KR100607901B1 - Method for identification and analysis of specific molecules using molecular beacons - Google Patents

Method for identification and analysis of specific molecules using molecular beacons Download PDF

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KR100607901B1
KR100607901B1 KR1020040021199A KR20040021199A KR100607901B1 KR 100607901 B1 KR100607901 B1 KR 100607901B1 KR 1020040021199 A KR1020040021199 A KR 1020040021199A KR 20040021199 A KR20040021199 A KR 20040021199A KR 100607901 B1 KR100607901 B1 KR 100607901B1
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nucleic acid
stranded nucleic
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김성천
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제노프라 주식회사
김성천
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Abstract

본 발명은 특정물질의 동정 및 정량방법에 관한 것으로, 보다 상세하게는 단일가닥 핵산 서열에 따른 분자비콘을 제조하고, 상기 제조된 분자비콘을 이용하여 특정물질의 동정 및 정량하는 방법에 관한 것이다. 분자비콘은 특정물질과 특이적으로 결합할 수 있으며 두 물질이 동시에 있으면 열역학적으로 안정한 복합체를 형성하는 성질이 있다. The present invention relates to a method for identifying and quantifying a specific substance, and more particularly, to a method for preparing a molecular beacon according to a single-stranded nucleic acid sequence, and identifying and quantifying a specific substance using the prepared molecular beacon. Molecular beacons can bind specifically to specific substances, and when two substances are present at the same time, they form a thermodynamically stable complex.

따라서, 본 발명에 의해 제조된 분자비콘 및 상기 분자비콘을 이용한 특정물질의 동정 및 정량분석은 미생물, 세포, 단백질을 포함하는 생체분자 등에 대한 탐색 및 분석 등에 사용할 수 있어 궁극적으로는 의학, 수의학, 환경공학, 식품공학, 농업 등에 광범위하게 응용될 수 있다.Therefore, the identification and quantitative analysis of the molecular beacon and the specific material using the molecular beacon prepared by the present invention can be used for the search and analysis of biomolecules including microorganisms, cells, proteins, etc. Ultimately, medicine, veterinary medicine, It can be widely applied to environmental engineering, food engineering and agriculture.

단일가닥핵산리간드, 분자비콘, 동정, 정량, 분석방법Single-stranded nucleic acid ligand, molecular beacon, identification, quantification, analysis method

Description

분자비콘을 이용한 특정물질의 동정 및 분석방법{Method for identification and analysis of specific molecules using molecular beacons} Method for identification and analysis of specific molecules using molecular beacons}             

도 1 은 본 발명의 분자비콘 구성을 나타낸 도면이고,1 is a view showing the configuration of the molecular beacon of the present invention,

도 2 은 단일가닥핵산의 다양한 염기서열 길이(a는 염기 15 개 이상, b는 염기 9 내지 11, c는 염기 7 이하의 경우) 및 온도(22℃ 와 37℃의 경우)에 대한 안정성 여부를 나타낸 도면이고,Figure 2 shows the stability of the various base sequence lengths (a is at least 15 bases, b is 9 to 11 bases, c is less than 7 bases) and temperature (for 22 ℃ and 37 ℃) of the single-stranded nucleic acid The figure shown,

도 3 은 분자비콘의 작용에 관한 개략도이고, 3 is a schematic diagram of the action of the molecular beacons,

도 4 은 분자비콘와 특정물질 복합체 형성에 있어서, 온도(22℃ 및 37℃)에 따른 반응안정성을 나타낸 도면이고,4 is a view showing the reaction stability according to the temperature (22 ℃ and 37 ℃) in the formation of the molecular beacon and specific material complex,

도 5 은 분자비콘으로 특정물질을 동정, 분석하는 방법을 나타낸 도면이다.5 is a view showing a method for identifying and analyzing specific substances by molecular beacons.

본 발명은 특정물질의 동정 및 정량적 분석방법에 관한 것이며, 보다 상세하게는 분자비콘의 복합체 형성능력을 이용하여 특정물질의 동정 및 정량적인 변화에 대한 탐색 및 분석하는 단순하고 새로운 방법에 관한 것이다. The present invention relates to a method for identifying and quantitatively analyzing a specific substance, and more particularly, to a simple and novel method for searching and analyzing identification and quantitative change of a specific substance using a complex beacon capacity of molecular beacons.

특정물질의 동정 및 정량적인 변화에 대한 탐색 및 분석하는 기술은 물리학 및 생화학의 발달로 많이 개발되었으나 기존 방법이나 기기의 사용 및 유지비, 용이성, 정확도, 민감도, 검사시간 및 과정의 자동화 등에 대한 문제로 효율적인 새로운 방법 및 기기에 대한 필요성이 매우 높다. 특정물질을 분석하는 방법은 그 자체가 궁극적인 목표가 아니라 전 과정의 한 부분이며 이런 방법은 미생물 및 바이러스 동정, 세포, 단백질, 유기물질 분석 등에 유용하게 사용할 수 있어 의학, 수의학, 환경공학, 식품공학, 농업 등에 광범위하게 응용되고 있다. The technology to search for and analyze the identification and quantitative change of a specific substance has been developed due to the development of physics and biochemistry, but the problems related to the use and maintenance cost, ease, accuracy, sensitivity, inspection time and process automation of existing methods or devices The need for efficient new methods and devices is very high. Analyzing specific substances is not an ultimate goal in itself, but rather a part of the whole process, which can be useful for microbial and viral identification, cell, protein, and organic material analysis, leading to medical, veterinary, environmental and food engineering. It is widely applied to agriculture and agriculture.

핵산은 뉴클레오타이드가 공유결합으로 연결된 선형적인 다합체이며 뉴클레오타이드는 작은 유기화합물로 인산, 당 및 퓨린(아데닌 혹은 구아닌) 혹은 피리미딘(사이토신, 티미딘, 우라실)이다. 단일가닥핵산은 상보적인 단일가닥핵산과는 염기쌍의 수소결합에 의해 이중가닥 이중가닥으로 존재하는데, 단일가닥은 특정한 물리적인 조건에서 뉴클레오타이드들간의 수소결합 및 상호작용에 의해 결합하여 독특한 입체구조를 형성하는데, 이런 구조는 단일가닥의 염기서열에 의해 결정된다. Nucleic acids are linear multimers in which nucleotides are covalently linked, and nucleotides are small organic compounds that are phosphoric acid, sugars and purines (adenine or guanine) or pyrimidine (cytosine, thymidine, uracil). Single-stranded nucleic acids exist as double-stranded double-stranded strands by base pair hydrogen bonds with complementary single-stranded nucleic acids. Single-stranded nucleic acids bind by hydrogen bonds and interactions between nucleotides under specific physical conditions to form unique conformations. This structure is determined by the nucleotide sequence of a single strand.

일반적으로 데옥시리보핵산(DNA) 및 리보핵산(RNA)과 같은 핵산들은 세포구조 및 효소 등의 활성을 가진 단백질들을 발현하기 위한 정보의 저장체이나, 1982년에 RNA가 특정한 구조를 형성함으로써 효소적 활성도 갖고 있다는 보고가 나온 후로 핵산이 구조적인 특성과 그에 따른 특정 기능에 대한 많은 보고가 있다. 핵산은 4개의 염기의 반복으로 구성되어 높은 다양성을 유지하여 많은 입체구조를 형성 하며 이런 입체구조는 특정물질과 상호작용을 하여 복합체를 형성하여 안정화된다. In general, nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are stores of information for expressing proteins with cell structure and enzyme activity, but in 1982, RNA forms a specific structure. Since the publication of its activity, there have been many reports on the structural properties of a nucleic acid and its specific function. Nucleic acid is composed of four base repeats to maintain a high diversity to form a large number of three-dimensional structure, these three-dimensional structure is stabilized by interacting with a specific material to form a complex.

핵산이 단백질을 포함하는 특정물질에 대하여 하나의 리간드(ligand)로서 작용할 수 있는 바, 다양한 염기순서로 배열된 단일가닥 핵산이 조합된 라이브러리로부터 일정한 선별과정과 염기서열결정을 통하여 높은 결합력과 특이성으로 특정물질과 결합하는 핵산들을 선별되고 있다. 특정물질과 결합하는 핵산을 선별하는 방법은 셀렉스 (SELEX, Systematic Evolution of Ligand by Exponential enrichment)라 하고, 선별된 산물인 단일가닥핵산 리간드를 압타머(aptamer)라 하며(Tuerk C. and Gold L.; Science, 249, pp505-510, 1990), SELEX를 통하여 자연상태에서 핵산과 결합할 수 있는 단백질뿐만 아니라 핵산과 결합하고 있지 않은 단백질을 비롯한 여러 생체분자와도 매우 높은 친화력으로 결합할 수 있는 분자들이 선별되고 있다. SELEX 방법은 압타머 리간드가 생체에서 안정성 및 운반성, 선택성 및 친화력 등을 고려하여 변형된 뉴클레오타이드를 이용하여 개발되고 있다.Nucleic acid can act as a ligand to a specific substance containing a protein, with high binding capacity and specificity through a constant screening process and sequencing from a library of single stranded nucleic acids arranged in various nucleotide sequences. Nucleic acids that bind to specific substances are being screened. The method of selecting nucleic acid binding to a specific substance is called SELEX (Systematic Evolution of Ligand by Exponential enrichment), and the single-stranded nucleic acid ligand, the selected product, is called aptamer (Tuerk C. and Gold L. Science , 249, pp 505-510, 1990), a molecule capable of binding with a high affinity to various biomolecules, including proteins that can not bind to nucleic acids in nature through SELEX, as well as proteins that do not bind to nucleic acids. Are being screened. The SELEX method is developed using nucleotides in which aptamer ligands are modified in consideration of stability and transportability, selectivity and affinity in vivo.

이와 같이 선별된 단일가닥 핵산리간드를 바탕으로 특정물질을 동정 및 분석하는 기술은 열량측정법(calorimetric method, Stojanovic MN, Landry DW.; J. AM. CHEM. SOC., 124, pp9678-9679, 2002), 전기화학형광법 및 효소법(Electrochemiluminescence and enzymatic method, Bruno JG, Kiel JL.; Biotechniques; 32(1), pp178-80, pp182-3, 2002), 바이오칩 및 신호증폭방법(미국 특허 US 6,544,776) 등이 개발되고 있으나 구체적으로 보도된 바는 없다. The technique for identifying and analyzing specific substances based on the single-stranded nucleic acid ligands thus selected is based on the calorimetric method (Strojanovic MN, Landry DW . ; J. AM. CHEM. SOC., 124, pp9678-9679, 2002). , Electrochemiluminescence and enzymatic methods, Bruno JG, Kiel JL .; Biotechniques ; 32 (1), pp178-80, pp182-3, 2002), biochips and signal amplification methods (US Pat. No. 6,544,776). It is being developed but has not been specifically reported.

특정물질의 동정 및 분석은 물리, 화학적인 성질을 이용하여 많이 수행되고 있으며 물질상호간의 특이성 및 친화력을 이용한 분석은 통상적으로 면역학적인 방 법을 바탕으로 개발된 방법으로 수행되고 있다. 면역학적인 방법은 항원-항체 반응에 따라 항체가 시료에 있는 특정한 항원과 결합하여 복합체를 형성하며 이 때 표지된 이차 항체를 사용하여 항원-항체 복합체를 분석하는 기법이다. 예를 들면, ELISA는 일차 항체를 고체매질에 고정하며 항원이 있는 시료와 반응시킨 후, 이 복합체를 인지하는 표지된 이차 항체를 처리하여 특정항원에 대한 분석하는 방법이다. 표지된 이차 항체가 고체매질에 고정된 일차항체-항원 복합체를 인지하여 결합하면 표지체가 고체매질에 결합하게 된다. ELISA에 사용하는 프로브는 형광지시약, 디옥시제닌(dioxygenin), 호오스래디쉬 퍼록시데이즈(horseradish peroxidase), 알칼라인 포스파타아제(alkaline phosphatase) 등으로 표지한다. 단백질 분석기술은 단백질과 단백질간의 상호작용에서 발생하는 매우 약한 신호를 감지할 수 있어야 한다. 현재까지 검출방법은 주로 형광법이 많이 쓰이고 있으며 전기화학적 검출 방법 등이 개발되고 있다. 상기와 같이 다양한 과정을 통해 특정 단백질의 구분 및 정량에 대한 탐색 및 분석하는 방법들이 개발되어 있지만 고가의 기기 및 시약을 사용하고 있으며 복잡한 과정을 수행해야 하는 등의 문제가 있다.The identification and analysis of specific substances are frequently performed using physical and chemical properties, and the analysis using specificity and affinity between materials is usually carried out by methods developed based on immunological methods. An immunological method is a technique in which an antibody binds to a specific antigen in a sample to form a complex according to an antigen-antibody reaction, and then the antigen-antibody complex is analyzed using a labeled secondary antibody. For example, ELISA is a method of analyzing a specific antigen by immobilizing a primary antibody in a solid medium and reacting with a sample containing an antigen, followed by processing a labeled secondary antibody that recognizes the complex. When the labeled secondary antibody recognizes and binds to the primary antibody-antigen complex immobilized on the solid medium, the label binds to the solid medium. Probes used in ELISA are labeled with a fluorescent indicator, dioxygenin, horseradish peroxidase, alkaline phosphatase and the like. Protein analysis technology must be able to detect very weak signals from proteins and their interactions. Until now, the detection method is mainly used fluorescence method and the electrochemical detection method has been developed. As described above, methods for searching and analyzing the identification and quantification of specific proteins through various processes have been developed, but there are problems such as using expensive instruments and reagents and performing complicated processes.

이에 본 발명자들은 분자비콘(molecular beacon)을 사용하여 반응을 수행한 후, 형광법으로 특정물질 동정, 정량적인 변화를 분석하여 분자비콘을 이용한 특정물질 분석 방법을 확인하여 본 발명을 완성하였다. Therefore, the present inventors completed the present invention by performing a reaction using a molecular beacon (molecular beacon), identifying a specific substance by fluorescence and analyzing the quantitative change to identify a specific substance analysis method using the molecular beacon.

본 발명의 목적은, 분자비콘 및 특정물질이 존재할 경우, 열역학적으로 안정한 복합체를 일차적으로 형성하는 성질을 이용하여 특정물질을 동정, 정량적인 변화를 확인함에 따라 이들 이용하여 미생물, 세포, 단백질을 포함한 다양한 생체분자에 대한 탐색 및 분석하는 시스템을 제공하고자 하는 것이다.
An object of the present invention is to identify microorganisms and to identify quantitative changes by using a property that primarily forms a thermodynamically stable complex when molecular beacons and specific substances are present, including microorganisms, cells, and proteins. It is an object of the present invention to provide a system for searching and analyzing various biomolecules.

상기 목적을 달성하기 위하여, 본 발명은 특정물질의 분석을 위한 단일가닥 핵산(nucleic acid, NA) 리간드(R-NA), 5' 말단부분에 형광물질(Fluorophore, F)이 결합된 단일가닥핵산(F-NA), 3'말단부분에 형광신호를 제어하는 물질(Quencher, Q)이 결합된 단일가닥핵산(Q-NA) 및 상기 R-NA와 F-NA 사이 또는 R-NA와 Q-NA 사이를 연결하는 단일가닥핵산(H-NA)으로 구성된 분자비콘(molecular beacon)을 제공한다.In order to achieve the above object, the present invention is a single-stranded nucleic acid (nucleic acid, NA) ligand (R-NA) for the analysis of a specific substance, a single-stranded nucleic acid (F) is coupled to the fluorescent material (Fluorophore, F) at the 5 'end (F-NA), single-stranded nucleic acid (Q-NA) combined with a substance (Quencher, Q) that controls the fluorescence signal at the 3 'end and between the R-NA and F-NA or between R-NA and Q- It provides a molecular beacon consisting of a single stranded nucleic acid (H-NA) connecting between NA.

또한, 본 발명은 (i) 단일가닥 핵산의 염기서열 및 길이를 설계하여 분자비콘을 제조하는 제 1단계 ; (ⅱ) 제 1단계에서 제조된 분자비콘과 특정물질이 포함된 시료와 반응하여 복합체를 형성하는 제 2단계; 및 (ⅲ) 제 2단계에서 형성된 복합체를 탐색하는 제 3단계를 포함하는 분자비콘을 이용한 특정물질의 동정 및 정량방법을 제공한다.In addition, the present invention (i) a first step of preparing a molecular beacon by designing the nucleotide sequence and length of the single-stranded nucleic acid; (Ii) a second step of forming a complex by reacting with the sample containing the molecular beacon prepared in the first step and the specific material; And (iii) provides a method for identifying and quantifying a specific substance using a molecular beacon comprising a third step of searching for the complex formed in the second step.

또한, 본 발명은 특정물질에 대한 특이적 염기서열을 갖는 단일가닥핵산 리간드을 포함하는 분자비콘 및 반응용액을 포함하는 특정물질의 동정 및 정량적 분석을 위한 분석키트를 제공한다.The present invention also provides an analysis kit for identification and quantitative analysis of a specific substance including a molecular beacon and a reaction solution comprising a single-stranded nucleic acid ligand having a specific base sequence for the specific substance.

이하, 본 발명을 더욱 상세히 설명한다.Hereinafter, the present invention will be described in more detail.

본 발명은 특정물질의 분석을 위한 단일가닥 핵산 리간드(R-NA), 5' 말단부분에 형광물질(Fluorophore, F)이 결합된 단일가닥핵산(F-NA), 3'말단부분에 형광신호를 제어하는 물질(Quencher, Q)이 결합된 단일가닥핵산(Q-NA) 및 상기 R-NA와 F-NA 사이 또는 R-NA와 Q-NA 사이를 연결하는 단일가닥핵산(H-NA)으로 구성된 분자비콘(molecular beacon)을 제공한다(도 1 참조).The present invention is a single-stranded nucleic acid ligand (R-NA) for analysis of a specific substance, a single-stranded nucleic acid (F-NA) in which a fluorescent substance (F-Fluorphore, F) is bound to the 5 'end, a fluorescent signal at the 3' end A single stranded nucleic acid (Q-NA) bound to a substance controlling (Quencher, Q) and a single stranded nucleic acid (H-NA) connecting between the R-NA and F-NA or between R-NA and Q-NA. It provides a molecular beacon consisting of (see Figure 1 ).

이때, F-NA 및 Q-NA는 상기 단일가닥 핵산 리간드와 상보적인 서열이고, H-NA는 5 내지 15개의 핵산서열이다. 또한, 상기 F-NA와 Q-NA는 1 내지 10 bp의 거리를 두고 단일가닥 핵산 리간드와 상보적인 결합을 이루는 것이 바람직하다. 본 발명의 F-NA와 Q-NA는 8 내지 12 bp의 염기서열을 갖는 단일가닥핵산인 것이 바람직하다.In this case, F-NA and Q-NA is a sequence complementary to the single-stranded nucleic acid ligand, H-NA is 5 to 15 nucleic acid sequences. In addition, the F-NA and Q-NA is preferably a complementary bond with the single-stranded nucleic acid ligand at a distance of 1 to 10 bp. F-NA and Q-NA of the present invention is preferably a single-stranded nucleic acid having a nucleotide sequence of 8 to 12 bp.

본 발명에 있어서, "분자비콘(Molecular beacon)"이라 함은 단일가닥 핵산 리간드와 상보적인 단일가닥 핵산으로 형광신호를 조절할 수 있는 시스템을 말한다. 분자비콘은 F-NA의 형광을 제어하는 시스템을 제공하는데, 단일가닥핵산 리간드의 염기서열에 상보적인 단일가닥 즉, 분자비콘에서 형광물질(Fluorophore, F)과 결합된 핵산가닥(F-NA) 과 형광신호를 제어하는 물질(Quencher, Q)과 결합된 핵산가닥(Q-NA)으로 구성되고 이들 도메인을 연결하는 두개의 연결단일가닥핵산(H-NA)으로 구성되어 있으며, 단일가닥 핵산리간드에 F-NA와 Q-NA가 결합하여 서로 인접하며 Q에 의해 F의 형광을 제어하게 되고 두 물질이 해리되어 F에 의해 형광이 발 생하는 시스템인 분자비콘이다(도 3 참조).In the present invention, the term "molecular beacon" refers to a system capable of controlling the fluorescence signal with a single-stranded nucleic acid complementary to the single-stranded nucleic acid ligand. Molecular beacons provide a system for controlling the fluorescence of F-NA, a single strand complementary to the nucleotide sequence of a single-stranded nucleic acid ligand, ie, a nucleic acid strand (F-NA) bound to a fluorophore (F) in a molecular beacon And a nucleic acid strand (Q-NA) coupled with a substance (Quencher, Q) that controls fluorescence signals, and two linked single-stranded nucleic acids (H-NA) linking these domains. F-NA and Q-NA are adjacent to each other to control the fluorescence of F by Q and the two materials are dissociated and the molecular beacon is a system that generates fluorescence by F (see Fig. 3 ).

상기 분자비콘은 특정물질과 결합하는 염기서열인 핵심부분을 포함하는 염기서열로 구성되는 단일가닥핵산리간드인 R-NA, 상기와 상보적인 단일가닥핵산인 F-NA 및 Q-NA, 그리고 이들을 연결하는 H-NA로 구성된 단일분자로 구성하는데, 상보적인 단일가닥핵산인 F-NA 및 Q-NA는 복합체를 형성하는 단일가닥핵산인 R-NA의 염기서열을 고려하여 설계한다. 혹은 상보적인 단일가닥핵산중 하나만 R-NA에 연결하고 나머지는 독립분자로 제작하여 분자비콘을 구성할 수도 있다. The molecular beacon is a single-stranded nucleic acid ligand R-NA consisting of a nucleotide sequence including a core portion that binds to a specific substance, F-NA and Q-NA, and complementary single-stranded nucleic acids, and linking them It consists of a single molecule consisting of H-NA, complementary single-stranded nucleic acids F-NA and Q-NA is designed in consideration of the base sequence of the single-stranded nucleic acid R-NA forming a complex. Alternatively, only one of the complementary single-stranded nucleic acids can be linked to R-NA, and the rest can be produced as independent molecules to form molecular beacons.

상기 단일가닥핵산 리간드의 염기서열은 SELEX에 의해 선별된 단일가닥핵산의 염기서열로, 특정물질을 인식하는 부위를 포함하며, 15 내지 100 bp의 염기서열을 갖는 단일가닥핵산이다. 예를들면, 본 발명의 바람직한 실시예에서는 단일가닥핵산 리간드는 기보고된 TNF-α와 특이적으로 결합하는 단일가닥핵산(미국특허 제5,972,599호; 서열번호 1)의 염기서열을 포함한다.The nucleotide sequence of the single-stranded nucleic acid ligand is a nucleotide sequence of the single-stranded nucleic acid selected by SELEX, including a site for recognizing a specific substance, and has a nucleotide sequence of 15 to 100 bp. For example, in a preferred embodiment of the present invention, the single-stranded nucleic acid ligand comprises a nucleotide sequence of a single-stranded nucleic acid (US Pat. No. 5,972,599; SEQ ID NO: 1 ) that specifically binds to previously reported TNF-α.

또한, 상기 F-NA와 Q-NA은 단일가닥핵산 리간드의 염기서열과 상보적인 가닥으로, F-NA와 Q-NA는 단일가닥핵산 리간드상에서 1 내지 10 염기정도 이격되어, 사이가 떨어져 결합할 수 있으며, 단일가닥핵산 리간드와 형성하는 구조체의 안정성을 고려한 부위로 8 내지 12 bp의 염기서열을 갖는 단일가닥핵산 혹은 5 내지 20 펩티드 핵산(peptide nucleic acid) 서열을 갖는 물질 등을 포함한다.In addition, the F-NA and Q-NA are strands complementary to the nucleotide sequence of the single-stranded nucleic acid ligand, F-NA and Q-NA are separated by about 1 to 10 bases on the single-stranded nucleic acid ligand, the bond will be separated And a single stranded nucleic acid having a nucleotide sequence of 8 to 12 bp or a substance having a 5 to 20 peptide nucleic acid sequence as a site considering stability of a structure to be formed with a single stranded nucleic acid ligand.

또한, 상기 반응부위는 F 및 Q이며, F-NA 및 Q-NA가 단일가닥핵산 리간드에 결합하여 F-NA의 F 형광을 Q-NA의 Q가 제어하는 분자비콘으로, Q 반응기는 3'- DABCYL(4-(4-dimethyl-aminophenylazo)benzoic acid) 또는 블랙 홀 소진제(Black Hole Quencher), F 반응기는 5'- 플로레신(Fluorescein), Cy3, Cy5, 텍사스 레드(Texas red) 등을 사용할 수 있다. 예를들면, 본 발명의 바람직한 F-NA는 단일가닥핵산 리간드에 상보적으로 결합하고 핵산의 5'에 Cy5가 결합된 단일가닥염기서열(서열번호 2), Q-NA는 단일가닥핵산 리간드에 상보적으로 결합하고 핵산의 3'에 DABCYL이 결합된 단일가닥염기서열(서열번호 3)이며 유기 합성할 수도 있다.In addition, the reaction site is F and Q, the F-NA and Q-NA is a molecular beacon to control the F-fluorescence of F-NA by Q-NA Q binding to the single-stranded nucleic acid ligand, the Q reactor is 3 ' -DABCYL (4- (4-dimethyl-aminophenylazo) benzoic acid) or Black Hole Quencher, F reactor uses 5'-Fluorescein, Cy3, Cy5, Texas red, etc. Can be used. For example, a preferred F-NA of the present invention binds to a single-stranded nucleic acid ligand complementary to a single-stranded base sequence ( SEQ ID NO: 2 ) wherein Cy5 is bound to 5 'of the nucleic acid, and Q-NA to a single-stranded nucleic acid ligand. It is a single-stranded base sequence ( SEQ ID NO: 3 ) that complementarily binds to and binds DABCYL to 3 ′ of the nucleic acid, and may be organically synthesized.

또한, 본 발명은 (i) 단일가닥 핵산의 염기서열 및 길이를 설계하여 분자비콘을 제조하는 제 1단계 ; (ⅱ) 제 1단계에서 제조된 분자비콘과 특정물질이 포함된 시료와 반응하여 복합체를 형성하는 제 2단계; 및 (ⅲ) 제 2단계에서 형성된 복합체의 형광성을 탐색하는 제 3단계를 포함하는 분자비콘을 이용한 특정물질의 동정 및 정량방법을 제공한다.In addition, the present invention (i) a first step of preparing a molecular beacon by designing the nucleotide sequence and length of the single-stranded nucleic acid; (Ii) a second step of forming a complex by reacting with the sample containing the molecular beacon prepared in the first step and the specific material; And (iii) a third step of searching for fluorescence of the complex formed in the second step.

본 발명의 제 1단계는 단일가닥 핵산의 염기서열 및 길이를 설계하여 분자비콘을 제조하는 단계이다. 이때, 단일가닥 핵산을 구성하는 단일가닥핵산 리간드 서열은 셀렉스(SELEX, Systematic Evolution of Ligand by Exponential enrichment)법에 의해 선별되는 것이 바람직하다. 예를 들면, 본 발명의 바람직한 실시예에서는 기보고된 TNF-α와 특이적으로 결합하는 단일가닥 핵산리간드(서열번호 1)의 염기서열을 포함한다. The first step of the present invention is to prepare a molecular beacon by designing the nucleotide sequence and length of the single-stranded nucleic acid. In this case, the single-stranded nucleic acid ligand sequence constituting the single-stranded nucleic acid is preferably selected by the SELEX (Systematic Evolution of Ligand by Exponential enrichment) method. For example, a preferred embodiment of the present invention includes the nucleotide sequence of a single stranded nucleic acid ligand (SEQ ID NO: 1) that specifically binds to previously reported TNF-α.

본 발명의 제 2단계는 상기 제 1단계에서 제조된 분자비콘과 특정물질이 포함된 시료와 반응하여 복합체를 형성하는 단계이다. 이때, 특정물질은 이에 한정하 는 것은 아니나, 세균, 진균류, 바이러스, 세포주, 조직, 이로부터 분리된 단백질, 탄수화물, 지질, 다당체, 당단백, 호르몬, 수용체, 항원, 항체 및 효소로 이루어진 군에서 선택된 하나 이상인 것이 바람직하다. 상기 세포주 및 조직은 원핵생물 또는 진핵생물에서 유래한 것을 포함하며, 진핵생물은 인간, 동물, 식물을 의미한다. The second step of the present invention is a step of forming a complex by reacting with the sample containing the molecular beacon and the specific material prepared in the first step. At this time, the specific substance is not limited thereto, but is selected from the group consisting of bacteria, fungi, viruses, cell lines, tissues, proteins, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies and enzymes isolated therefrom. It is preferred that there is at least one. The cell lines and tissues include those derived from prokaryotes or eukaryotes, and eukaryotes refer to humans, animals and plants.

상기 제 2단계의 반응을 위한 온도는 SELEX 수행온도보다 낮은 온도에서 수행하며, 15 ~ 40℃인 것이 바람직하다. 제 1단계에서 제조된 분자비콘의 온도에 대한 반응성은 22℃에서는 F-NA의 형광물질을 Q-NA의 소진제(Quencher)가 제어하여 형광정도가 매우 낮으나, 37℃에서는 F-NA 또는 Q-NA가 서로 해리되어 형광정도가 상대적으로 증가한다(도 4 참조). 따라서, 본 발명의 분자비콘은 특정물질과 복합체를 형성하는 성질, 주어진 환경에서 분자비콘 그리고 단일가닥핵산-특정물질의 복합체 간에 자유에너지의 차이, 즉, 분자비콘의 자유에너지가 단일가닥핵산-특정물질의 복합체보다 높게 설계, 제조하여 특정물질을 동정 및 정량적인 분석이 가능하다.The temperature for the reaction of the second step is carried out at a temperature lower than the SELEX temperature, preferably 15 ~ 40 ℃. Reactivity to the temperature of the molecular beacon prepared in the first step is very low fluorescence by controlling the fluorescent material of F-NA at Q-NA at 22 ℃, but F-NA or Q at 37 ℃ -NA dissociates with each other and the degree of fluorescence increases relatively (see FIG. 4 ). Therefore, the molecular beacon of the present invention is characterized in that the free energy of the molecular beacon, that is, the complex energy and the complex energy of the beacon and single-stranded nucleic acid-specific material in a given environment, that is, the free energy of the molecular beacon is single-stranded nucleic acid-specific It is possible to identify and quantitatively identify specific materials by designing and manufacturing higher than composites of materials.

또한 상기 제 2단계의 반응용액은 염의 조성, 비특이적 결합을 방지하는 요소 등으로 구성되며, 바람직하게는 50mM 트리스(Tris)·염산(pH 7.4), 5mM 염화칼륨, 100mM 염화나트륨, 1mM 염화마그네슘, 0.1% 소듐아지드를 함유한 선택버퍼와 0.2% 우혈청알부민 포함하는 선택버퍼를 사용한다.In addition, the reaction solution of the second step is composed of a salt composition, an element that prevents non-specific binding, etc., preferably 50mM Tris, hydrochloric acid (pH 7.4), 5mM potassium chloride, 100mM sodium chloride, 1mM magnesium chloride, 0.1% A selection buffer containing sodium azide and a selection buffer containing 0.2% bovine serum albumin are used.

본 발명의 제 3단계는 상기 제 2단계에서 형성된 복합체의 형광성을 탐색하는 단계이다. 이때, 탐색은 해리된 F-NA의 형광물질을 탐지하는 것이다. 즉, 분자비콘 및 단일가닥핵산-특정물질의 복합체는 주어진 환경에서 이들 간의 특이성 및 특정물질 양에 따라 형성되는 복합체의 종류 및 형성정도가 결정되며 이로부터 특정물질을 동정하여 정량할 수 있다. 즉, Q-NA에 의해 F-NA의 형광이 제어되는 분자비콘에 특정물질을 처리하면 단일가닥핵산리간드와 특정물질이 반응하여 단일가닥핵산-특정물질 복합체가 형성되면서 단일가닥핵산리간드에서 Q-NA와 F-NA가 해리되어 F-NA에 의해 형광이 발생한다. 즉, 단일가닥핵산리간드와 특정물질의 특이성 및 양에 의해 결정되는 형광정도를 분석하여 특정물질의 종류 및 정량적인 변화를 확인할 수 있게 된다. The third step of the present invention is a step of searching for fluorescence of the complex formed in the second step. At this time, the search is to detect the fluorescent material of the dissociated F-NA. In other words, the complex of the molecular beacon and the single-stranded nucleic acid-specific material is determined according to the specificity and specific substance amount between them in a given environment, and the degree of formation of the complex can be identified and quantified from the specific material. In other words, when a specific substance is treated to a molecular beacon in which fluorescence of F-NA is controlled by Q-NA, the single-stranded nucleic acid ligand reacts with a specific substance to form a single-stranded nucleic acid-specific substance complex, and NA and F-NA dissociate to cause fluorescence by F-NA. That is, by analyzing the degree of fluorescence determined by the specificity and amount of the single-stranded nucleic acid ligand and the specific substance, it is possible to confirm the type and quantitative change of the specific substance.

또한, 본 발명은 특정물질에 대한 특이적 염기서열을 갖는 단일가닥핵산 리간드을 포함하는 분자비콘 및 반응용액을 포함하는 특정물질의 동정 및 정량적 분석을 위한 분석키트를 제공한다. 이때, 분석키트는 단일가닥핵산 어레이의 형태로 제작이 가능하다.The present invention also provides an analysis kit for identification and quantitative analysis of a specific substance including a molecular beacon and a reaction solution comprising a single-stranded nucleic acid ligand having a specific base sequence for the specific substance. At this time, the analysis kit can be manufactured in the form of a single stranded nucleic acid array.

이하, 본 발명을 하기의 실시예에 의거하여 더욱 상세히 설명하나, 본 발명이 이에 의해 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

실시예 1. 단일가닥핵산 분자비콘의 제조 및 형광측정 Example 1. Preparation and Fluorescence Measurement of Single-stranded Nucleic Acid Molecular Beacons

특정물질의 동정 및 정량을 위한 단일가닥핵산 리간드(이하, R-NA라 함), 5'말단에 형광물질이 표지된 단일가닥핵산(이하, F-NA라 함), 3'말단에 소진제(Quencher)가 부착된 단일가닥핵산(이하, Q-NA라 함) 및 두가지 핵산을 연결 하는 단일가닥핵산(이하, H-NA라 함)로 구성된 단일가닥핵산 분자비콘을 제조하였다(도 1).Single-stranded nucleic acid ligand (hereinafter referred to as R-NA) for identification and quantification of specific substances, single-stranded nucleic acid (hereinafter referred to as F-NA) labeled with fluorescent substance at 5 'end, and exhausting agent at 3' end A single-stranded nucleic acid molecular beacon consisting of a single-stranded nucleic acid (hereinafter referred to as Q-NA) and a single-stranded nucleic acid (hereinafter referred to as H-NA) connecting two nucleic acids (Quencher) was prepared ( FIG. 1 ). .

1-1. 단일가닥핵산 분자비콘 (1)의 제조1-1. Preparation of single-stranded nucleic acid molecular beacon (1)

특정물질로서 TNF-α와 특이적으로 결합하는 서열번호 1로 기재되는 단일가닥핵산 리간드인 R-NA (미국특허등록 제5,972,599호; 5'-GUC GAA CUA GCG CUG GAG CGU GCG UUG GU-3'), 상기 단일가닥핵산 리간드에 상보적인 서열번호 2로 기재되는 단일가닥 핵산서열인 F-NA (5'- CCA GCG CTA G-3')과 서열번호 3으로 기재되는 단일가닥 핵산서열인 Q-NA (5'-CTT CGC ACG C-3') 및 상기 R-NA와 F-NA 및 R-NA와 Q-NA사이를 연결하는 연결 단일가닥 핵산서열인 U8의 H-NA로 구성된 단일가닥핵산서열을 설계하여 분자비콘 (1)을 제작하였다. 이때, F-NA의 5' 말단에는 Cy3, Q-NA의 3' 말단에는 DABCYL(4-(4-dimethyl-aminophenylazo)benzoic acid)을 각각 표지하여 분자비콘을 제작하였다.R-NA, a single-stranded nucleic acid ligand described in SEQ ID NO: 1 that specifically binds TNF-α as a specific substance (US Pat. No. 5,972,599; 5'-GUC GAA CUA GCG CUG GAG CGU GCG UUG GU-3 ' ), F-NA (5'- CCA GCG CTA G-3 '), a single-stranded nucleic acid sequence described in SEQ ID NO: 2 complementary to the single-stranded nucleic acid ligand, and Q-, a single-stranded nucleic acid sequence described in SEQ ID NO: 3 Single strand consisting of NA (5'-CTT CGC ACG C-3 ') and H-NA of U 8 which is a connecting single-stranded nucleic acid sequence connecting R-NA and F-NA and R-NA and Q-NA Molecular Beacon (1) was produced by designing nucleic acid sequences. In this case, molecular beacon was prepared by labeling Cy3 at the 5 'end of F-NA and DABCYL (4- (4-dimethyl-aminophenylazo) benzoic acid) at the 3' end of Q-NA, respectively.

1-2. 단일가닥핵산 분자비콘 (2)의 제조1-2. Preparation of single-stranded nucleic acid molecular beacon (2)

특정물질로서 TNF-α와 특이적으로 결합하는 서열번호 1로 기재되는 단일가닥핵산 리간드인 R-NA, 상기 단일가닥핵산 리간드에 상보적인 서열번호 2로 기재되는 단일가닥 핵산서열인 F-NA와 서열번호 3으로 기재되는 단일가닥 핵산서열인 Q-NA 및 상기 R-NA와 F-NA 사이를 연결하는 연결 단일가닥 핵산서열인 U8의 H-NA로 구성된 단일가닥핵산서열을 설계한 후, Q-NA를 독립분자로 설계하여 분자비콘 (2)를 제조하였다. 이때, F-NA의 5' 말단에는 Cy3, Q-NA의 3' 말단에는 DABCYL(4-(4- dimethyl-aminophenylazo)benzoic acid)을 각각 표지하여 분자비콘을 제작하였다.R-NA, which is a single-stranded nucleic acid ligand as described in SEQ ID NO: 1 that specifically binds to TNF-α, and F-NA, which is a single-stranded nucleic acid sequence as set forth in SEQ ID NO: 2 , which is complementary to the single-stranded nucleic acid ligand, After designing a single-stranded nucleic acid sequence consisting of Q-NA, a single-stranded nucleic acid sequence as set forth in SEQ ID NO: 3 , and H-NA of U 8 , a linkage single-stranded nucleic acid sequence connecting between the R-NA and F-NA, Molecular Beacon (2) was prepared by designing Q-NA as an independent molecule. At this time, the molecular beacon was prepared by labeling Cy3 at the 5 'end of F-NA and DABCYL (4- (4-dimethyl-aminophenylazo) benzoic acid) at the 3' end of Q-NA, respectively.

실시예 2. 단일가닥핵산 분자비콘의 서열길이의 영향 Example 2 Effect of Sequence Length of Single-stranded Nucleic Acid Molecular Beacons

분자비콘의 형광측정을 위해 길이조건 및 온도조건을 달리하여 형광치를 조사하였다.For fluorescence measurement of molecular beacons, fluorescence values were investigated by varying the length and temperature conditions.

구체적으로, 분자비콘을 구성하는 상보적인 단일가닥핵산(F-NA 또는 Q-NA)의 염기서열 길이가 염기 15개 이상인 경우, 9개 내지 11개인 경우 및 7개 이하인 경우의 분자비콘을 제조하여 온도에 대한 안정성을 살펴보았다(도 2).Specifically, when the base sequence length of the complementary single-stranded nucleic acid (F-NA or Q-NA) constituting the molecular beacon is 15 or more bases, the molecular beacon of 9 to 11 cases and 7 or less to prepare a The stability to temperature was examined ( FIG. 2 ).

그 결과, 염기서열의 길이가 15개 이상인 경우에는 22℃ 및 37℃ 모두에서 안정성이 유지되었고, 9개 내지 11개인 경우에는 22℃에서는 안정성이 유지되나 37 ℃ 경우는 구조체가 불안정하였으며, 7개 이하인 경우에는 22℃ 및 37℃ 모두에서 구조체가 불안정하였는 바, 상보적인 단일가닥핵산(F-NA 또는 Q-NA)의 염기서열 길이는 9개 내지 11개인 경우를 사용하는 것이 바람직함을 확인하였다.As a result, when the length of the base sequence is 15 or more, stability was maintained at both 22 ° C. and 37 ° C., and stability was maintained at 22 ° C. in the case of 9 to 11, but the structure was unstable at 37 ° C., and 7 In the case of the following, the structure was unstable at both 22 ℃ and 37 ℃, it was confirmed that the base sequence length of complementary single-stranded nucleic acid (F-NA or Q-NA) is preferably used 9 to 11 .

실시예 3. 분자비콘의 형광측정 Example 3 Fluorescence Measurement of Molecular Beacons

상기 실시예 1-1에서 제조된 100 nM의 분자비콘(1)을 버퍼에 용해시키고, 94℃에 5분, 22℃ 및 37℃에서 30분간 처리한 다음, 형광측정장치(Turner Biosystem 사, PicofluorTM, Handheld Dual Channel Fluorometer)로 형광을 측정하였다(도 4). 이때, 사용한 반응용액은 50mM의 트리스(Tris)·염산(pH 7.4), 5mM의 염화칼륨, 100mM의 염화나트륨, 1mM의 염화마그네슘, 0.1% 소듐아지드를 함유한 선택버퍼 및 0.2% 우혈청알부민을 포함하는 선택버퍼를 사용하였다.The 100 nM molecular beacon (1) prepared in Example 1-1 was dissolved in a buffer, treated at 94 ° C. for 5 minutes, 22 ° C. and 37 ° C. for 30 minutes, followed by a fluorescence measuring apparatus (Turner Biosystem, Picofluor Fluorescence was measured by TM , Handheld Dual Channel Fluorometer ( FIG. 4 ). The reaction solution used included 50 mM Tris and hydrochloric acid (pH 7.4), 5 mM potassium chloride, 100 mM sodium chloride, 1 mM magnesium chloride, 0.1% sodium azide, and 0.2% bovine serum albumin. A selection buffer was used.

또한, 상기 실시예 1-2에서 제조된 분자비콘(2)의 경우, 100 nM의 단일가닥핵산(F-NA - H-NA - R-NA) 및 300 nM의 Q-NA를 버퍼에 용해시키고, 94℃에 5분, 22℃ 및 37℃에서 30분간 처리한 다음, 형광측정장치(Turner Biosystem 사, PicofluorTM, Handheld Dual Channel Fluorometer)로 형광을 측정하였다. 이때, 사용한 반응용액은 50mM의 트리스(Tris)·염산(pH 7.4), 5mM의 염화칼륨, 100mM의 염화나트륨, 1mM의 염화마그네슘, 0.1% 소듐아지드를 함유한 선택버퍼 및 0.2% 우혈청알부민을 포함하는 선택버퍼를 사용하였다.In addition, in the molecular beacon (2) prepared in Example 1-2, 100 nM single-stranded nucleic acid (F-NA-H-NA-R-NA) and 300 nM Q-NA was dissolved in a buffer , 5 minutes at 94 ℃, 22 ℃ and 37 ℃ 30 minutes, and then measured the fluorescence with a fluorescence measuring device (Turner Biosystem, Picofluor TM , Handheld Dual Channel Fluorometer). The reaction solution used included 50 mM Tris and hydrochloric acid (pH 7.4), 5 mM potassium chloride, 100 mM sodium chloride, 1 mM magnesium chloride, 0.1% sodium azide, and 0.2% bovine serum albumin. A selection buffer was used.

그 결과, 대조군인 40 nM F-NA의 형광치는 1020이었다. 한편, 분자비콘(1)의 경우 22℃에서는 40 형광치이고, 37℃에서는 120 형광치를 나타내었으며, 분자비콘(2)의 경우 또한 유사한 결과를 얻었다. 22℃에서는 F-NA의 5'말단에 표지된 형광을 인접한 Q-NA의 3'말단에 표지된 소진제(Quencher)가 소멸시키기 때문이며, 분자비콘의 안정성이 낮아지는 37℃에서는 R-NA에 상보적으로 결합되어 있던 F-NA 또는 Q-NA가 해리되어 이로인해 F-NA의 5' 말단에 표지된 형광이 증가되기 때문이다.As a result, the fluorescence value of 40 nM F-NA as a control was 1020. On the other hand, the molecular beacon (1) showed 40 fluorescence at 22 ° C, 120 fluorescence at 37 ° C, and similar results were obtained for the molecular beacon (2). This is because the fluorescence labeled at the 5 'end of the F-NA is extinguished at 22 ° C and the quencher labeled at the 3' end of the adjacent Q-NA disappears. At 37 ° C, the stability of the molecular beacon is lowered. This is because the complementary bound F-NA or Q-NA is dissociated, thereby increasing the labeled fluorescence at the 5 'end of the F-NA.

실시예 4. 단일가닥핵산 분자비콘을 이용한 특정물질의 분석Example 4 Analysis of Specific Substances Using Single-stranded Nucleic Acid Molecular Beacons

본 발명에 의해 제조된 분자비콘을 이용하여 특정물질의 동정 및 정량분석을 실시하였다.Molecular beacons prepared by the present invention were used to identify and quantify specific substances.

구체적으로, 상기 실시예 1-1에서 제조된 80 nM의 분자비콘(1) 및 1μM의 TNF-α (Genzyme Inc., Cambridge Mass.)시료를 동시에 37℃에서 30분간 및 22℃에서 10분간 반응시킨 후, 형광측정장치로 형광을 측정하였다. 이때, 사용된 반응용액은 50mM의 트리스(Tris)·염산(pH 7.4), 5mM의 염화칼륨, 100mM의 염화나트륨, 1mM의 염화마그네슘, 0.1% 소듐아지드를 함유한 선택버퍼 및 0.2% 우혈청알부민을 포함하는 선택버퍼를 사용하였다. 상기 실험은 3번 반복 수행하였으며, 시료 처리 전후의 형광치 변화를 관찰하였다(도 5). Specifically, the 80 nM molecular beacon (1) prepared in Example 1-1 and 1 μM of TNF-α (Genzyme Inc., Cambridge Mass.) Sample at the same time for 30 minutes at 37 ℃ and 10 minutes at 22 ℃ After fluorescence was measured using a fluorescence measuring device. At this time, the reaction solution used was 50 mM Tris, hydrochloric acid (pH 7.4), 5 mM potassium chloride, 100 mM sodium chloride, 1 mM magnesium chloride, 0.1% sodium azide, and a selection buffer containing 0.2% bovine serum albumin. An optional selection buffer was used. The experiment was repeated three times, and the change in fluorescence value before and after sample treatment was observed ( FIG. 5 ).

그 결과, 첫 번째는 910, 두 번째는 900, 세 번째는 930 형광치를 보였다. 즉, 시료의 TNF-α가 분자비콘과 결합하여 복합체를 형성하였으며, 분자비콘-TNF-α 복합체에 의해 형광을 발생하는 양이 증가하여 형광치가 상대적으로 증가되었음을 확인하였다. 따라서, 특정시료의 확인 및 정량적인 분석이 가능함을 확인하였다.As a result, the first showed 910, the second 900, and the third 930 fluorescence. That is, the TNF-α of the sample was combined with the molecular beacon to form a complex, and the amount of fluorescence generated by the molecular beacon-TNF-α complex was confirmed to be relatively increased. Therefore, it was confirmed that the identification and quantitative analysis of specific samples were possible.

상기에서 언급한 바와 같이, 본 발명에 의해 제조된 분자비콘 및 상기 분자 비콘을 이용한 특정물질의 동정 및 정량분석은 미생물, 세포, 단백질을 포함하는 생체분자 등에 대한 탐색 및 분석 등에 사용할 수 있어 궁극적으로는 의학, 수의학, 환경공학, 식품공학, 농업 등에 광범위하게 응용될 수 있다.As mentioned above, the identification and quantitative analysis of the molecular beacon prepared by the present invention and a specific substance using the molecular beacon can be used for searching and analyzing biomolecules including microorganisms, cells, and proteins, and the like. It can be widely applied in medicine, veterinary medicine, environmental engineering, food engineering, agriculture and so on.

<110> GenoProt Inc. KIM, Sung Chun <120> Method for identification and analysis of specific molecules using molecular beacons <130> DIF/2004-03-0021/CHC <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> single strand nucleic acid ligand for TNF alpha <400> 1 gucgaacuag cgcuggagcg ugcguuggu 29 <210> 2 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Fluorescence-tagged complementary sequence of single strand nucleic acid ligand for TNF alpha <400> 2 ccagcgctag 10 <210> 3 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Quencher-tagged complementary sequence of single strand nucleic acid ligand for TNF alpha <400> 3 cttcgcacgc 10 <110> GenoProt Inc.          KIM, Sung Chun <120> Method for identification and analysis of specific molecules          using molecular beacons <130> DIF / 2004-03-0021 / CHC <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> single strand nucleic acid ligand for TNF alpha <400> 1 gucgaacuag cgcuggagcg ugcguuggu 29 <210> 2 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Fluorescence-tagged complementary sequence of single strand          nucleic acid ligand for TNF alpha <400> 2 ccagcgctag 10 <210> 3 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Quencher-tagged complementary sequence of single strand nucleic acid          acid ligand for TNF alpha <400> 3 cttcgcacgc 10

Claims (10)

특정물질의 분석을 위한 단일가닥 핵산 리간드(R-NA), 5' 말단부분에 형광물질(Fluorophore, F)이 결합된 단일가닥핵산(F-NA), 3'말단부분에 형광신호를 제어하는 물질(Quencher, Q)이 결합된 단일가닥핵산(Q-NA) 및 상기 R-NA와 F-NA 사이 또는 R-NA와 Q-NA 사이를 연결하는 단일가닥핵산(H-NA)으로 구성되며, F-NA 및 Q-NA는 R-NA와 상보적인 서열로서 R-NA와 1 내지 10 bp의 거리를 두고 상보적인 결합을 이루며, H-NA는 5 내지 15개의 핵산서열이며, F-NA와 Q-NA는 8 내지 12 bp의 염기서열을 갖는 단일가닥핵산인것을 특징으로 하는 분자비콘(molecular beacon).Single-stranded nucleic acid ligand (R-NA) for analysis of specific substances, single-stranded nucleic acid (F-NA) in which fluorescent material (F) is bound to 5 'end, and fluorescent signal at 3' end It consists of a single stranded nucleic acid (Q-NA) to which a substance (Quencher, Q) is bound and a single stranded nucleic acid (H-NA) connecting between the R-NA and F-NA or between R-NA and Q-NA. , F-NA and Q-NA are complementary to the R-NA and form a complementary bond at a distance of 1 to 10 bp with the R-NA, and the H-NA is 5 to 15 nucleic acid sequences, and the F-NA And Q-NA is a molecular beacon (molecular beacon) characterized in that the single-stranded nucleic acid having a base sequence of 8 to 12 bp. 삭제delete 삭제delete 삭제delete 셀렉스(SELEX, Systematic Evolution of Ligand by Exponential enrichment)법에 의해 선정된 단일가닥핵산리간드에 기초하여 (i) 단일가닥 핵산의 염기서열 및 길이를 설계하여 제 1항의 분자비콘을 제조하는 제 1단계 ; (ⅱ) 제 1단계에서 제작된 분자비콘과 특정물질이 포함된 시료가 15~40℃에서 반응하여 복합체를 형성하는 제 2단계; 및 (ⅲ) 해리된 F-NA의 형광물질을 탐지하여 제 2단계에서 형성된 복합체를 탐색하는 제 3단계를 포함하는 분자비콘을 이용한 특정물질의 동정 및 정량방법.Based on a single-stranded nucleic acid ligand selected by the SELEX (Systematic Evolution of Ligand by Exponential enrichment) method (i) a first step of preparing the molecular beacon of claim 1 by designing the nucleotide sequence and length of the single-stranded nucleic acid; (Ii) a second step of forming a complex by reacting the sample containing the molecular beacon prepared in the first step and the specific material at 15 ~ 40 ℃; And (iii) detecting a fluorescent substance of the dissociated F-NA and searching for the complex formed in the second step. 삭제delete 제 5 항에 있어서, 상기 제 2단계의 특정물질은 세균, 진균류, 바이러스, 세포주, 조직, 이로부터 분리된 단백질, 탄수화물, 지질, 다당체, 당단백, 호르몬, 수용체, 항원, 항체 및 효소로 이루어진 군에서 선택된 하나 이상인 것인 분석방법.The method of claim 5, wherein the specific substance of the second step is a group consisting of bacteria, fungi, viruses, cell lines, tissues, proteins, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies and enzymes isolated therefrom. At least one selected from the assay methods. 삭제delete 삭제delete 삭제delete
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KR100923048B1 (en) * 2007-06-11 2009-10-22 김성천 Nucleic Acid Chip for Obtaining Bind Profile of Single Strand Nucleic Acid and Unknown Biomolecule, Manufacturing Method Thereof, and Analysis Method of Unknown Biomolecule Using Nucleic Acid Chip

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WO2012070863A3 (en) * 2010-11-23 2012-10-11 한국과학기술원 Method for detecting nucleic acids using a nucleic acid enzyme/molecular beacon
KR101249493B1 (en) * 2010-11-23 2013-04-01 한국과학기술원 Method for Detecting Nucleic Acid Using DNAzyme-Molecular Beacon

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