000 rpm for 10 minutes at 4°C to remove the supernatant, and then washed by adding 1 ml of washing solution (500 mM NaCl, 60 mM imidazole, and 20 mM Tris-HCl). The process was repeated 5 times. Remove all supernatants to remove DNA aptamers that did not bind to ApoB100, add 200 ⁇ l of DNA aptamer elution solution (300 mM NaCl, 20 mM Tris-HCl, and 250 mM imidazole) and incubate at 4°C for 1 hour. The reaction was carried out while stirring.
- DNA aptamer candidates from the pool of DNA aptamers specifically binding to ApoB100 were confirmed as follows using the nanodrop method above.
- PCR was performed using the selected DNA aptamer pool as a template using primers (SEQ ID NOs. 17 and 18) as described above, and the obtained PCR product was cloned using Solgent's T-blunt PCR cloning kit. Used for T-vector cloning. T-vector cloning was performed by mixing 1 ⁇ l of T-blunt vector (10 ng/ ⁇ l), 4 ⁇ l of PCR product, and 1 ⁇ l of 6 ⁇ T-blunt buffer and reacting at 25°C for 5 minutes. The reaction solution was mixed with 100 ⁇ l of DH5 ⁇ soluble strain (competent cell), heat shocked at 42°C for 30 seconds, and left on ice for 2 minutes.
- SOC medium 2% tryptone, 0.5% yeast extract, 10mM NaCl, 2.5mM KCl, 10mM MgCl 2 , 10mM MgSO 4 , 20mM glucose
- SOC medium 2% tryptone, 0.5% yeast extract, 10mM NaCl, 2.5mM KCl, 10mM MgCl 2 , 10mM MgSO 4 , 20mM glucose
- the affinity of the 16 types of aptamers selected above for ApoB100 was confirmed using a conventional surface plasmon resonance (SPR) method.
- DNA aptamers 16 types were prepared by diluting them in HBS-EP buffer (GE Healthcare, BR-1001-88) to a concentration of 0, 100, 200, or 500 nM. At this time, the 200 nM concentration was tested twice to reduce errors in the results. An experiment was performed using the prepared DNA aptamer and BIAcore T200 according to the manufacturer's protocol, and the difference in the binding affinity of the DNA aptamer to the sensor chip CM5 coated with ApoB100 and the binding affinity of the DNA aptamer to the sensor chip CM5 without any coating was performed. was confirmed.
- the speed variable was obtained and quantified using the BIA evaluation program (BIACORE), and after each experiment, the sensor chip was regenerated using 0.5% SDS.
- the affinity of the DNA aptamer for ApoB100 is shown in Table 3.
- the final binding force was calculated by calculating the average and standard deviation from the affinity values for each concentration at which the DNA aptamer was treated.
- the THP-1 (KCLB 40202, Korea) cell line a human-derived mononuclear cell line
- RPMI 1640 culture medium containing 10% FBS (fetal bovine serum).
- FBS fetal bovine serum
- the cultured cells were distributed in a 96-well plate at 4 ⁇ 10 4 cells per well and cultured overnight at 37°C and 5% CO 2 conditions.
- Cultured cells were treated with 10 ⁇ l of DNA aptamer to a concentration of 0.32, 1.6, 8, 40, 200, 1,000, or 5,000 nM and cultured for another 24 hours.
- the cell culture medium was used to perform the CyQUANTTM LDH cytotoxicity assay kit (Invitrogen, USA) according to the manufacturer's protocol.
- PBS buffer was used as the negative control.
- cytotoxicity % was calculated from the obtained values and shown in Figure 2.
- the cholesterol-lowering activity of the nine types of DNA aptamers selected above was confirmed using a cholesterol assay kit (Abcam, UK) as follows.
- LDL low-density lipoprotien, Sigma aldrich, USA
- aptamer for ApoB100 50 ⁇ l of LDL (low-density lipoprotien, Sigma aldrich, USA) and the same amount of aptamer for ApoB100 were mixed, 100 ⁇ l of 2 ⁇ precipitation buffer was added, and reacted at room temperature for 10 minutes.
- the aptamers used were AB100_1-12, AB100_2-1, AB100_3-1, AB100_3-2, AB100_3-8, AB100_3-10, AB100_3-11, AB100_3-19, or AB100_3-20, and 1.14, 3.08, and 5.03 ⁇ g of aptamers were used. Three concentrations were tested. At this time, a sample to which no aptamer was added was used as a control.
- the mixture was centrifuged at 5,000 rpm for 10 minutes to recover the pellet.
- AB100_1-12, AB100_2-1, AB100_3-1, and AB100_3-8 aptamers significantly reduced cholesterol even at a low concentration of 1.14 ⁇ g.
- the cholesterol-reducing effect was confirmed when AB100_1-12 aptamer, which showed the most significant activity above, was treated in combination with lecithin, a treatment for cholesterol reduction. Specifically, the experiment was performed in the same manner as described in Example 6, and the experiment was performed by adding various concentrations (0.1, 0.5, 1, 5, 10, or 50 ⁇ M) of AB100_1-12 aptamer to 1 mg of lecithin. did. As a result, the results of confirming the LDL/vLDL concentration are shown in Figure 4.
- the DNA aptamer according to the present invention reduces cholesterol by specifically binding to ApoB100 without showing cytotoxicity, and can be usefully used in the treatment of diseases that can be caused by high cholesterol.
Abstract
The present invention relates to a DNA aptamer that specifically binds to ApoB100 and to a use of the DNA aptamer. Specifically, the DNA aptamer according to the present invention can bind specifically with high affinity to ApoB100 and reduce cholesterol, whereby the aptamer can be advantageously used for detecting ApoB100 or for the diagnosis or treatment of diseases such as dyslipidemia and vascular diseases.
Description
본 발명은 아포지단백(apolipoprotein B100, ApoB100)에 특이적으로 결합하는 DNA 앱타머 및 상기 DNA 앱타머의 용도에 관한 것이다.The present invention relates to a DNA aptamer that specifically binds to apolipoprotein B100 (ApoB100) and the use of the DNA aptamer.
이상지질혈증(dyslipidemia)이란 혈중에 총 콜레스테롤, LDL(low density lipoprotein) 콜레스테롤, 중성지방이 증가된 상태이거나, HDL(high density lipoprotein) 콜레스테롤이 감소된 상태를 말한다. 이상지질혈증의 대부분은 비만, 당뇨병, 음주와 같은 원인에 의해 발생하나, 유전적 요인으로 혈액 내 특정 지질이 증가하여 발생하는 경우도 있다.Dyslipidemia refers to a state in which total cholesterol, low density lipoprotein (LDL) cholesterol, and triglycerides in the blood are increased, or the level of HDL (high density lipoprotein) cholesterol is decreased. Most cases of dyslipidemia are caused by causes such as obesity, diabetes, and drinking, but in some cases, it is caused by an increase in specific lipids in the blood due to genetic factors.
이상지질혈증은 특별한 증세를 보이지 않는 경우가 대부분이나 이를 조절하지 않고 장기간 방치하면 콜레스테롤이 혈관 벽에 쌓여 혈관을 좁히거나 막아 협심증, 심근경색, 뇌졸중(뇌경색)과 같은 심뇌혈관질환을 일으킬 수 있다.In most cases, dyslipidemia does not show any special symptoms, but if left uncontrolled for a long period of time, cholesterol can build up on the walls of blood vessels, narrowing or blocking them, causing cardiovascular and cerebrovascular diseases such as angina, myocardial infarction, and stroke (cerebral infarction).
이상지질혈증은 1차성과 2차성으로 그 원인을 크게 나눌 수 있다. 1차성 원인은 지방 위주의 식생활, 운동 부족, 유전적인 요인 등에 의해 발생하는 원발성 이상지질혈증을 의미하고, 2차성 원인은 갑상성기능저하증, 만성 간질환, 신증후군 등의 기저질환 또는 임산, 약물 복용 등에 의해 유발되는 이상지질혈증을 나타낸다. 가족성 고콜레스테롤혈증이나 가족성 고중성지방혈증 등 유전적 소인에 의한 경우 황색종, 황색판종, 간비대, 신장비대 등이 나타날 수 있지만, 대개 무증상으로 대부분 혈액검사로 발견된다.Dyslipidemia can be broadly divided into primary and secondary causes. The primary cause is primary dyslipidemia caused by a fat-centered diet, lack of exercise, and genetic factors, and the secondary cause is underlying diseases such as hypothyroidism, chronic liver disease, nephrotic syndrome, pregnancy, or drug use. It refers to dyslipidemia caused by factors such as: In cases of genetic predisposition such as familial hypercholesterolemia or familial hypertriglyceridemia, xanthomas, xanthomatoma, hepatomegaly, and renal enlargement may appear, but they are usually asymptomatic and are mostly discovered through blood tests.
이에, 이상지질혈증을 경제적으로 진단할 수 있는 연구가 진행되고 있으며, 대한민국 등록특허 제10-2155306호는 유전자의 변이를 검출하여 고밀도지질단백질콜레스테롤의 수준을 예측하기 위한 조성물 및 키트, 및 이를 이용한 개체의 고밀도지질단백질콜레스테롤 수준을 예측하기 위한 정보를 제공하는 방법을 개시한다. 상기 문헌은 개체의 고밀도지질단백질(HDL) 수준을 조기에 예측하여 심혈관 질환의 발병을 예방하거나 개인 맞춤의학적 치료에 적용할 수 있다.Accordingly, research is underway to economically diagnose dyslipidemia, and Republic of Korea Patent No. 10-2155306 discloses compositions and kits for predicting the level of high-density lipoprotein cholesterol by detecting genetic mutations, and using the same. Disclosed is a method for providing information for predicting an individual's high-density lipoprotein cholesterol level. The above literature can be applied to early prediction of an individual's high-density lipoprotein (HDL) level to prevent the onset of cardiovascular disease or to personalized medical treatment.
본 발명의 목적은 특정 염기서열로 구성된 아포지단백(apolipoprotein B100, ApoB100)에 특이적으로 결합하는 DNA 앱타머를 제공하는 것이다.The purpose of the present invention is to provide a DNA aptamer that specifically binds to apolipoprotein B100 (ApoB100) composed of a specific base sequence.
본 발명의 다른 목적은, 상기 DNA 앱타머를 포함하는 ApoB100 검출용 조성물 및 키트, 및 상기 DNA 앱타머를 이용하여 ApoB100을 검출하는 방법을 제공하는 것이다.Another object of the present invention is to provide a composition and kit for detecting ApoB100 containing the DNA aptamer, and a method for detecting ApoB100 using the DNA aptamer.
본 발명의 또 다른 목적은, 상기 DNA 앱타머를 포함하는 이상지질혈증 또는 혈관질환 진단용 조성물 및 키트, 및 상기 DNA 앱타머를 이용하여 이상지질혈증 또는 혈관질환 진단을 위한 정보를 제공하는 방법을 제공하는 것이다.Another object of the present invention is to provide a composition and kit for diagnosing dyslipidemia or vascular disease containing the DNA aptamer, and a method of providing information for diagnosing dyslipidemia or vascular disease using the DNA aptamer. It is done.
본 발명의 또 다른 목적은 상기 DNA 앱타머의 이상지질혈증 또는 혈관질환 치료 용도를 제공하는 것이다.Another object of the present invention is to provide a use of the DNA aptamer for treating dyslipidemia or vascular disease.
상기 목적을 달성하기 위하여, 본 발명은 서열번호 1 내지 16으로 각각 기재된 염기서열로 구성된 군으로부터 선택된 염기서열로 구성되는 아포지단백(apolipoprotein B100, ApoB100)에 특이적으로 결합하는 DNA 앱타머를 제공한다.In order to achieve the above object, the present invention provides a DNA aptamer that specifically binds to apolipoprotein B100 (ApoB100) consisting of a base sequence selected from the group consisting of the base sequences shown in SEQ ID NOs: 1 to 16, respectively. .
또한, 본 발명은 상기 DNA 앱타머를 포함하는 ApoB100 검출용 조성물 또는 키트를 제공한다.Additionally, the present invention provides a composition or kit for detecting ApoB100 containing the DNA aptamer.
또한, 본 발명은 상기 DNA 앱타머를 시료와 반응시키는 단계를 포함하는 ApoB100 검출방법을 제공한다.Additionally, the present invention provides a method for detecting ApoB100 including the step of reacting the DNA aptamer with a sample.
또한, 본 발명은 상기 DNA 앱타머를 포함하는 이상지질혈증 또는 혈관질환 진단용 조성물 또는 키트를 제공한다.Additionally, the present invention provides a composition or kit for diagnosing dyslipidemia or vascular disease containing the DNA aptamer.
또한, 본 발명은 상기 DNA 앱타머를 시료와 반응시키는 단계를 포함하는 이상지질혈증 또는 혈관질환 진단의 정보를 제공하기 위한 방법을 제공한다.Additionally, the present invention provides a method for providing information on diagnosing dyslipidemia or vascular disease, including the step of reacting the DNA aptamer with a sample.
또한, 본 발명은 상기 DNA 앱타머를 유효성분으로 포함하는 이상지질혈증 또는 혈관질환의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating dyslipidemia or vascular disease containing the DNA aptamer as an active ingredient.
또한, 본 발명은 상기 DNA 앱타머를 유효성분으로 포함하는 이상지질혈증 또는 혈관질환의 예방 또는 개선용 건강기능식품을 제공한다.In addition, the present invention provides a health functional food for preventing or improving dyslipidemia or vascular disease containing the DNA aptamer as an active ingredient.
또한, 본 발명은 상기 DNA 앱타머를 개체에 투여하는 단계를 포함하는 이상지질혈증 또는 혈관질환의 예방, 개선 또는 치료방법을 제공한다.Additionally, the present invention provides a method for preventing, improving, or treating dyslipidemia or vascular disease, comprising administering the DNA aptamer to an individual.
나아가, 본 발명은 이상지질혈증 또는 혈관질환의 예방, 개선 또는 치료방법에 사용하기 위한 상기 DNA 앱타머의 용도를 제공한다.Furthermore, the present invention provides the use of the DNA aptamer for use in a method of preventing, improving, or treating dyslipidemia or vascular disease.
본 발명에 따른 DNA 앱타머는 ApoB100에 특이적으로 높은 친화도록 결합하고, 콜레스테롤을 저감시킴으로써 ApoB100을 검출하거나, 이상지질혈증 및 혈관질환 등과 같은 질환의 진단 또는 치료에 유용하게 사용될 수 있다.The DNA aptamer according to the present invention specifically binds to ApoB100 with high affinity and can be usefully used to detect ApoB100 by reducing cholesterol, or to diagnose or treat diseases such as dyslipidemia and vascular disease.
도 1은 본 발명의 일 실시예에서 SELEX 횟수에 따른 ApoB100에 대한 결합력을 확인한 결과 그래프이다.Figure 1 is a graph showing the results of confirming the binding affinity to ApoB100 according to the number of SELEX times in one embodiment of the present invention.
도 2는 본 발명의 일 실시예에서 DNA 앱타머의 세포독성을 확인한 결과 그래프이다.Figure 2 is a graph showing the results of confirming the cytotoxicity of the DNA aptamer in an example of the present invention.
도 3은 본 발명의 일 실시예에서 DNA 앱타머의 콜레스테롤 저감 효과를 확인한 결과 그래프이다.Figure 3 is a graph showing the results of confirming the cholesterol-reducing effect of DNA aptamers in one embodiment of the present invention.
도 4는 본 발명의 일 실시예에서 DNA 앱타머 및 레시틴의 병용처리에 의한 콜레스테롤 저감 효과를 확인한 결과 그래프이다.Figure 4 is a graph showing the results of confirming the cholesterol reduction effect by combined treatment of DNA aptamer and lecithin in one embodiment of the present invention.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 서열번호 1 내지 16으로 각각 기재된 염기서열로 구성된 군으로부터 선택된 염기서열로 구성되는 아포지단백(apolipoprotein B100, ApoB100)에 특이적으로 결합하는 DNA 앱타머를 제공한다.The present invention provides a DNA aptamer that specifically binds to apolipoprotein B100 (ApoB100) consisting of a base sequence selected from the group consisting of the base sequences shown in SEQ ID NOs: 1 to 16, respectively.
본 명세서에서 사용된 용어, "아포지단백(apolipoprotein B100, ApoB100)"은 간이나 장의 콜레스테롤을 조직으로 운반하는 지단백질 중의 하나로서, 다양한 종류의 세포에서 LDL 수용체에 인식된다. ApoB100은 4,563개의 아미노산으로 구성된 가장 큰 ApoB 그룹에 포함되는 단백질로 혈장에서 ApoB100 및 ApoB48의 두가지 아형(isoform)으로 존재한다. ApoB100의 수준은 콜레스테롤에 의해 발생하는 혈관질환과 관련있으며, LDL-C보다 더 나은 예측인자로서 알려져 있다.As used herein, the term “apolipoprotein B100 (ApoB100)” is one of the lipoproteins that transport liver or intestinal cholesterol to tissues, and is recognized by LDL receptors in various types of cells. ApoB100 is a protein included in the largest ApoB group, consisting of 4,563 amino acids, and exists in two isoforms in plasma: ApoB100 and ApoB48. The level of ApoB100 is related to vascular disease caused by cholesterol and is known to be a better predictor than LDL-C.
상기 ApoB100은 통상의 기술분야에 ApoB100이라고 알려진 모든 폴리펩티드 서열을 포함할 수 있다.The ApoB100 may include all polypeptide sequences known as ApoB100 in the art.
본 명세서에서 사용된 용어, "앱타머(aptamer)"는 특정 물질에 고친화성 및 특이성으로 결합할 수 있는 핵산분자를 의미한다. 상기 앱타머는 통상의 기술분야에 잘 알려진 방법으로 제조될 수 있고, 상기 방법은 필요에 따라 적절히 변형될 수 있다.As used herein, the term “aptamer” refers to a nucleic acid molecule that can bind to a specific substance with high affinity and specificity. The aptamer can be prepared by a method well known in the art, and the method can be appropriately modified as needed.
본 발명에 따른 DNA 앱타머는 서열번호 1 내지 16으로 각각 기재된 염기서열로 구성된 군으로부터 선택될 수 있다. 상기 DNA 앱타머는 ApoB100에 특이적으로 결합하는 특성을 유지하는 한, 상기 서열번호 1 내지 16으로 각각 기재된 염기서열과 80% 이상, 90% 이상, 95% 이상, 97% 이상 또는 99% 이상 상동성을 가질 수 있다.The DNA aptamer according to the present invention may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16, respectively. As long as the DNA aptamer maintains the characteristic of specifically binding to ApoB100, it is at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% homologous to the base sequences shown in SEQ ID NOs: 1 to 16, respectively. You can have
상기 DNA 앱타머는 표적 물질에 대한 결합성 및 안정성을 높이기 위해, 각 뉴클레오티드의 당 잔기, 구체적으로 리보오스 또는 디옥시리보스가 수식될 수 있다. 상기 수식은 당 잔기의 2'위치, 3'위치 및 4'위치로 구성된 군으로부터 선택되는 어느 하나 이상의 산소 원자를 다른 원자로 치환한 것일 수 있다. 수식의 예로는 플루오로화, O-알킬화, O-알릴화, S-알킬화, S-알릴화, 아미노화 등이 포함될 수 있다. 상기와 같은 당 잔기의 변형은 통상적인 방법으로 수행될 수 있다.The DNA aptamer may be modified with the sugar residue of each nucleotide, specifically ribose or deoxyribose, to increase binding and stability to the target substance. The above formula may be one in which one or more oxygen atoms selected from the group consisting of the 2', 3', and 4' positions of the sugar residue are replaced with another atom. Examples of modifications may include fluorination, O-alkylation, O-allylation, S-alkylation, S-allylation, amination, etc. Modification of the sugar residue as described above can be performed by conventional methods.
또한, 상기 DNA 앱타머는 표적 물질에 대한 결합성을 높이기 위해, 핵산염기가 변형될 수 있다. 상기 핵산염기의 변형은 5위치의 피리미딘 변형, 6위치의 푸린 변형, 8위치의 푸린 변형, 환외(環外) 아민에서의 변형, 4-티오우리딘으로의 치환, 5-브로모로의 치환 또는 5-요오드-우리실로의 치환 등을 포함할 수 있다.Additionally, the nucleotide base of the DNA aptamer may be modified to increase binding to the target substance. The modification of the nucleotide base includes pyrimidine modification at position 5, purine modification at position 6, purine modification at position 8, modification at extracyclic amine, substitution with 4-thiouridine, and substitution with 5-bromo. Or it may include substitution with 5-iodine-uricyl, etc.
또한, 상기 DNA 앱타머는 뉴클레아제 및 가수분해효소 등에 대해 내성을 갖도록 인산기가 변형될 수 있다. 예를 들면, 상기 인산기는 티오에이트, 디티오에이트, 아미데이트, 포름아세탈, 3'-아민 등으로 치환될 수 있다. 나아가, 상기 DNA 앱타머는 3' 말단 또는 5' 말단의 변형을 포함할 수 있고, 상기 변형은 캡핑이나, 바이오티닐, 폴리에틸글리콜, 아미노산, 펩티드, 핵산, 뉴클레오시드, 미리스토일(myristoyl), 리소콜릭-올레일(lithocolic-oleyl), 도코사닐(docosanyl), 라우로일(lauroyl), 스테아로일(stearoyl), 팔미토일(palmitoyl), 올레오일(oleoyl), 리놀레오일(linoleoyl), 지질, 스테로이드, 콜레스테롤, 카페인, 비타민, 색소, 형광물질, 항암제, 독소, 효소, 방사성 물질, 비오틴 등이 부가된 것일 수 있다.Additionally, the phosphate group of the DNA aptamer may be modified to be resistant to nucleases, hydrolases, etc. For example, the phosphate group may be substituted with thioate, dithioate, amidate, formacetal, 3'-amine, etc. Furthermore, the DNA aptamer may include modifications at the 3' end or 5' end, and the modifications include capping, biotinyl, polyethyl glycol, amino acids, peptides, nucleic acids, nucleosides, and myristoyl. , lithocolic-oleyl, docosanyl, lauroyl, stearoyl, palmitoyl, oleoyl, linoleoyl , lipids, steroids, cholesterol, caffeine, vitamins, pigments, fluorescent substances, anticancer drugs, toxins, enzymes, radioactive substances, biotin, etc. may be added.
또한, 본 발명은 상기 DNA 앱타머를 포함하는 ApoB100 검출용 조성물 또는 키트를 제공한다.Additionally, the present invention provides a composition or kit for detecting ApoB100 containing the DNA aptamer.
본 발명에 따른 ApoB100 검출용 조성물 및 키트에 포함되는 DNA 앱타머는 상술한 바와 같은 특징을 가질 수 있다. 일례로, 상기 DNA 앱타머는 서열번호 1 내지 16으로 기재된 염기서열로 구성된 군으로부터 선택될 수 있다.The DNA aptamer included in the composition and kit for ApoB100 detection according to the present invention may have the characteristics described above. For example, the DNA aptamer may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16.
본 발명에 따른 ApoB100 검출용 조성물에 포함되는 DNA 앱타머는 발색효소, 형광물질, 방사성 동위원소 또는 콜로이드 등의 검출체로 표지된 접합체일 수 있다. 발색효소는 퍼록시다제, 알칼라인 포스파타제 또는 산성 포스파타제일 수 있고, 형광물질은 티오우레아(FTH), 7-아세톡시쿠마린-3-일, 플루오레신-5-일, 플루오레신-6-일, 2',7'-디클로로플루오레신-5-일, 2',7'-디클로로플루오레신-6-일, 디하이드로 테트라메틸로사민-4-일, 테트라메틸로다민-5-일, 테트라메틸로다민-6-일, 4,4-디플루오로-5,7-디메틸-4-보라-3a,4a-디아자-s-인다센-3-에틸 또는 4,4-디플루오로-5,7-디페닐-4-보라-3a,4a-디아자-s-인다센-3-에틸, Cy3, Cy5, 폴리 L-라이신-플루오레세인 이소티오시아네이트(FITC), 로다민-B-이소티오시아네이트(RITC), PE(Phycoerythrin) 또는 로다민일 수 있다.The DNA aptamer included in the composition for detecting ApoB100 according to the present invention may be a conjugate labeled with a detection agent such as a chromogenic enzyme, a fluorescent substance, a radioactive isotope, or a colloid. The chromogenic enzyme may be peroxidase, alkaline phosphatase, or acid phosphatase, and the fluorescent substance may be thiourea (FTH), 7-acetoxycoumarin-3-yl, fluorescein-5-yl, or fluorescein-6-yl. , 2',7'-dichlorofluorescein-5-yl, 2',7'-dichlorofluorescein-6-yl, dihydro tetramethylrosamine-4-yl, tetramethylrhodamine-5-yl , tetramethylrhodamine-6-yl, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-ethyl or 4,4-difluor rho-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-ethyl, Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), rhoda It may be Min-B-isothiocyanate (RITC), Phycoerythrin (PE), or Rhodamine.
또한, 상기 DNA 앱타머는 상기 DNA 앱타머에 특이적으로 결합할 수 있는 리간드를 포함할 수 있다. 상기 리간드는 발색효소, 형광물질, 방사성 동위원소 또는 콜로이드 등의 검출체로 표지된 접합체, 및 스트렙타비딘 또는 아비딘이 처리된 리간드일 수 있다. 본 발명의 검출용 조성물은 상기 서술한 바와 같은 시약 외에도 이들의 구조를 안정하게 유지시키는 증류수 또는 완충액을 더 포함할 수 있다.Additionally, the DNA aptamer may include a ligand that can specifically bind to the DNA aptamer. The ligand may be a conjugate labeled with a detector such as a chromogenic enzyme, a fluorescent substance, a radioactive isotope, or a colloid, and a ligand treated with streptavidin or avidin. In addition to the reagents described above, the detection composition of the present invention may further include distilled water or a buffer solution to keep their structures stable.
본 발명에 따른 검출용 조성물을 포함하는 키트는 이에 포함되는 DNA 앱타머의 세척이나 복합체의 분리 등과 같은 이후 단계를 용이하게 하기 위해 고형 기질에 결합될 수 있다. 이때, 고형 기질로는 합성수지, 니트로셀룰로오스, 유리기판, 금속기판, 유리섬유, 미세구체 또는 미세비드가 사용될 수 있다. 또한, 합성수지로는 폴리에스터, 폴리염화비닐, 폴리스티렌, 폴리프로필렌, PVDF 또는 나일론 등이 사용될 수 있다.A kit containing the detection composition according to the present invention can be bound to a solid substrate to facilitate subsequent steps, such as washing the DNA aptamer contained therein or separating the complex. At this time, synthetic resin, nitrocellulose, glass substrate, metal substrate, glass fiber, microspheres, or microbeads may be used as the solid substrate. Additionally, polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF, or nylon may be used as the synthetic resin.
또한, 상기 키트는 당업자에게 알려진 종래의 제조방법에 의해 제조될 수 있으며, 버퍼, 안정화제, 불활성 단백질 등을 더 포함할 수 있다. 상기 키트는 시약의 양을 탐색하기 위해 검출체로서 부착된 형광물질의 형광을 검출함으로써 수행되는 형광법 또는 검출체로서 부착된 방사선 동위원소의 방사선을 검출함으로써 수행되는 방사선법을 통한 초고속 스크리닝(high throughput screening, HTS) 시스템, 검출체의 표지 없이 표면의 플라즈몬 공명 변화를 실시간으로 측정하는 SPR(surface plasmon resonance) 방법 또는 SPR 시스템을 영상화하여 확인하는 SPRI(surface plasmon resonance imaging) 방법을 이용할 수 있다.Additionally, the kit may be manufactured by conventional manufacturing methods known to those skilled in the art, and may further include buffers, stabilizers, inactive proteins, etc. The kit is used for ultra-high throughput screening through a fluorescence method performed by detecting the fluorescence of a fluorescent substance attached as a detector to detect the amount of reagent, or a radiography method performed by detecting the radiation of a radioisotope attached as a detector. screening (HTS) system, the SPR (surface plasmon resonance) method that measures plasmon resonance changes on the surface in real time without labeling the detector, or the SPRI (surface plasmon resonance imaging) method that confirms the SPR system by imaging it.
또한, 본 발명은 상기 DNA 앱타머를 시료와 반응시키는 단계를 포함하는 ApoB100 검출방법을 제공한다.Additionally, the present invention provides a method for detecting ApoB100 including the step of reacting the DNA aptamer with a sample.
본 발명에 따른 ApoB100 검출방법에 사용되는 DNA 앱타머는 상술한 바와 같은 특징을 가질 수 있다. 일례로, 상기 DNA 앱타머는 서열번호 1 내지 16으로 기재된 염기서열로 구성된 군으로부터 선택될 수 있다.The DNA aptamer used in the ApoB100 detection method according to the present invention may have the characteristics described above. For example, the DNA aptamer may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16.
상기 시료는 ApoB100을 검출하고자 하는 시료라면 모든 시료를 포함할 수 있다. 또한, 상기 시료는 음이온 교환 크로마토그래피, 친화도 크로마토그래피, 크기별 배제 크로마토그래피, 액체 크로마토그래피, 연속추출, 원심분리 또는 젤 전기영동 등의 방법을 이용하여 전처리될 수 있다.The sample may include any sample for which ApoB100 is to be detected. Additionally, the sample may be pretreated using methods such as anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, continuous extraction, centrifugation, or gel electrophoresis.
또한, 본 발명은 상기 DNA 앱타머를 포함하는 이상지질혈증 또는 혈관질환 진단용 조성물 또는 키트를 제공한다.Additionally, the present invention provides a composition or kit for diagnosing dyslipidemia or vascular disease containing the DNA aptamer.
본 발명에 따른 이상지질혈증 또는 혈관질환 진단용 조성물에 포함되는 DNA 앱타머는 상술한 바와 같은 특징을 가질 수 있다. 일례로, 상기 DNA 앱타머는 서열번호 1 내지 16으로 기재된 염기서열로 구성된 군으로부터 선택될 수 있다. 또한, 상기 조성물 및 키트 또한 상술한 바와 같은 특징을 가질 수 있다.The DNA aptamer included in the composition for diagnosing dyslipidemia or vascular disease according to the present invention may have the characteristics described above. For example, the DNA aptamer may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16. Additionally, the compositions and kits may also have the characteristics described above.
본 명세서에서 사용된 용어, "이상지질혈증(dyslipidemia)"은 혈중에 총콜레스테롤, LDL 콜레스테롤 및 중성지방이 증가된 상태이거나 HDL 콜레스테롤이 감소된 상태를 의미하며, 심혈관계질환이 발생할 수 있는 위험요인 중 하나로 알려져 있다. 상기 이상지질혈증은 통상의 기술분야에 알려진 모든 종류의 이상지질혈증을 포함할 수 있다. 일례로, 상기 이상지질혈증은 고지혈증, 고콜레스테롤혈증 또는 고중성지방혈증을 포함할 수 있다.As used herein, the term “dyslipidemia” refers to a state in which total cholesterol, LDL cholesterol, and triglycerides in the blood are increased or HDL cholesterol is decreased, and is a risk factor for developing cardiovascular disease. Known as one of the The dyslipidemia may include all types of dyslipidemia known in the art. For example, the dyslipidemia may include hyperlipidemia, hypercholesterolemia, or hypertriglyceridemia.
본 명세서에서 사용된 용어, "혈관질환(vascular disease)"은 혈전 등의 혈액응고 덩어리 또는 지방의 축적으로 혈관이 좁아져 발생하는 질환을 의미한다. 상기 혈관질환은 통상의 기술분야에 알려진 모든 종류의 혈관질환을 포함할 수 있다. 또한, 상기 혈관질환은 혈관이 좁아져 발생하는 질환뿐 아니라, 혈관이 터져서 발생하는 질환도 포함할 수 있다. 일례로, 상기 혈관질환은 동맥경화, 죽상경화증, 죽상동맥경화증, 뇌경색, 협심증, 말초혈관 폐색성 질환, 심근경색, 당뇨성 망막증 또는 고혈압성 망막증을 포함할 수 있다.As used herein, the term “vascular disease” refers to a disease that occurs when blood vessels become narrowed due to accumulation of fat or blood clots such as blood clots. The vascular disease may include all types of vascular diseases known in the art. In addition, the vascular disease may include not only diseases caused by narrowing of blood vessels, but also diseases caused by rupture of blood vessels. For example, the vascular disease may include arteriosclerosis, atherosclerosis, atherosclerosis, cerebral infarction, angina pectoris, peripheral vascular occlusive disease, myocardial infarction, diabetic retinopathy, or hypertensive retinopathy.
나아가, 본 발명은 상기 DNA 앱타머를 시료와 반응시키는 단계를 포함하는 이상지질혈증 또는 혈관질환 진단의 정보를 제공하기 위한 방법을 제공한다.Furthermore, the present invention provides a method for providing information on diagnosing dyslipidemia or vascular disease, including the step of reacting the DNA aptamer with a sample.
본 발명에 따른 이상지질혈증 또는 혈관질환 진단의 정보를 제공하기 위한 방법에 사용되는 DNA 앱타머는 상술한 바와 같은 특징을 가질 수 있다. 일례로, 상기 DNA 앱타머는 서열번호 1 내지 16으로 기재된 염기서열로 구성된 군으로부터 선택될 수 있다. 또한, 상기 이상지질혈증 또는 혈관질환은 상술한 바와 같은 특징을 가질 수 있다.The DNA aptamer used in the method for providing information for diagnosing dyslipidemia or vascular disease according to the present invention may have the characteristics described above. For example, the DNA aptamer may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16. Additionally, the dyslipidemia or vascular disease may have the characteristics described above.
상기 시료는 이상지질혈증 또는 혈관질환을 진단하고자 하는 시료라면 모든 시료를 포함할 수 있다. 구체적으로, 상기 시료는 포유동물 유래 시료일 수 있고, 구체적으로 인간 유래 시료일 수 있다. 또한, 상기 시료는 혈액, 타액, 누액, 요액, 활액, 점액, 세포 및 조직으로 구성된 군으로부터 선택되는 어느 하나 이상일 수 있다. 또한, 상기 시료는 본 발명에 따른 방법에 사용되기 전에 상술한 바와 같은 방법으로 전처리될 수 있다.The sample may include any sample that is intended to diagnose dyslipidemia or vascular disease. Specifically, the sample may be a mammal-derived sample, and specifically, it may be a human-derived sample. Additionally, the sample may be any one or more selected from the group consisting of blood, saliva, tear fluid, urine, synovial fluid, mucus, cells, and tissues. Additionally, the sample may be pretreated in the manner described above before being used in the method according to the present invention.
또한, 본 발명은 상기 DNA 앱타머를 유효성분으로 포함하는 이상지질혈증 또는 혈관질환의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating dyslipidemia or vascular disease containing the DNA aptamer as an active ingredient.
본 발명에 따른 이상지질혈증 또는 혈관질환의 예방 또는 치료용 약학적 조성물에 포함되는 DNA 앱타머는 상술한 바와 같은 특징을 가질 수 있다. 일례로, 상기 DNA 앱타머는 서열번호 1 내지 16으로 기재된 염기서열로 구성된 군으로부터 선택될 수 있다. 또한, 상기 이상지질혈증 또는 혈관질환은 상기 서술한 바와 같은 특징을 가질 수 있다.The DNA aptamer included in the pharmaceutical composition for preventing or treating dyslipidemia or vascular disease according to the present invention may have the characteristics described above. For example, the DNA aptamer may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16. Additionally, the dyslipidemia or vascular disease may have the characteristics described above.
본 발명에 따른 약학적 조성물은 조성물 전체 중량에 대하여 유효성분인 본 발명에 따른 DNA 앱타머를 10 내지 95 중량%로 포함할 수 있다. 또한, 본 발명의 약학적 조성물은 상기 유효성분 이외에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 추가로 포함할 수 있다.The pharmaceutical composition according to the present invention may contain 10 to 95% by weight of the DNA aptamer according to the present invention, which is an active ingredient, based on the total weight of the composition. In addition, the pharmaceutical composition of the present invention may further include one or more active ingredients that exhibit the same or similar functions in addition to the above-mentioned effective ingredients.
본 발명의 약학적 조성물은 생물학적 제제에 통상적으로 사용되는 담체, 희석제, 부형제 또는 이의 혼합물을 포함할 수 있다. 약학적으로 허용가능한 담체는 조성물을 생체 내에 전달하는데 적합한 것이면 모두 사용할 수 있다. 구체적으로, 상기 담체는 Merck Index, 13th ed., Merck & Co. Inc.에 기재된 화합물, 식염수, 멸균수, 링거액, 덱스트로스 용액, 말토덱스트린 용액, 글리세롤, 에탄올 또는 이의 혼합물일 수 있다. 또한, 필요에 따라 항산화제, 완충액, 정균제 등과 같은 통상의 첨가제를 첨가할 수 있다.The pharmaceutical composition of the present invention may include carriers, diluents, excipients, or mixtures thereof commonly used in biological products. Any pharmaceutically acceptable carrier can be used as long as it is suitable for delivering the composition in vivo. Specifically, the carrier is Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or mixtures thereof. Additionally, conventional additives such as antioxidants, buffers, bacteriostatic agents, etc. can be added as needed.
상기 조성물을 제제화하는 경우, 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 첨가할 수 있다.When formulating the composition, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be added.
본 발명의 조성물은 경구용 제제 또는 비경구용 제제로 제형화될 수 있다. 경구용 제제로는 고형 제제 및 액상 제제가 포함될 수 있다. 상기 고형 제제는 정제, 환제, 산제, 과립제, 캡슐제 또는 트로키제일 수 있고, 이러한 고형 제제는 상기 조성물에 적어도 하나 이상의 부형제를 첨가하여 조제할 수 있다. 상기 부형제는 전분, 탄산칼슘, 수크로스, 락토오스, 젤라틴 또는 이의 혼합물일 수 있다. 또한, 상기 고형 제제는 윤활제를 포함할 수 있고, 그 예로는 마그네슘 스티레이트, 탈크등이 있다. 한편, 상기 액상 제제는 현탁제, 내용액제, 유제 또는 시럽제일 수 있다. 이때, 상기 액상 제제에는 습윤제, 감미제, 방향제, 보존제 등과 같은 부형제가 포함될 수 있다.The composition of the present invention may be formulated as an oral preparation or a parenteral preparation. Oral preparations may include solid preparations and liquid preparations. The solid preparation may be a tablet, pill, powder, granule, capsule, or troche, and such solid preparation may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin, or mixtures thereof. Additionally, the solid preparation may contain a lubricant, examples of which include magnesium styrate and talc. Meanwhile, the liquid preparation may be a suspension, oral solution, emulsion, or syrup. At this time, the liquid formulation may contain excipients such as wetting agents, sweeteners, fragrances, preservatives, etc.
상기 비경구용 제제는 주사제, 좌제, 호흡기 흡입용 분말, 스프레이용 에어로졸제, 파우더 및 크림 등을 포함할 수 있다. 상기 주사제는 멸균된 수용액, 비수성용제, 현탁용제, 유제 등을 포함할 수 있다. 이때, 비수성용제 또는 현탁용제로서는 프로필렌글리콜, 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 기름이나, 에틸올레이트와 같이 주사가능한 에스테르 등이 사용될 수 있다.The parenteral preparations may include injections, suppositories, powders for respiratory inhalation, aerosols for sprays, powders and creams. The injection may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, etc. At this time, the non-aqueous solvent or suspension may be propylene glycol, polyethylene glycol, vegetable oil such as olive oil, or injectable ester such as ethyl oleate.
나아가, 본 발명은 상기 DNA 앱타머를 유효성분으로 포함하는 이상지질혈증 또는 혈관질환의 예방 또는 개선용 건강기능식품을 제공한다.Furthermore, the present invention provides a health functional food for preventing or improving dyslipidemia or vascular disease containing the DNA aptamer as an active ingredient.
본 발명에 따른 이상지질혈증 또는 혈관질환의 예방 또는 개선용 건강기능식품에 포함되는 DNA 앱타머는 상술한 바와 같은 특징을 가질 수 있다. 일례로, 상기 DNA 앱타머는 서열번호 1 내지 16으로 기재된 염기서열로 구성된 군으로부터 선택될 수 있다. 또한, 상기 이상지질혈증 또는 혈관질환은 상기 서술한 바와 같은 특징을 가질 수 있다.The DNA aptamer included in the health functional food for preventing or improving dyslipidemia or vascular disease according to the present invention may have the characteristics described above. For example, the DNA aptamer may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16. Additionally, the dyslipidemia or vascular disease may have the characteristics described above.
본 발명의 DNA 앱타머는 식품에 그대로 첨가하거나, 다른 식품 또는 식품 성분과 함께 사용될 수 있다. 이때, 첨가되는 유효성분의 함량은 목적에 따라 결정될 수 있고, 일반적으로는 전체 식품 중량의 0.01 내지 90 중량부일 수 있다.The DNA aptamer of the present invention can be added as is to food or used together with other foods or food ingredients. At this time, the content of the active ingredient added may be determined depending on the purpose, and may generally be 0.01 to 90 parts by weight of the total weight of the food.
건강기능식품의 형태 및 종류는 특별히 제한되지 않는다. 구체적으로, 상기 건강기능식품은 정제, 캅셀, 분말, 과립, 액상 및 환의 형태일 수 있다. 상기 건강기능식품은 추가성분으로서 여러 가지 향미제, 감미제 또는 천연 탄수화물을 포함할 수 있다. 상기 감미제는 천연 또는 합성 감미제일 수 있고, 천연 감미제의 예로는 타우마틴, 스테비아 추출물 등이 있다. 한편, 합성 감미제의 예로는 사카린, 아스파르탐 등이 있다. 또한, 상기 천연 탄수화물은 모노사카라이드, 디사카라이드, 폴리사카라이드, 올리고당 및 당알코올 등일 수 있다.The form and type of health functional food are not particularly limited. Specifically, the health functional food may be in the form of tablets, capsules, powders, granules, liquids, and pills. The health functional food may contain various flavors, sweeteners, or natural carbohydrates as additional ingredients. The sweetener may be a natural or synthetic sweetener, and examples of natural sweeteners include thaumatin and stevia extract. Meanwhile, examples of synthetic sweeteners include saccharin and aspartame. Additionally, the natural carbohydrate may be monosaccharide, disaccharide, polysaccharide, oligosaccharide, sugar alcohol, etc.
본 발명의 건강기능식품은 상기 서술한 추가성분 외에, 영양제, 비타민, 전해질, 풍미제, 착색제, 펙스탄 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올 등을 더 포함할 수 있다. 이러한 성분은 독립적으로 또는 조합으로 사용될 수 있다. 상기 첨가제의 비율은 본 발명의 조성물의 100 중량부당 0.01 내지 0.1 중량부의 범위에서 선택될 수 있다.In addition to the additional ingredients described above, the health functional food of the present invention contains nutrients, vitamins, electrolytes, flavors, colorants, pexane and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, and preservatives. , glycerin, alcohol, etc. may be further included. These ingredients can be used independently or in combination. The ratio of the additive may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.
또한, 본 발명은 상기 DNA 앱타머를 개체에 투여하는 단계를 포함하는 이상지질혈증 또는 혈관질환의 예방, 개선 또는 치료방법을 제공한다.Additionally, the present invention provides a method for preventing, improving, or treating dyslipidemia or vascular disease, comprising administering the DNA aptamer to an individual.
본 발명에 따른 이상지질혈증 또는 혈관질환의 예방, 개선 또는 치료방법에 사용되는 DNA 앱타머는 상술한 바와 같은 특징을 가질 수 있다. 일례로, 상기 DNA 앱타머는 서열번호 1 내지 16으로 기재된 염기서열로 구성된 군으로부터 선택될 수 있다.The DNA aptamer used in the method for preventing, improving, or treating dyslipidemia or vascular disease according to the present invention may have the characteristics described above. For example, the DNA aptamer may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16.
상기 이상지질혈증 또는 혈관질환은 상술한 바와 같은 특징을 가질 수 있다.The dyslipidemia or vascular disease may have the characteristics described above.
상기 개체는 포유동물일 수 있고, 구체적으로 인간일 수 있다. 상기 투여는 목적하는 방법에 따라 경구 투여하거나 비경구 투여일 수 있으며, 비경구 투여는 피부 외용 또는 복강내 주사, 직장내 주사, 피하주사, 정맥주사, 근육내 주사 또는 흉부내 주사 주입방식중 선택될 수 있다.The subject may be a mammal, and may specifically be a human. The above administration may be oral administration or parenteral administration depending on the desired method. Parenteral administration may be selected from the following injection methods: external dermal application, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. It can be.
또한, 상기 투여는 약제학적으로 유효한 양으로 투여되는 것일 수 있다. 이는 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물 등에 따라 달라질 수 있다. 상기 투여는 단독투여 또는 다른 치료제와의 병용투여일 수 있다. 병용 투여시, 투여는 순차적 또는 동시일 수 있다. Additionally, the administration may be administered in a pharmaceutically effective amount. This may vary depending on the type and severity of the disease, drug activity, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, drugs used simultaneously, etc. The administration may be single administration or combined administration with other therapeutic agents. When administered in combination, administration may be sequential or simultaneous.
그러나, 바람직한 효과를 위해서, 상기 투여의 양은 0.001 내지 10,000 mg/㎏, 구체적으로는 0.1 g 내지 5 g/kg 일 수 있다. 이는 하루에 1회일 수 있고, 수회로 나뉠 수도 있다.However, for a desirable effect, the amount to be administered may be 0.001 to 10,000 mg/kg, specifically 0.1 g to 5 g/kg. This may be once a day, or it may be divided into several sessions.
나아가, 본 발명은 이상지질혈증 또는 혈관질환의 예방, 개선 또는 치료방법에 사용하기 위한 상기 DNA 앱타머의 용도를 제공한다.Furthermore, the present invention provides the use of the DNA aptamer for use in a method of preventing, improving, or treating dyslipidemia or vascular disease.
본 발명에 따른 이상지질혈증 또는 혈관질환의 예방, 개선 또는 치료방법에 사용하기 위한 상기 DNA 앱타머는 상술한 바와 같은 특징을 가질 수 있다. 일례로, 상기 DNA 앱타머는 서열번호 1 내지 16으로 기재된 염기서열로 구성된 군으로부터 선택될 수 있다. 또한, 상기 이상지질혈증 또는 혈관질환은 상술한 바와 같은 특징을 가질 수 있다.The DNA aptamer for use in the method for preventing, improving, or treating dyslipidemia or vascular disease according to the present invention may have the characteristics described above. For example, the DNA aptamer may be selected from the group consisting of base sequences shown in SEQ ID NOs: 1 to 16. Additionally, the dyslipidemia or vascular disease may have the characteristics described above.
이하, 본 발명을 하기 실시예에 의해 상세히 설명한다, 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 이들에 의해 본 발명이 제한되는 것은 아니다. 본 발명의 청구범위에 기재된 기술적 사상과 실질적으로 동일한 구성을 갖고 동일한 작용 효과를 이루는 것은 어떠한 것이라도 본 발명의 기술적 범위에 포함된다.Hereinafter, the present invention will be described in detail through the following examples. However, the following examples are only for illustrating the present invention and are not intended to limit the present invention thereto. Anything that has substantially the same structure as the technical idea described in the claims of the present invention and achieves the same operation and effect is included in the technical scope of the present invention.
실시예 1. DNA 앱타머의 제작Example 1. Construction of DNA aptamer
ApoB100(apolipoprotein B100)에 특이적으로 결합하는 DNA 앱타머를 제작하기 위해, 먼저 하기 기재된 바와 같이 DNA 라이브러리를 제작하였다.To construct a DNA aptamer that specifically binds to ApoB100 (apolipoprotein B100), a DNA library was first prepared as described below.
1-1. 랜덤 DNA의 증폭1-1. Amplification of random DNA
먼저, 40개의 랜덤 염기서열을 포함하는 76 bp 크기의 주형 DNA(서열번호 17: 5'-ATACCAGCTTATTCAATT-N40-AGATAGTAAGTGCAATCT-3')와 이에 대한 정방향 프라이머(서열번호 18: 5'-ATACCAGCTTATTCAATT-3') 및 역방향 프라이머(서열번호 19: 5'-AGATTGCACTTACTATCT-3')를 코스모진텍 사(한국)에 주문제작하였다. 이후, 1 ㎕의 주형 DNA, 5 ㎕의 10x PCR 완충용액, 4 ㎕의 dNTP 혼합물, 2 ㎕의 10 μM 정방향 프라이머, 2 ㎕의 10 μM 역방향 프라이머, 0.3 ㎕의 ExTaq 중합효소(Takara, 일본)(1 unit/㎕), 및 35.7 ㎕의 증류수를 혼합하여 PCR 반응물을 준비하였다. 준비된 반응물을 하기 표 1에 기재된 바와 같은 조건으로 PCR을 수행하였다.First, a 76 bp template DNA containing 40 random base sequences (SEQ ID NO: 17: 5'-ATACCAGCTTATTCAATT-N 40 -AGATAGTAAGTGCAATCT-3') and a forward primer for it (SEQ ID NO: 18: 5'-ATACCAGCTTATTCAATT-3) ') and reverse primer (SEQ ID NO: 19: 5'-AGATTGCACTTACTATCT-3') were custom-made by Cosmogenetech (Korea). Then, 1 μl of template DNA, 5 μl of 10x PCR buffer, 4 μl of dNTP mixture, 2 μl of 10 μM forward primer, 2 μl of 10 μM reverse primer, 0.3 μl of ExTaq polymerase (Takara, Japan) ( 1 unit/㎕), and 35.7 ㎕ of distilled water were mixed to prepare a PCR reaction. PCR was performed on the prepared reaction product under the conditions described in Table 1 below.
| 초기 변성 단계early metamorphic stage |
94℃, 5분94℃, 5 |
1회1 time |
| 변성 단계denaturation stage |
94℃, 30초94℃, 30 |
5회5 times |
| 어닐링(annealing) 단계Annealing step | 52℃, 30초52℃, 30 seconds | |
| 연신(elongation) 단계Elongation step | 72℃, 30초72℃, 30 seconds | |
| 후기 연신 단계Late elongation stage |
72℃, 5분72℃, 5 |
1회1 time |
수득된 PCR 산물을 아가로스 겔 전기영동을 통해 확인하고, 상기 PCR 산물을 PCR 정제키트(Qiagen, 미국)를 사용하여 50 ㎕의 증류수를 사용하여 수득하였다. 수득된 산물은 아가로스 겔 전기영동으로 확인하였다.The obtained PCR product was confirmed through agarose gel electrophoresis, and the PCR product was obtained using a PCR purification kit (Qiagen, USA) using 50 μl of distilled water. The obtained product was confirmed by agarose gel electrophoresis.
1-2. ssDNA 증폭1-2. ssDNA amplification
실시예 1-1에서 수득된 PCR 산물을 주형으로 비대칭(assymetric) PCR을 수행하여 dsDNA를 ssDNA로 증폭시켰다.Asymmetric PCR was performed using the PCR product obtained in Example 1-1 as a template to amplify dsDNA into ssDNA.
구체적으로, 1 ㎕의 PCR 산물, 10 ㎕의 10x 완충용액, 8 ㎕의 dNTP 혼합물, 2 ㎕의 10 μM 정방향 프라이머(서열번호 17), 2 ㎕의 10 μM 역방향 프라이머(서열번호 18), 0.5 ㎕의 ExTaq 중합효소(Takara, 일본)(1 unit/㎕), 및 76.5 ㎕의 증류수를 혼합하여 PCR 반응물을 준비하였다. 이때, 역방향 프라이머는 비오틴이 결합된 프라이머를 사용하였다. 준비된 반응물을 변성, 어닐링 및 연신 단계를 5회 반복하는 대신 15회 반복하는 것을 제외하고는 상기 표 1에 기재된 바와 같은 조건으로 PCR을 수행하였다. 수득된 PCR 산물을 아가로스 겔 전기영동을 통해 확인하였다. 이후, PCR 산물에 이에 3배 부피의 100% 에탄올과, 1/1000배 부피의 tRNA를 첨가하여 -70℃에서 1시간 동안 반응시켰다. 이를 4℃에서 13,000 rpm으로 20분 동안 원심분리하여 ssDNA만을 수득하였다.Specifically, 1 μl of PCR product, 10 μl of 10x buffer, 8 μl of dNTP mixture, 2 μl of 10 μM forward primer (SEQ ID NO: 17), 2 μl of 10 μM reverse primer (SEQ ID NO: 18), 0.5 μl A PCR reaction was prepared by mixing ExTaq polymerase (Takara, Japan) (1 unit/㎕) and 76.5 ㎕ of distilled water. At this time, a biotin-conjugated primer was used as the reverse primer. PCR was performed under the conditions described in Table 1, except that the denaturation, annealing, and elongation steps for the prepared reaction were repeated 15 times instead of 5 times. The obtained PCR product was confirmed through agarose gel electrophoresis. Afterwards, 3 times the volume of 100% ethanol and 1/1000 times the volume of tRNA were added to the PCR product and reacted at -70°C for 1 hour. This was centrifuged at 13,000 rpm for 20 minutes at 4°C to obtain only ssDNA.
1-3. ssDNA의 선별 및 회수1-3. Selection and recovery of ssDNA
상기 확보된 ssDNA 산물 중 비오틴(Biotin)이 결합되어 있는 ssDNA를 제거하기 위하여, 상기 실시예에서 획득한 100 ㎕의 PCR 산물을 85℃에서 5분 동안 가열하였다. 이를 5분 동안 얼음에 넣어 식히고, 50 ㎕의 스트렙트아비딘(streptavidin, Pierce, USA)을 첨가하여 4℃에서 1시간 동안 반응시켰다. 반응액을 4℃, 13,000 rpm의 조건 하에서 10분간 원심분리한 후, 상층액만을 회수하여 상기 서술한 바와 같이 100% 에탄올 및 tRNA를 이용하여 ssDNA만을 수득하였다.In order to remove ssDNA bound to biotin among the obtained ssDNA products, 100 μl of the PCR product obtained in the above example was heated at 85°C for 5 minutes. This was cooled in ice for 5 minutes, and 50 ㎕ of streptavidin (Pierce, USA) was added and reacted at 4°C for 1 hour. After centrifuging the reaction solution for 10 minutes at 4°C and 13,000 rpm, only the supernatant was recovered, and only ssDNA was obtained using 100% ethanol and tRNA as described above.
1-4. 3차원 구조의 DNA 앱타머 제작1-4. Production of DNA aptamer with 3D structure
50 ㎕의 ssDNA 앱타머에 50 ㎕의 2× TBS 완충액을 첨가하고, 85℃에서 5분동안 끓여 변성시킨 후, 상온에서 1시간 이상 서서히 식혀 ssDNA 앱타머의 안정한 3차원 구조를 형성시켰다.50 μl of 2× TBS buffer was added to 50 μl of ssDNA aptamer, denatured by boiling at 85°C for 5 minutes, and then slowly cooled at room temperature for over 1 hour to form a stable three-dimensional structure of the ssDNA aptamer.
실시예 2. ApoB100에 특이적으로 결합하는 DNA 앱타머 선별Example 2. Selection of DNA aptamers that specifically bind to ApoB100
ApoB100에 특이적으로 결합하는 ssDNA 앱타머를 SELEX 방법으로 선별하였다.ssDNA aptamers that specifically bind to ApoB100 were selected using the SELEX method.
먼저, 200 ㎕의 Ni-NTA 아가로즈에 200 ㎕의 1× 앱타머 선별용액(500 mM NaCl, 20 mM Tris-HCl 및 5 mM 이미다졸)을 첨가하고, 4℃에서 30분 동안 교반하여 반응시켰다. 이후, 이를 4℃에서 13,000 rpm으로 10분 동안 원심분리하여 상층액을 제거하고, 이를 2회 반복함으로써 활성화된 Ni-NTA 아가로즈를 수득하였다. 활성화된 Ni-NTA 아가로즈에 ApoB100 및 DNA 앱타머를 혼합하여 첨가하고, 이를 4℃에서 1시간 동안 교반하면서 반응시켰다. 반응이 끝난 후, 반응물을 4℃에서 13,000 rpm으로 10분 동안 원심분리하여 상층액을 제거하고, 1 ㎖의 세척용액(500 mM NaCl, 60 mM 이미다졸 및 20 mM Tris-HCl)을 첨가하여 세척하는 과정을 5회 반복하였다. 상층액을 모두 제거하여 ApoB100과 결합하지 못한 DNA 앱타머를 제거하고, 200 ㎕의 DNA 앱타머 용출용액(300 mM NaCl, 20 mM Tris-HCl 및 250 mM 이미다졸)을 첨가하여 4℃에서 1시간 동안 교반하면서 반응시켰다. 이후, 상기 반응액을 ℃에서 30분 동안 상층액을 수득하였다. 수득된 상층액으로부터 상기 서술한 바와 같이 100% 에탄올 및 tRNA를 이용하여 ssDNA만을 수득하였다. 수득된 펠렛은 건조시킨 후, 50 ㎕의 핵산분해효소(nuclease)가 포함되지 않은 증류수에 희석하였다. 수득된 ssDNA를 주형으로 사용하여 SELEX를 7회 반복수행하였다. 이후, ApoB100 없이 Ni-NTA 아가로즈만 사용하여 네가티브 SELEX를 수행한 후, 다시 SELEX를 3회 수행하였다. SELEX를 반복수행하는 과정에서 수득된 각 풀(pool)에 포함된 DNA 앱타머의 ApoB100에 대한 친화성을 통상적인 나노드롭(nano drop) 방법으로 분광광도계를 사용하여 확인하고, 그 결과를 도 1에 나타내었다.First, 200 μl of 1× aptamer selection solution (500 mM NaCl, 20 mM Tris-HCl, and 5 mM imidazole) was added to 200 μl of Ni-NTA agarose, and the reaction was stirred at 4°C for 30 minutes. . Afterwards, this was centrifuged at 13,000 rpm for 10 minutes at 4°C to remove the supernatant, and this was repeated twice to obtain activated Ni-NTA agarose. ApoB100 and DNA aptamer were mixed and added to activated Ni-NTA agarose, and reacted with stirring at 4°C for 1 hour. After the reaction was completed, the reaction product was centrifuged at 13,000 rpm for 10 minutes at 4°C to remove the supernatant, and then washed by adding 1 ml of washing solution (500 mM NaCl, 60 mM imidazole, and 20 mM Tris-HCl). The process was repeated 5 times. Remove all supernatants to remove DNA aptamers that did not bind to ApoB100, add 200 ㎕ of DNA aptamer elution solution (300 mM NaCl, 20 mM Tris-HCl, and 250 mM imidazole) and incubate at 4°C for 1 hour. The reaction was carried out while stirring. Afterwards, the supernatant was obtained from the reaction solution at ℃ for 30 minutes. Only ssDNA was obtained from the obtained supernatant using 100% ethanol and tRNA as described above. The obtained pellet was dried and then diluted in 50 μl of distilled water that does not contain nuclease. SELEX was repeated 7 times using the obtained ssDNA as a template. Afterwards, negative SELEX was performed using only Ni-NTA agarose without ApoB100, and then SELEX was performed again three times. The affinity for ApoB100 of the DNA aptamer contained in each pool obtained during repeated SELEX was confirmed using a spectrophotometer using a conventional nano drop method, and the results are shown in Figure 1 shown in
도 1에 나타난 바와 같이, SELEX를 9 및 10회 반복하여 수득된 풀이 ApoB100에 가장 특이적으로 결합하는 것을 확인하였다.As shown in Figure 1, it was confirmed that the pool obtained by repeating SELEX 9 and 10 times most specifically bound to ApoB100.
실시예 3. ApoB100에 특이적으로 결합하는 DNA 앱타머의 서열 확인Example 3. Confirmation of the sequence of DNA aptamer that specifically binds to ApoB100
상기에서 나노드랍 방법으로 ApoB100에 특이적으로 결합하는 DNA 앱타머 풀로부터 DNA 앱타머 후보군의 서열을 다음과 같이 확인하였다.The sequences of DNA aptamer candidates from the pool of DNA aptamers specifically binding to ApoB100 were confirmed as follows using the nanodrop method above.
구체적으로, 선별된 DNA 앱타머 풀을 주형으로 상기 서술한 바와 같이 프라이머(서열번호 17 및 18)를 사용하여 PCR을 수행하고, 수득된 PCR 산물은 솔젠트사의 T-블런트 PCR 클로닝 키트를 이용하여 T-벡터 클로닝에 사용되었다. T-벡터 클로닝은 1㎕의 T-블런트 벡터(10 ng/㎕), 4 ㎕의 PCR 산물 및 1 ㎕의 6× T-블런트 버퍼를 혼합하여 25℃에서 5분 동안 반응시켜 수행하였다. 상기 반응액을 100 ㎕의 DH5α 수용성 균주(competent cell)와 섞은 후 42℃에서 30초 동안 열 충격(heat shock)을 가한 후 얼음에서 2분 동안 방치하였다. 반응액에 900 ㎕의 SOC 배지(2% 트립톤, 0.5% 효모추출물, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM 글루코즈)를 첨가한 후 37℃에서 1시간 동안 배양하였다. 이후, 200 ㎕의 배양액을 취하여 암피실린(ampicillin, 50 ㎍/㎖), 카나마이신(kanamycin, 50 ㎍/㎖), X-gal(50 ㎍/㎖) 및 IPTG(5 ㎍/㎖)가 포함되어 있는 LB 배양 플레이트(LB plate)에 도말하고, 37℃에서 15시간 동안 배양시킨 후, 24개의 흰색 콜로니만을 선별하여 확인된 16개의 각각 다른 DNA 앱타머 염기서열(Solgent, Korea)을 하기 표 2에 나타내었다.Specifically, PCR was performed using the selected DNA aptamer pool as a template using primers (SEQ ID NOs. 17 and 18) as described above, and the obtained PCR product was cloned using Solgent's T-blunt PCR cloning kit. Used for T-vector cloning. T-vector cloning was performed by mixing 1 μl of T-blunt vector (10 ng/μl), 4 μl of PCR product, and 1 μl of 6× T-blunt buffer and reacting at 25°C for 5 minutes. The reaction solution was mixed with 100 ㎕ of DH5α soluble strain (competent cell), heat shocked at 42°C for 30 seconds, and left on ice for 2 minutes. Add 900 ㎕ of SOC medium (2% tryptone, 0.5% yeast extract, 10mM NaCl, 2.5mM KCl, 10mM MgCl 2 , 10mM MgSO 4 , 20mM glucose) to the reaction solution and incubate at 37°C for 1 hour. cultured for a while. Afterwards, 200 ㎕ of culture medium was taken and LB containing ampicillin (50 ㎍/㎖), kanamycin (50 ㎍/㎖), X-gal (50 ㎍/㎖) and IPTG (5 ㎍/㎖). After smearing on a culture plate (LB plate) and culturing at 37°C for 15 hours, only 24 white colonies were selected, and the 16 different DNA aptamer base sequences (Solgent, Korea) identified are shown in Table 2 below. .
| 이름name | 서열order | 서열번호sequence number |
| AB100_1-12AB100_1-12 | ATACCAGCTTATTCAATTCCGTGAGGTG GCGTATCAAC GGATATTAGG GAGAGGGGGGAGATAGTAAGTGCAATCTATACCAGCTTATTCAATTCCGTGAGGTG GCGTATCAAC GGATATTAGG GAGAGGGGGGAGATAGTAAGTGCAATCT | 서열번호 1SEQ ID NO: 1 |
| AB100_2-1AB100_2-1 | ATACCAGCTTATTCAATTGTGACTGGCC ATCGATTTTA ACCCTTTTTC CTTGTGCCACAGATAGTAAGTGCAATCTATACCAGCTTATTCAATTGTGACTGGCC ATCGATTTTA ACCCTTTTTC CTTGTGCCACAGATAGTAAGTGCAATCT | 서열번호 2SEQ ID NO: 2 |
| AB100_3-1AB100_3-1 | ATACCAGCTTATTCAATTGCGGTAGCAG GGCGCATTCG GATGAACATC GCCGGTGACGAGATAGTAAGTGCAATCTATACCAGCTTATTCAATTGCGGTAGCAG GGCGCATTCG GATGAACATC GCCGGTGACGAGATAGTAAGTGCAATCT | 서열번호 3SEQ ID NO: 3 |
| AB100_3-2AB100_3-2 | ATACCAGCTTATTCAATTCCTATTCCTTATTATATTTTCTTTTTTTGTAATTTGGTCGAGATAGTAAGTGCAATCTATACCAGCTTATTCAATTCCTATTCCTTATTATATTTTCTTTTTTTGTAATTTGGTCGAGATAGTAAGTGCAATCT | 서열번호 4SEQ ID NO: 4 |
| AB100_3-3AB100_3-3 | ATACCAGCTTATTCAATTCACGCTATCG GCCTTGAGAG GGTCTAAGCA ACGTATCCCAAGATAGTAAGTGCAATCTATACCAGCTTATTCAATTCACGCTATCG GCCTTGAGAG GGTCTAAGCA ACGTATCCCAAGATAGTAAGTGCAATCT | 서열번호 5SEQ ID NO: 5 |
| AB100_3-8AB100_3-8 | ATACCAGCTTATTCAATTTGGGCTGGCC GAGAGTTGAC GATTTGTGCA GTCGTTAGGCAGATAGTAAGTGCAATCTATACCAGCTTATTCAATTTGGGCTGGCC GAGAGTTGAC GATTTGTGCA GTCGTTAGGCAGATAGTAAGTGCAATCT | 서열번호 6SEQ ID NO: 6 |
| AB100_3-10AB100_3-10 | ATACCAGCTTATTCAATTCGTGAAAGGC GAACGACCAA CGGTGGAGTC TGGTTGGTGGAGATAGTAAGTGCAATCTATACCAGCTTATTCAATTCGTGAAAGGC GAACGACCAA CGGTGGAGTC TGGTTGGTGGAGATAGTAAGTGCAATCT | 서열번호 7SEQ ID NO: 7 |
| AB100_3-11AB100_3-11 | ATACCAGCTTATTCAATTCGCCGAAAGACAGTTAGTGTTCACTCTCGTGATGATGGCAAGATAGTAAGTGCAATCTATACCAGCTTATTCAATTCGCCGAAAGACAGTTAGTGTTCACTCTCGTGATGGATGGCAAGATAGTAAGTGCAATCT | 서열번호 8SEQ ID NO: 8 |
| AB100_3-13AB100_3-13 | ATACCAGCTTATTCAATTGGCGAGGGAG TATGTACGAC TACGTTGTAG GGTTGCTCTAAGATAGTAAGTGCAATCTATACCAGCTTATTCAATTGGCGAGGGAG TATGTACGAC TACGTTGTAG GGTTGCTCTAAGATAGTAAGTGCAATCT | 서열번호 9SEQ ID NO: 9 |
| AB100_3-18AB100_3-18 | ATACCAGCTTATTCAATTGGGCGCCTGC GCTATTTGTG ATTGAAGTAA TTGAAGTGCGAGATAGTAAGTGCAATCTATACCAGCTTATTCAATTGGGCGCCTGC GCTATTTGTG ATTGAAGTAA TTGAAGTGCGAGATAGTAAGTGCAATCT | 서열번호 10SEQ ID NO: 10 |
| AB100_3-19AB100_3-19 | ATACCAGCTTATTCAATTTGTCGTACGT GTACAGGGTA CAAATCTCCA ATCTTGGTTAAGATAGTAAGTGCAATCTATACCAGCTTATTCAATTTGTCGTACGT GTACAGGGTA CAAAATCTCCA ATCTTGGTTAAGATAGTAAGTGCAATCT | 서열번호 11SEQ ID NO: 11 |
| AB100_3-20AB100_3-20 | ATACCAGCTTATTCAATTCGGAAGGGGC ATGTAAGGAC CCTGTCTGAG TGAGAACATGAGATAGTAAGTGCAATCTATACCAGCTTATTCAATTCGGAAGGGGC ATGTAAGGAC CCTGTCTGAG TGAGAACATGAGATAGTAAGTGCAATCT | 서열번호 12SEQ ID NO: 12 |
| AB100_4-3AB100_4-3 | ATACCAGCTTATTCAATTCGGGCGAGGA GATTGAAATC GGTAGGACCT CCTGGCCTGC GAGATAGTAAGTGCAATCTATACCAGCTTATTCAATTCGGGCGAGGA GATTGAAATC GGTAGGACCT CCTGGCCTGC GAGATAGTAAGTGCAATCT | 서열번호 13SEQ ID NO: 13 |
| AB100_4-4AB100_4-4 | ATACCAGCTTATTCAATTCTAAAAAGTT TCGCTCTCTC GCTCTTTATG TATGGTTTCAAGATAGTAAGTGCAATCTATACCAGCTTATTCAATTCTAAAAAGTT TCGCTCTCTC GCTCTTTATG TATGGTTTCAAGATAGTAAGTGCAATCT | 서열번호 14SEQ ID NO: 14 |
| AB100_4-5AB100_4-5 | ATACCAGCTTATTCAATTCCTATTCCTT ATTATATTTT CTTTTTTTGT AATTTGGTCGAGATAGTAAGTGCAATCTATACCAGCTTATTCAATTCCTATTCCTT ATTATATTTT CTTTTTTTGT AATTTGGTCGAGATAGTAAGTGCAATCT | 서열번호 15SEQ ID NO: 15 |
| AB100_4-20AB100_4-20 | ATACCAGCTTATTCAATTCTATTCCTTA TTATATTTTC TTTTTTTGTA ATTTGGTCGAGATAGTAAGTGCAATCTATACCAGCTTATTCAATTCTATTCCTTA TTATATTTTC TTTTTTTGTA ATTTGGTCGAGATAGTAAGTGCAATCT | 서열번호 16SEQ ID NO: 16 |
실시예 4. ApoB100에 대한 결합력의 정량분석Example 4. Quantitative analysis of binding affinity to ApoB100
상기에서 선별된 16종류의 앱타머의 ApoB100에 대한 친화력을 통상적인 표면 플라즈몬 공명(Surface Plasmon Resonance, SPR) 방법으로 확인하였다.The affinity of the 16 types of aptamers selected above for ApoB100 was confirmed using a conventional surface plasmon resonance (SPR) method.
먼저, 16종류의 DNA 앱타머를 HBS-EP 완충액(GE Healthcare, BR-1001-88)에 0, 100, 200 또는 500 nM의 농도가 되도록 희석하여 준비하였다. 이때, 200 nM 농도인 경우를 2회 실험하여 결과에 대한 오차를 줄일 수 있도록 준비하였다. 준비된 DNA 앱타머와 BIAcore T200을 사용하여 제조사의 프로토콜에 따라 실험을 수행하였으며, ApoB100이 코팅된 센서칩 CM5에 대한 DNA 앱타머의 결합력과 아무것도 코팅되지 않은 센서칩 CM5에 대한 DNA 앱타머의 결합력 차이를 확인하였다. 이때, 속도변수는 BIA 평가프로그램(BIACORE)을 이용하여 수득 및 정량하였고, 각 실험 후, 센서칩은 0.5% SDS를 이용하여 재생시켰다. 그 결과, DNA 앱타머의 ApoB100에 대한 친화력을 표 3에 나타내었다. 최종 결합력은 DNA 앱타머가 처리된 각각의 농도에 대한 친화도 값으로부터 평균 및 표준편차를 계산하여 산출하였다.First, 16 types of DNA aptamers were prepared by diluting them in HBS-EP buffer (GE Healthcare, BR-1001-88) to a concentration of 0, 100, 200, or 500 nM. At this time, the 200 nM concentration was tested twice to reduce errors in the results. An experiment was performed using the prepared DNA aptamer and BIAcore T200 according to the manufacturer's protocol, and the difference in the binding affinity of the DNA aptamer to the sensor chip CM5 coated with ApoB100 and the binding affinity of the DNA aptamer to the sensor chip CM5 without any coating was performed. was confirmed. At this time, the speed variable was obtained and quantified using the BIA evaluation program (BIACORE), and after each experiment, the sensor chip was regenerated using 0.5% SDS. As a result, the affinity of the DNA aptamer for ApoB100 is shown in Table 3. The final binding force was calculated by calculating the average and standard deviation from the affinity values for each concentration at which the DNA aptamer was treated.
| 100 nM100 nM | 200 nM(1)200 nM(1) | 500 nM500 nM | 200 nM(2)200 nM(2) | 평균average | 결합력(nM)Binding force (nM) | |
| AB100_1-12AB100_1-12 | 3.79E-093.79E-09 | 1.23E-081.23E-08 | 5.21E-095.21E-09 | 4.37E-094.37E-09 | 6.42E-096.42E-09 | 6.42±1.726.42±1.72 |
| AB100_2-1AB100_2-1 | 1.68E-101.68E-10 | 1.10E-101.10E-10 | 1.33E-091.33E-09 | 1.38E-091.38E-09 | 7.47E-107.47E-10 | 0.747±0.3040.747±0.304 |
| AB100_3-1AB100_3-1 | 2.18E-102.18E-10 | 1.70E-091.70E-09 | 7.09E-097.09E-09 | 4.40E-084.40E-08 | 1.32E-081.32E-08 | 13.2±8.9513.2±8.95 |
| AB100_3-2AB100_3-2 | 1.29E-081.29E-08 | 2.51E-072.51E-07 | 2.10E-092.10E-09 | 1.54E-071.54E-07 | 1.05E-071.05E-07 | 105±51.6105±51.6 |
| AB100_3-3AB100_3-3 | 1.28E-071.28E-07 | 4.03E-084.03E-08 | 6.76E-096.76E-09 | 1.09E-081.09E-08 | 4.65E-084.65E-08 | 46.5±24.446.5±24.4 |
| AB100_3-8AB100_3-8 | 4.27E-104.27E-10 | 3.15E-103.15E-10 | 3.05E-103.05E-10 | 2.01E-102.01E-10 | 3.12E-103.12E-10 | 0.312±0.03990.312±0.0399 |
| AB100_3-10AB100_3-10 | 3.97E-103.97E-10 | 1.10E-101.10E-10 | 4.06E-104.06E-10 | 4.01E-104.01E-10 | 3.28E-103.28E-10 | 0.328±0.06320.328±0.0632 |
| AB100_3-11AB100_3-11 | 3.24E-103.24E-10 | 3.24E-103.24E-10 | 1.31E-101.31E-10 | 2.67E-102.67E-10 | 2.62E-102.62E-10 | 0.262±0.03950.262±0.0395 |
| AB100_3-13AB100_3-13 | 8.89E-088.89E-08 | 9.06E-119.06E-11 | 1.15E-091.15E-09 | 1.38E-091.38E-09 | 2.29E-082.29E-08 | 22.9±19.0522.9±19.05 |
| AB100_3-18AB100_3-18 | 4.18E-094.18E-09 | 2.24E-082.24E-08 | 1.85E-081.85E-08 | 1.99E-111.99E-11 | 1.13E-081.13E-08 | 13.8±4.7013.8±4.70 |
| AB100_3-19AB100_3-19 | 3.93E-093.93E-09 | 3.43E-093.43E-09 | 1.41E-081.41E-08 | 1.23E-081.23E-08 | 8.43E-098.43E-09 | 8.43±2.408.43±2.40 |
| AB100_3-20AB100_3-20 | 6.47E-076.47E-07 | 6.73E-096.73E-09 | 7.85E-097.85E-09 | 8.19E-098.19E-09 | 1.67E-071.67E-07 | 167±138167±138 |
| AB100_4-3AB100_4-3 | 1.03E-081.03E-08 | 1.02E-081.02E-08 | 1.03E-081.03E-08 | 1.04E-081.04E-08 | 1.03E-081.03E-08 | 10.3±0.037610.3±0.0376 |
| AB100_4-4AB100_4-4 | 5.20E-095.20E-09 | 9.35E-099.35E-09 | 4.41E-094.41E-09 | 2.06E-092.06E-09 | 5.25E-095.25E-09 | 5.25±1.315.25±1.31 |
| AB100_4-5AB100_4-5 | 3.48E-093.48E-09 | 5.03E-095.03E-09 | 5.36E-095.36E-09 | 4.60E-094.60E-09 | 4.62E-094.62E-09 | 4.62±0.3564.62±0.356 |
| AB100_4-20AB100_4-20 | 1.12E-081.12E-08 | 1.06E-081.06E-08 | 1.05E-081.05E-08 | 1.05E-081.05E-08 | 1.07E-081.07E-08 | 10.7±0.14610.7±0.146 |
표 3에 나타난 바와 같이, 선별된 16종류의 DNA 앱타머는 모두 ApoB100에 특이적으로 결합하였다.As shown in Table 3, all 16 types of selected DNA aptamers specifically bound to ApoB100.
실시예 5. DNA 앱타머의 세포독성 확인Example 5. Confirmation of cytotoxicity of DNA aptamer
상기에서 선별된 DNA 앱타머 중 9개를 선별하여 LDH 분석방법으로 세포독성을 확인하였다.Nine of the DNA aptamers selected above were selected and their cytotoxicity was confirmed using the LDH analysis method.
먼저, 인간유래 단핵구 세포인 THP-1(KCLB 40202, 한국) 세포주를 10% FBS(fetal bovine serum)이 포함된 RPMI 1640 배양배지를 사용하여 배양하였다. 배양된 세포를 96웰 플레이트에 웰당 4×104개가 되도록 분주하고, 37℃ 및 5% CO2 조건에서 하룻밤 동안 배양하였다. 배양된 세포에 DNA 앱타머를 0.32, 1.6, 8, 40, 200, 1,000 또는 5,000 nM의 농도가 되도록 10 ㎕ 처리하고 다시 24시간 동안 배양하였다. 배양 후, 세포 배양액을 사용하여 CyQUANT™ LDH 세포독성 분석 키트(Invitrogen, 미국)를 사용하여 제조사의 프로토콜에 따라 수행되었다. 이때, 음성 대조군은 PBS 완충액을 사용하였다. 그 결과, 수득된 값으로부터 세포독성(%)을 계산하여 도 2에 나타내었다.First, the THP-1 (KCLB 40202, Korea) cell line, a human-derived mononuclear cell line, was cultured using RPMI 1640 culture medium containing 10% FBS (fetal bovine serum). The cultured cells were distributed in a 96-well plate at 4×10 4 cells per well and cultured overnight at 37°C and 5% CO 2 conditions. Cultured cells were treated with 10 μl of DNA aptamer to a concentration of 0.32, 1.6, 8, 40, 200, 1,000, or 5,000 nM and cultured for another 24 hours. After incubation, the cell culture medium was used to perform the CyQUANT™ LDH cytotoxicity assay kit (Invitrogen, USA) according to the manufacturer's protocol. At this time, PBS buffer was used as the negative control. As a result, cytotoxicity (%) was calculated from the obtained values and shown in Figure 2.
도 2에 나타난 바와 같이, 9종류의 DNA 앱타머는 모두 세포독성을 나타내지 않았다. 다만, AB100_3-2 앱타머는 2.5~5 μM의 고농도에서만 약 6~9%의 세포독성을 보였다.As shown in Figure 2, all nine types of DNA aptamers did not show cytotoxicity. However, AB100_3-2 aptamer showed about 6-9% cytotoxicity only at high concentrations of 2.5-5 μM.
실시예 6. 콜레스테롤 저감Example 6. Cholesterol Reduction
상기에서 선별된 9종의 DNA 앱타머의 콜레스테롤 저감 활성을 콜레스테롤 어세이 키트(cholesterol assay kit, Abcam, 영국)를 사용하여 다음과 같이 확인하였다.The cholesterol-lowering activity of the nine types of DNA aptamers selected above was confirmed using a cholesterol assay kit (Abcam, UK) as follows.
먼저, 50 ㎕의 LDL(low-density lipoprotien, Sigma aldrich, 미국)과 동량의 ApoB100에 대한 앱타머를 혼합하고, 100 ㎕의 2× 침전 완충액을 첨가하고 실온에서 10분 동안 반응시켰다. 이때, 앱타머는 AB100_1-12, AB100_2-1, AB100_3-1, AB100_3-2, AB100_3-8, AB100_3-10, AB100_3-11, AB100_3-19 또는 AB100_3-20을 사용하였고, 1.14, 3.08 및 5.03 ㎍의 세 농도로 실험하였다. 이때, 대조군으로서 앱타머를 첨가하지 않은 샘플을 사용하였다. 반응이 끝난 후, 상기 혼합물을 5,000 rpm에서 10분 동안 원심분리하여 펠렛을 회수하였다. 회수된 펠렛을 200 ㎕의 1× PBS에 현탁하였다. 상기 현탁액을 96웰 플레이트에 50 ㎕씩 분주하고, 45.6 ㎕의 콜레스테롤 어세이 완충액, 0.4 ㎕의 콜레스테롤 프로브, 2 ㎕의 효소 혼합물, 2 ㎕의 콜레스테롤 에스테라제(cholesterol esterase)를 첨가하였다. 이를 37℃에서 1시간 동안 반응시킨 후, Ex/Em=535/587 ㎚ 조건에서 형광세기를 측정하여 샘플 내 LDL/vLDL 농도를 제조사의 프로토콜에 따라 확인하였다. 그 결과, 앱타머를 첨가하지 않은 샘플의 LDL/vLDL 농도를 기준으로 앱타머를 처리한 경우의 LDL/vLDL 농도를 상대적인 값으로 나타낸 결과를 도 3에 나타내었다.First, 50 ㎕ of LDL (low-density lipoprotien, Sigma aldrich, USA) and the same amount of aptamer for ApoB100 were mixed, 100 ㎕ of 2× precipitation buffer was added, and reacted at room temperature for 10 minutes. At this time, the aptamers used were AB100_1-12, AB100_2-1, AB100_3-1, AB100_3-2, AB100_3-8, AB100_3-10, AB100_3-11, AB100_3-19, or AB100_3-20, and 1.14, 3.08, and 5.03 μg of aptamers were used. Three concentrations were tested. At this time, a sample to which no aptamer was added was used as a control. After the reaction was completed, the mixture was centrifuged at 5,000 rpm for 10 minutes to recover the pellet. The recovered pellet was suspended in 200 μl of 1×PBS. 50 μl of the suspension was dispensed into 96-well plates, and 45.6 μl of cholesterol assay buffer, 0.4 μl of cholesterol probe, 2 μl of enzyme mixture, and 2 μl of cholesterol esterase were added. After reacting at 37°C for 1 hour, the fluorescence intensity was measured under the condition of Ex/Em=535/587 nm to confirm the LDL/vLDL concentration in the sample according to the manufacturer's protocol. As a result, the results showing the relative values of the LDL/vLDL concentration when treated with the aptamer based on the LDL/vLDL concentration of the sample without the addition of the aptamer are shown in Figure 3.
도 3에 나타난 바와 같이, ApB100에 대한 앱타머는 모두 콜레스테롤 저감 효과를 나타내었다. 특히, AB100_1-12, AB100_2-1, AB100_3-1 및 AB100_3-8 앱타머는 1.14 ㎍의 낮은 농도에서도 유의적으로 콜레스테롤을 저감시켰다.As shown in Figure 3, all aptamers for ApB100 showed a cholesterol-reducing effect. In particular, AB100_1-12, AB100_2-1, AB100_3-1, and AB100_3-8 aptamers significantly reduced cholesterol even at a low concentration of 1.14 μg.
실시예 7. 병용 처리에 의한 콜레스테롤 저감Example 7. Cholesterol reduction by combination treatment
상기에서 가장 유의적인 활성을 나타낸 AB100_1-12 앱타머를 콜레스테롤 저감용 치료제인 레시틴(lecithin)과 병용처리한 경우의 콜레스테롤 저감 효과를 확인하였다. 구체적으로, 실험은 실시예 6에 기재된 바와 동일한 방법으로 수행되었고, 1 ㎎의 레시틴에 다양한 농도(0.1, 0.5, 1, 5, 10 또는 50 μM)의 AB100_1-12 앱타머를 첨가하여 실험을 수행하였다. 그 결과, LDL/vLDL 농도를 확인한 결과를 도 4에 나타내었다.The cholesterol-reducing effect was confirmed when AB100_1-12 aptamer, which showed the most significant activity above, was treated in combination with lecithin, a treatment for cholesterol reduction. Specifically, the experiment was performed in the same manner as described in Example 6, and the experiment was performed by adding various concentrations (0.1, 0.5, 1, 5, 10, or 50 μM) of AB100_1-12 aptamer to 1 mg of lecithin. did. As a result, the results of confirming the LDL/vLDL concentration are shown in Figure 4.
도 4에 나타난 바와 같이, 레시틴을 단독으로 처리한 경우, LDL 콜레스테롤의 농도가 약물 미처리군과 비교하여 약 63% 감소하였으며, 여기에 AB100_1-12 앱타머를 병용처리한 경우에는 73 내지 78%까지 콜레스테롤 농도가 감소하였다.As shown in Figure 4, when treated with lecithin alone, the concentration of LDL cholesterol decreased by about 63% compared to the drug-untreated group, and when treated in combination with AB100_1-12 aptamer, it decreased by 73 to 78%. Cholesterol concentration decreased.
따라서, 상기 결과로부터 본 발명에 따른 DNA 앱타머가 세포독성을 보이지 않으면서 ApoB100에 특이적으로 결합하여 콜레스테롤을 저감시킴으로써, 고콜레스테롤에 의해 발생할 수 있는 질환의 치료에 유용하게 사용될 수 있다.Therefore, from the above results, the DNA aptamer according to the present invention reduces cholesterol by specifically binding to ApoB100 without showing cytotoxicity, and can be usefully used in the treatment of diseases that can be caused by high cholesterol.
Claims (15)
- 서열번호 1 내지 16으로 각각 기재된 염기서열로 구성된 군으로부터 선택된 염기서열로 구성되는 ApoB100(apolipoprotein B100)에 특이적으로 결합하는 DNA 앱타머.A DNA aptamer that specifically binds to ApoB100 (apolipoprotein B100) consisting of a base sequence selected from the group consisting of the base sequences shown in SEQ ID NOs: 1 to 16, respectively.
- 제1항의 DNA 앱타머를 포함하는 ApoB100 검출용 조성물.A composition for detecting ApoB100 comprising the DNA aptamer of claim 1.
- 제1항의 DNA 앱타머를 포함하는 ApoB100 검출용 키트.A kit for detecting ApoB100 containing the DNA aptamer of claim 1.
- 제1항의 DNA 앱타머를 시료와 반응시키는 단계를 포함하는 ApoB100 검출방법.ApoB100 detection method comprising reacting the DNA aptamer of claim 1 with a sample.
- 제1항의 DNA 앱타머를 포함하는 이상지질혈증 또는 혈관질환 진단용 조성물.A composition for diagnosing dyslipidemia or vascular disease comprising the DNA aptamer of claim 1.
- 제5항에 있어서, 상기 이상지질혈증은 고지혈증, 고콜레스테롤혈증 또는 고중성지방혈증인, 이상지질혈증 또는 혈관질환 진단용 조성물.The composition for diagnosing dyslipidemia or vascular disease according to claim 5, wherein the dyslipidemia is hyperlipidemia, hypercholesterolemia, or hypertriglyceridemia.
- 제5항에 있어서, 상기 혈관질환은 동맥경화, 죽상경화증, 죽상동맥경화증, 뇌경색, 협심증, 말초혈관 폐색성 질환, 심근경색, 당뇨성 망막증 또는 고혈압성 망막증인, 이상지질혈증 또는 혈관질환 진단용 조성물.The method of claim 5, wherein the vascular disease is arteriosclerosis, atherosclerosis, atherosclerosis, cerebral infarction, angina pectoris, peripheral vascular occlusive disease, myocardial infarction, diabetic retinopathy or hypertensive retinopathy, dyslipidemia, or a composition for diagnosing vascular disease. .
- 제1항의 DNA 앱타머를 포함하는 이상지질혈증 또는 혈관질환 진단용 키트.A kit for diagnosing dyslipidemia or vascular disease comprising the DNA aptamer of claim 1.
- 제1항의 DNA 앱타머를 시료와 반응시키는 단계를 포함하는 이상지질혈증 또는 혈관질환 진단의 정보를 제공하기 위한 방법.A method for providing information on the diagnosis of dyslipidemia or vascular disease, comprising reacting the DNA aptamer of claim 1 with a sample.
- 제1항의 DNA 앱타머를 유효성분으로 포함하는 이상지질혈증 또는 혈관질환의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating dyslipidemia or vascular disease, comprising the DNA aptamer of claim 1 as an active ingredient.
- 제10항에 있어서, 상기 이상지질혈증은 고지혈증, 고콜레스테롤혈증 또는 고중성지방혈증인, 이상지질혈증 또는 혈관질환의 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating dyslipidemia or vascular disease according to claim 10, wherein the dyslipidemia is hyperlipidemia, hypercholesterolemia, or hypertriglyceridemia.
- 제10항에 있어서, 상기 혈관질환은 동맥경화, 죽상경화증, 죽상동맥경화증, 뇌경색, 협심증, 말초혈관 폐색성 질환, 심근경색, 당뇨성 망막증 또는 고혈압성 망막증인, 이상지질혈증 또는 혈관질환의 예방 또는 치료용 약학적 조성물.The method of claim 10, wherein the vascular disease is arteriosclerosis, atherosclerosis, atherosclerosis, cerebral infarction, angina pectoris, peripheral vascular occlusive disease, myocardial infarction, diabetic retinopathy or hypertensive retinopathy, dyslipidemia, or prevention of vascular disease. or therapeutic pharmaceutical compositions.
- 제1항의 DNA 앱타머를 유효성분으로 포함하는 이상지질혈증 또는 혈관질환의 예방 또는 개선용 건강기능식품.A health functional food for preventing or improving dyslipidemia or vascular disease containing the DNA aptamer of claim 1 as an active ingredient.
- 제1항의 DNA 앱타머를 개체에 투여하는 단계를 포함하는 이상지질혈증 또는 혈관질환의 예방, 개선 또는 치료방법Method for preventing, improving or treating dyslipidemia or vascular disease comprising administering the DNA aptamer of claim 1 to an individual.
- 이상지질혈증 또는 혈관질환의 예방, 개선 또는 치료방법에 사용하기 위한 제1항에 따른 DNA 앱타머의 용도.Use of the DNA aptamer according to paragraph 1 for use in a method of preventing, improving, or treating dyslipidemia or vascular disease.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150105440A1 (en) * | 2001-08-01 | 2015-04-16 | Genzyme Corporation | Antisense modulation of apolipoprotein b expression |
| KR20150090284A (en) * | 2007-03-24 | 2015-08-05 | 젠자임 코포레이션 | Administering antisense oligonucleotides complementary to human apolipoprotein b |
| WO2019010341A1 (en) * | 2017-07-07 | 2019-01-10 | Innamed, Inc. | Aptamers for measuring lipoprotein levels |
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| KR102155306B1 (en) | 2020-02-17 | 2020-09-11 | 연세대학교 산학협력단 | Compositions, kits for predicting high density lipoprotein cholesterol levels, and method using the same |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150105440A1 (en) * | 2001-08-01 | 2015-04-16 | Genzyme Corporation | Antisense modulation of apolipoprotein b expression |
| KR20150090284A (en) * | 2007-03-24 | 2015-08-05 | 젠자임 코포레이션 | Administering antisense oligonucleotides complementary to human apolipoprotein b |
| WO2019010341A1 (en) * | 2017-07-07 | 2019-01-10 | Innamed, Inc. | Aptamers for measuring lipoprotein levels |
Non-Patent Citations (2)
| Title |
|---|
| CAO JIANFANG, LV PEIYING, SHU YANG, WANG JIANHUA: "Aptamer/AuNPs encoders endow precise identification and discrimination of lipoprotein subclasses", BIOSENSORS AND BIOELECTRONICS, ELSEVIER SCIENCE LTD, UK, AMSTERDAM , NL, vol. 196, 1 January 2022 (2022-01-01), Amsterdam , NL , pages 113743, XP093091020, ISSN: 0956-5663, DOI: 10.1016/j.bios.2021.113743 * |
| KLAPAK DANIEL, BROADFOOT SARAH, PENNER GREGORY, SINGH ANUP, INAPURI ESHWAR: "Development of novel aptamers for low-density lipoprotein particle quantification", PLOS ONE, vol. 13, no. 10, 11 October 2018 (2018-10-11), pages e0205460, XP093087942, DOI: 10.1371/journal.pone.0205460 * |
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