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WO2018117333A1 - Bioprobe set capable of self-amplification of fluorescence signal for detecting microrna and use thereof - Google Patents

Bioprobe set capable of self-amplification of fluorescence signal for detecting microrna and use thereof Download PDF

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WO2018117333A1
WO2018117333A1 PCT/KR2017/002103 KR2017002103W WO2018117333A1 WO 2018117333 A1 WO2018117333 A1 WO 2018117333A1 KR 2017002103 W KR2017002103 W KR 2017002103W WO 2018117333 A1 WO2018117333 A1 WO 2018117333A1
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probe
bioprobe
bio
alexa
micro rna
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French (fr)
Korean (ko)
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임은경
정주연
국경혜
황슬기
임재우
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
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    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/10—Detection mode being characterised by the assay principle
    • C12Q2565/101—Interaction between at least two labels

Definitions

  • the present invention relates to a bioprobe set for micro RNA detection capable of amplifying autofluorescence signals and a use thereof, and more particularly, a first probe for detecting a microRNA including a fluorescent substance and a quencher, and at least three of the first probes.
  • a second probe comprising a base sequence complementarily binding to 20 to 20 sequences, a third probe comprising a base sequence complementarily binding to at least 3 to 15 sequences of the second probe, and a third probe;
  • a bioprobe set for microRNA detection capable of amplifying autofluorescence signal comprising a fourth probe including 6 to 30 base sequences complementarily binding to the first probe, a gene chip including the bioprobe set, and the bio Annealing the kit for detecting a microRNA comprising a probe set, and a bioprobe set according to the present invention; Contacting and hybridizing the annealed bioprobe set with a biological sample obtained from a subject; And detecting a fluorescence signal generated from the hybridization.
  • nucleic acid-based detection technology is based on molecular beacon (MB), a U-shaped DNA probe labeled with fluorescent and quencher.
  • the molecular beacon is a probe in the form of a stem structure for forming a loop and a hairpin structure of a base sequence complementary to a target nucleotide.
  • the technique using the molecular beacon is made by confirming the presence or absence of fluorescence signal generation due to the structural change of the molecular beacon due to the presence of the target nucleic acid. Since the target nucleic acid can be rapidly analyzed without separating the nucleic acid, various forms of It has been applied to the development of molecular beacon based nucleic acid analysis technology.
  • the molecular beacon-based analysis technology since the molecular beacon-based analysis technology generates a fluorescent signal by reacting the target nucleic acid and the molecular beacon in a 1: 1 ratio, it is impossible to detect a low amount of miRNA in the cell, or the sensitivity is high, resulting in high sensitivity. It has the disadvantage of being difficult to do. Therefore, there is a demand for a detection method capable of detecting miRNA with high sensitivity as a biomarker important for disease diagnosis.
  • Still another object of the present invention is to provide a kit for detecting miRNA comprising the bioprobe set and hybridization solution.
  • the present invention provides a first probe for detecting miRNA comprising a fluorescent material and a quencher, a base sequence complementarily binding to at least 3 to 20 sequences of the first probe
  • a second probe comprising, a third probe comprising a base sequence complementarily binding to at least 3 to 15 sequences of the second probe, and 6 to 30 complementarily binding to the third probe and the first probe
  • a bioprobe set for detecting miRNA capable of autofluorescence signal amplification including a fourth probe including a nucleotide sequence.
  • fluorescence molecule refers to a substance that absorbs and emits light of a specific wavelength and emits fluorescence, and may be labeled on a probe to confirm whether hybridization between the target nucleic acid and the probe has been performed and in vitro ( A substance capable of providing a miRNA detection image signal in vitro and / or in vivo .
  • the target nucleic acid may be miRNA.
  • quencher molecule refers to a material that absorbs light generated by a fluorescent material and reduces fluorescence intensity.
  • the target miRNA refers to miRNAs involved in various diseases and cancer regulation.
  • the target miRNA may be a miRNA associated with a disease selected from the group consisting of gastric cancer, breast cancer, brain cancer and viral infections.
  • the region capable of hybridizing with the target miRNA refers to a sequence capable of hybridizing with the target miRNA by having a sequence complementary to a portion of the target miRNA.
  • the term 'complementary' means that the probe is complementary enough to selectively hybridize to a target miRNA sequence under certain annealing or stringent conditions.
  • the region capable of hybridizing with the target miRNA may be one having at least 90%, preferably at least 95% complementary sequence with a portion of the target miRNA.
  • the region that can hybridize with the target miRNA may be appropriately adjusted depending on the target miRNA and is not limited to a specific length.
  • Nucleic acid molecules such as DNA and RNA have specific properties of binding to each other depending on the nucleotide sequences constituting the molecule, and the reaction can be controlled by appropriately designing the nucleotide sequences.
  • the fluorescent material bound to one end of the first probe is not particularly limited as long as it is a material capable of emitting fluorescence, and any fluorescent material commonly known in the art may be used without limitation.
  • the fluorescent material is, for example, Cy3, Cy5, Cy5.5, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, Alexa 680, Rhodamine, TAMRA, FAM, FITC, TRITC , Fluor X, DAB, ROX, Texas Red, Orange green 488X, Orange green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodify 630/650, Bodify 650/665, CAL Fluor orange 546, CAL Fluor red 610 , Quasar 670, biotin, and the like can be used, but is not limited thereto.
  • the fluorescent material in the present invention may be FAM, Cy3 or Cy5.
  • the matting material bonded to the other end of the first probe is also a material capable of controlling fluorescence
  • a specific kind is not particularly limited, and any kind of matting material commonly known in the art may be used without limitation.
  • the matting material may include, for example, BHQ (Biosearch Technologies, Inc.) such as DABCYL (DAB), DABSYL, BHQ1, BHQ2, and BHQ3, but is not limited thereto.
  • the matting material in the present invention may be DAB or BHQ2.
  • the bioprobe according to the present invention takes a manner in which on-off of the fluorescence signal is controlled according to the presence of the target miRNA in the cell.
  • the first probe When the target miRNA is present in the sample when the sample is contacted with the bioprobe according to the present invention including the first probe, the second probe, the third probe, and the fourth probe, the first probe has a sequence complementary to a portion of the target miRNA.
  • One probe hybridizes with the target miRNA to produce a fluorescence signal.
  • a second probe including a base sequence complementarily binding to at least 3 to 20 sequences of the first probe by a hybrid hybridization reaction between probe molecules is hybridized to the first probe, and then, The first probe and the second probe are separated while the third probe including the base sequence complementarily binding to at least 3 to 15 sequences hybridizes to the second probe, and complementarily binds to the third probe and the first probe.
  • a fourth probe including 6 to 30 nucleotide sequences may be amplified by a fluorescence signal through a self-cycling process in which the fourth probe binds to the third probe and the first probe again (FIG. 1).
  • the bioprobe set including the first probe, the second probe, the third probe, and the fourth probe is annealed under predetermined conditions before the target miRNA treatment to maintain the hairpin structure. Thereafter, the biomimetic set comprising the annealed first probe, the second probe, the third probe and the fourth probe is treated with a target miRNA to hybridize under a predetermined condition to generate a fluorescent signal.
  • the gene chip for detecting miRNA of the present invention may be configured in a form in which a set of bio probes for detecting miRNA is fixed at a high density on a substrate. Such configurations are well known in the art.
  • the substrate can be any substrate to which the oligonucleotide probe can be coupled under conditions that retain hybridization properties and keep the background level of hybridization low.
  • the substrate may be a microtiter plate, membrane (eg nylon or nitrocellulose) or microspheres (beads) or chips.
  • the hybridization solution may use a solution known in the art as a buffer that allows the probe and the nucleic acid sample to hybridize.
  • the kit of the present invention may further comprise a user manual describing the conditions for performing the optimal reaction.
  • the annealing step anneals the bioprobe set according to the present invention to inhibit the fluorescence of the probe, thereby allowing the fluorescence signal to recover and to measure the fluorescence intensity when hybridized with the target miRNA.
  • the annealing conditions are preferably heated at 95 ° C. for 5 minutes and slowly cooled to room temperature for at least 12 hours.
  • the hybridization is preferably performed at room temperature or 37 ° C., and the higher the temperature, the more open the hairpin structure of the probe, and thus, hybridization between probes may occur even when no miRNA is present.
  • biological sample refers to any sample that contains a target miRNA.
  • the biological sample can be any tissue or body fluid obtained from a subject with a disease of interest.
  • FIG. 1 is a view schematically illustrating a process of amplifying an autofluorescence signal by a bioprobe set according to the present invention.
  • FIG. 2 is a graph showing the annealing effect of the bioprobe set according to the present invention.
  • 3 is a graph showing the results of evaluating miRNA (0.1X) detectability of the bioprobe set according to the present invention.
  • FIG. 4 is a graph showing the results of evaluating miRNA (0.2X) detectability of the bioprobe set according to the present invention.
  • 5 is a graph showing the results of evaluating miRNA (0.4X) detectability of the bioprobe set according to the present invention.
  • FIG. 6 is a graph showing the results of evaluating miRNA (0.6X) detectability of the bioprobe set according to the present invention.
  • FIG. 7 is a graph showing the results of evaluating miRNA (0.8X) detectability of the bioprobe set according to the present invention.
  • FIG. 8 is a graph showing the results of evaluating miRNA (1X) detectability of the bioprobe set according to the present invention.
  • FIG. 9 is a graph showing the result of comparing the fluorescence signal intensity before and after the miRNA treatment of the bioprobe set according to the present invention.
  • FIG. 10 is a diagram showing the result of detecting a target miRNA-34a using a bioprobe set according to the present invention in MDA-MB-231 cells with low expression of miRNA-34a.
  • Bio probes can be designed by targeting various miRNAs, and in this example, miRNA-34a related to human breast cancer was used as a target miRNA to be detected.
  • the base sequence (SEQ ID NO: 1) of the miRNA-34a is as follows, and in the present invention, a DNA base sequence substituted (SEQ ID NO: 2) was used to facilitate the synthesis of the oligomer under extracellular conditions.
  • a bioprobe set capable of complementarily binding to the miRNA was designed.
  • the prepared bioprobes were referred to as oligo 1, 2, 3, and 4, respectively.
  • Table 2 below shows the sequence numbers (SEQ ID NOS: 3 to 6) and nucleotide sequences of the bioprobe set according to the present invention.
  • the bioprobe set of the present invention consists of a base sequence each comprising a base sequence complementary to the target miRNA sequence.
  • the oligo 1 attached Cy5 as a fluorescent material at the 5'-end and BHQ2 as a quencher at the 3'-end.
  • the oligo 1 was prepared by concentration (12.5, 25, 50, 100 pmol, respectively) and heated at 95 ° C. for 5 minutes in a heater (heat block), and then the heater was turned off and then slowly turned to room temperature (RT) for at least 12 hours. Annealed while cooling. Then, the annealing was confirmed by measuring the fluorescence intensity before / after annealing of the oligo 1.
  • the fluorescence intensity was measured after reacting the target miRNA or the control (5'-T GGCAGAATCGGAGCTGGTCCT 3 ', SEQ ID NO: 7) for 2 hours at 37 °C for each concentration such as oligo 1. Fluorescence was measured for fluorescence intensity at excitation wavelength 620 nm and emission wavelength 668 nm. The results are shown in FIG.
  • oligo 1 specifically binds to the target miRNA was confirmed that the fluorescent signal is increased as the fluorescent material is far from the quencher.
  • Example 1 Each oligo 1, 2, 3 and 4 prepared in Example 1 was heated for 5 minutes at 95 °C based on 25 pmol (1X) and then anneal while slowly cooling to room temperature (RT) for at least 12 hours, and targeted by concentration The fluorescence signal was measured every 10 minutes at 37 ° C. by processing the miRNA sequence (0.1 ⁇ -1 ⁇ ). The results are shown in FIGS. 3 to 8 and 9.
  • the bio probe set according to the present invention can specifically detect the target miRNA.
  • the high fluorescence signal when oligo 1, oligo 1, 2, 3 and 4 are present. This is a result confirming that the bioprobe set according to the present invention can amplify the autofluorescence signal.
  • MDA-MB-231 cells were seeded at 10 5 cells / well. The next day, after heating for 5 minutes at 95 °C and slowly cooled to room temperature (RT) for more than 12 hours, annealed oligo 1 (80 ng) and 1.5 ⁇ L lipofectamin 2000 reagent in 100 ⁇ L Opti-MEM medium and mixed The reaction was carried out by incubating at room temperature for 5 minutes (condition 1). Oligos 1, 2, 3, and 4 (80 ng each) and 1.5 ⁇ L Lipofectamine 2000 reagent annealed under the same conditions as above were reacted in the same manner (condition 2). After 5 minutes, the prepared medium was removed, and the above prepared conditions (conditions 1 and 2) were treated, and then cultured at 37 ° C. for 4, 8, 16, and 24 hours. As a control, cells cultured in 200 ⁇ L Opti-MEM medium were used.

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Abstract

The present invention relates to a bioprobe set capable of self-amplification of fluorescence signals for detecting microRNA and the use thereof and, more particularly, to a bioprobe set capable of self-amplifying fluorescence signals for detecting microRNA, the bioprobe set comprising a first probe, a second probe, a third probe, and a fourth probe, a gene chip comprising the bioprobe set, a microRNA detecting kit comprising the bioprobe set, and a method for detecting miRNA, the method comprising the steps of: annealing a bioprobe set according to the present invention; contacting and hybridizing a biosample from a subject with the annealed bioprobe set; and detecting a fluorescence signal generated by the hybridization.

Description

자가 형광 신호 증폭이 가능한 마이크로 RNA 검출용 바이오 프로브 세트 및 이의 용도Bioprobe set for micro RNA detection capable of amplifying autofluorescence signal and use thereof

본 발명은 자가 형광 신호 증폭이 가능한 마이크로 RNA 검출용 바이오 프로브 세트 및 이의 용도에 관한 것으로서, 더욱 상세하게는 형광물질 및 소광물질을 포함하는 마이크로 RNA 검출용 제1프로브, 상기 제1프로브의 적어도 3 내지 20개의 서열에 상보적으로 결합하는 염기서열을 포함하는 제2프로브, 상기 제2프로브의 적어도 3 내지 15개의 서열에 상보적으로 결합하는 염기서열을 포함하는 제3프로브, 및 제3프로브와 제1프로브에 상보적으로 결합하는 6 내지 30개의 염기서열을 포함하는 제4프로브를 포함하는 자가 형광 신호 증폭이 가능한 마이크로 RNA 검출용 바이오 프로브 세트, 상기 바이오 프로브 세트를 포함하는 유전자 칩, 상기 바이오 프로브 세트를 포함하는 마이크로 RNA 검출용 키트, 및 본 발명에 따른 바이오 프로브 세트를 어닐링하는 단계; 대상으로부터 수득한 생물학적 시료와 상기 어닐링된 바이오 프로브 세트를 접촉시켜 혼성화시키는 단계; 및 상기 혼성화로부터 발생되는 형광 신호를 검출하는 단계;를 포함하는 miRNA를 검출하는 방법에 관한 것이다.The present invention relates to a bioprobe set for micro RNA detection capable of amplifying autofluorescence signals and a use thereof, and more particularly, a first probe for detecting a microRNA including a fluorescent substance and a quencher, and at least three of the first probes. A second probe comprising a base sequence complementarily binding to 20 to 20 sequences, a third probe comprising a base sequence complementarily binding to at least 3 to 15 sequences of the second probe, and a third probe; A bioprobe set for microRNA detection capable of amplifying autofluorescence signal comprising a fourth probe including 6 to 30 base sequences complementarily binding to the first probe, a gene chip including the bioprobe set, and the bio Annealing the kit for detecting a microRNA comprising a probe set, and a bioprobe set according to the present invention; Contacting and hybridizing the annealed bioprobe set with a biological sample obtained from a subject; And detecting a fluorescence signal generated from the hybridization.

마이크로 RNA(microRNA 또는 miRNA, 이하 ‘miRNA’로 약칭함)는 약 19-25개의 뉴클레오티드로 이루어진 짧은 단일 가닥 비번역(non-coding) RNA로, 전사단계에서 RNA 침묵(RNA silencing) 그리고 전사 후 단계에서 유전자 발현의 조절 등 다양한 생체 내 프로세스에서 중요한 역할을 한다. 특히, miRNA는 다양한 질병 및 암 조절과 연관이 있는 것으로 알려지고 있어, 질병의 진단이나 예후용 바이오마커로서 활발히 연구되고 있다.MicroRNA (microRNA or miRNA, hereinafter abbreviated as 'miRNA') is a short single-strand non-coding RNA consisting of about 19-25 nucleotides. RNA silencing and post-transcriptional steps Plays an important role in a variety of in vivo processes, including regulation of gene expression. In particular, miRNA is known to be associated with various diseases and cancer regulation, and has been actively studied as a biomarker for diagnosis and prognosis of diseases.

기존의 핵산 기반 검출 기술은 형광 및 소광물질이 표지된 U자형의 DNA 프로브인 분자비콘(molecular beacon, MB)에 기반을 두고 있다. 분자비콘은 표적 뉴클레오티드와 상보적인 염기서열의 루프(loop)와 헤어핀(hairpin) 구조를 형성하기 위한 스템(stem) 구조로 구성된 형태의 프로브이다. 이 분자비콘을 이용한 기술은 표적 핵산의 존재에 의한 분자비콘의 구조 변화에 따른 형광 신호 생성의 유무를 확인함으로써 이루어지는데, 핵산의 분리과정 없이 표적 핵산을 신속하게 분석할 수 있기 때문에, 다양한 형태의 분자비콘 기반 핵산 분석 기술 개발에 적용되어 왔다.Conventional nucleic acid-based detection technology is based on molecular beacon (MB), a U-shaped DNA probe labeled with fluorescent and quencher. The molecular beacon is a probe in the form of a stem structure for forming a loop and a hairpin structure of a base sequence complementary to a target nucleotide. The technique using the molecular beacon is made by confirming the presence or absence of fluorescence signal generation due to the structural change of the molecular beacon due to the presence of the target nucleic acid. Since the target nucleic acid can be rapidly analyzed without separating the nucleic acid, various forms of It has been applied to the development of molecular beacon based nucleic acid analysis technology.

하지만, 이러한 분자비콘 기반의 분석 기술은 표적 핵산과 분자비콘이 1:1로 반응하여 형광신호를 발생시키므로, 세포내 저발현된 미량의 miRNA의 경우에는 검출이 불가능하거나 민감도가 떨어져 높은 민감도를 구현하기 힘들다는 단점을 가지고 있다. 따라서, 질병 진단에 중요한 바이오마커로서 miRNA를 고감도로 검출할 수 있는 검출방법이 요구되고 있다.However, since the molecular beacon-based analysis technology generates a fluorescent signal by reacting the target nucleic acid and the molecular beacon in a 1: 1 ratio, it is impossible to detect a low amount of miRNA in the cell, or the sensitivity is high, resulting in high sensitivity. It has the disadvantage of being difficult to do. Therefore, there is a demand for a detection method capable of detecting miRNA with high sensitivity as a biomarker important for disease diagnosis.

이에, 본 발명자들은 이와 같은 문제점을 해결하기 위하여 예의 연구 노력한 결과, 자가 순환 과정에 의하여 형광 신호의 증폭이 가능한 바이오 프로브 군을 개발하였고, 해당 바이오 프로브 군이 자가 순환 과정에 의하여 형광 신호를 증폭시킴으로써 세포내 미량으로 발현된 miRNA를 초고감도로 검출 가능함을 확인하고 본 발명을 완성하였다.Accordingly, the present inventors have diligently researched to solve such problems. As a result, the present inventors have developed a bioprobe group capable of amplifying a fluorescent signal by a self-circulating process. It was confirmed that the miRNA expressed in the intracellular trace amount can be detected with high sensitivity and completed the present invention.

본 발명의 목적은 자가 형광 신호 증폭이 가능한 miRNA 검출용 바이오 프로브 세트를 제공하는 것이다. It is an object of the present invention to provide a bioprobe set for detecting miRNA capable of amplifying autofluorescence signals.

본 발명의 다른 목적은 상기 바이오 프로브 세트를 포함하는 miRNA 검출용 유전자 칩을 제공하는 것이다.Another object of the present invention is to provide a gene chip for detecting miRNA comprising the bioprobe set.

본 발명의 또 다른 목적은 상기 바이오 프로브 세트 및 혼성화 용액을 포함하는 miRNA 검출용 키트를 제공하는 것이다.Still another object of the present invention is to provide a kit for detecting miRNA comprising the bioprobe set and hybridization solution.

본 발명의 또 다른 목적은 본 발명에 따른 바이오 프로브 세트를 어닐링하는 단계; 대상으로부터 수득한 생물학적 시료와 상기 어닐링된 바이오 프로브 세트를 접촉시켜 혼성화시키는 단계; 및 상기 혼성화로부터 발생되는 형광 신호를 검출하는 단계;를 포함하는 miRNA를 검출하는 방법을 제공하는 것이다.Another object of the present invention is the step of annealing the bioprobe set according to the present invention; Contacting and hybridizing the annealed bioprobe set with a biological sample obtained from a subject; And it provides a method for detecting miRNA comprising a; detecting a fluorescence signal generated from the hybridization.

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

상기 목적을 달성하기 위한 하나의 실시양태로서, 본 발명은 형광물질 및 소광물질을 포함하는 miRNA 검출용 제1프로브, 상기 제1프로브의 적어도 3 내지 20개의 서열에 상보적으로 결합하는 염기서열을 포함하는 제2프로브, 상기 제2프로브의 적어도 3 내지 15개의 서열에 상보적으로 결합하는 염기서열을 포함하는 제3프로브, 및 제3프로브와 제1프로브에 상보적으로 결합하는 6 내지 30개의 염기서열을 포함하는 제4프로브를 포함하는, 자가 형광 신호 증폭이 가능한 miRNA 검출용 바이오 프로브 세트를 제공한다.As one embodiment for achieving the above object, the present invention provides a first probe for detecting miRNA comprising a fluorescent material and a quencher, a base sequence complementarily binding to at least 3 to 20 sequences of the first probe A second probe comprising, a third probe comprising a base sequence complementarily binding to at least 3 to 15 sequences of the second probe, and 6 to 30 complementarily binding to the third probe and the first probe; Provided is a bioprobe set for detecting miRNA capable of autofluorescence signal amplification, including a fourth probe including a nucleotide sequence.

본 발명에서 사용되는 용어 “바이오 프로브(bioprobe)”는 상보적인 단일가닥 표적 서열과 혼성화하여 이중가닥 분자(혼성체)를 형성하는 단일 가닥 핵산 서열을 말한다. 상기 바이오 프로브로서 이용되는 올리고뉴클레오티드는 뉴클레오티드 유사체(analogue), 예를 들면, 포스포로티오에이트(phosphorothioate), 알킬포스포로티오에이트(alkylphosphorothioate) 또는 펩티드 핵산(peptide nucleic acid)을 포함할 수 있으나, 이에 제한되지는 않는다.The term "bioprobe" as used herein refers to a single stranded nucleic acid sequence that hybridizes with a complementary single stranded target sequence to form a double stranded molecule (hybrid). Oligonucleotides used as the bio probe may include nucleotide analogues such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, but It is not limited.

본 발명에서 사용되는 용어 “혼성화(hybridization)”는 핵산의 2개의 상보적 가닥이 조합하여 이중가닥 분자, 이른바 혼성체를 형성하는 과정을 의미한다.As used herein, the term “hybridization” refers to a process in which two complementary strands of nucleic acid combine to form a double stranded molecule, a so-called hybrid.

본 발명에서 사용되는 용어 “형광물질(fluorescence molecule)”은 특정 파장의 빛을 흡수, 방출하여 형광을 발하는 물질로써, 프로브에 표지하여 표적 핵산과 프로브 사이의 혼성화가 이루어졌는지 확인할 수 있고 시험관내(in vitro) 및/또는 생체내(in vivo)에서 miRNA 검출 영상 신호를 제공할 수 있는 물질을 의미한다. 본 발명에서 상기 표적 핵산은 miRNA일 수 있다. As used herein, the term “fluorescence molecule” refers to a substance that absorbs and emits light of a specific wavelength and emits fluorescence, and may be labeled on a probe to confirm whether hybridization between the target nucleic acid and the probe has been performed and in vitro ( A substance capable of providing a miRNA detection image signal in vitro and / or in vivo . In the present invention, the target nucleic acid may be miRNA.

본 발명에서 사용되는 용어 “소광물질(quencher molecule)”은 형광물질이 발생시킨 빛을 흡수하여 형광세기를 감소시키는 물질을 의미한다.As used herein, the term “quencher molecule” refers to a material that absorbs light generated by a fluorescent material and reduces fluorescence intensity.

본 발명에서 상기 “표적 miRNA”는 다양한 질병 및 암 조절에 관련된 miRNA를 의미한다. 바람직하게는, 상기 표적 miRNA는 위암, 유방암, 뇌암 및 바이러스 감염병으로 이루어진 군으로부터 선택된 질병과 관련된 miRNA일 수 있다.In the present invention, the "target miRNA" refers to miRNAs involved in various diseases and cancer regulation. Preferably, the target miRNA may be a miRNA associated with a disease selected from the group consisting of gastric cancer, breast cancer, brain cancer and viral infections.

본 발명에서 사용되는 상기 제1프로브는 헤어핀 구조의 형태로, 일 말단에 형광물질이 결합되어 있고, 헤어핀 구조의 중앙 부분에 표적 miRNA와 혼성화될 수 있는 영역이 있으며, 타 말단 근처에 소광물질이 결합되어 있되, 상기 소광물질은 형광물질과 근접한 위치에 있도록 디자인될 수 있다.The first probe used in the present invention is in the form of a hairpin structure, in which a fluorescent material is bound at one end thereof, a central portion of the hairpin structure has a region capable of hybridizing with a target miRNA, and a matting material near the other end thereof. While coupled, the quencher may be designed to be in close proximity to the fluorescent material.

본 발명에서 상기 표적 miRNA와 혼성화될 수 있는 영역은 표적 miRNA의 일부분과 상보적인 서열을 가져 표적 miRNA와 혼성화될 수 있는 서열을 의미한다. 상기 용어 ‘상보적’은 소정의 어닐링 조건 또는 엄중 조건(stringent condition)하에서 프로브가 표적 miRNA 서열에 선택적으로 혼성화할 정도로 충분히 상보적인 것을 의미한다. 예를 들어, 상기 표적 miRNA와 혼성화될 수 있는 영역은 표적 miRNA의 일부분과 90% 이상, 바람직하게는 95% 이상 상보적인 서열을 갖는 것일 수 있다. 상기 표적 miRNA와 혼성화될 수 있는 영역은 표적 miRNA에 따라 적절히 그 길이를 조절할 수 있으며 특정 길이로 한정되는 것은 아니다. In the present invention, the region capable of hybridizing with the target miRNA refers to a sequence capable of hybridizing with the target miRNA by having a sequence complementary to a portion of the target miRNA. The term 'complementary' means that the probe is complementary enough to selectively hybridize to a target miRNA sequence under certain annealing or stringent conditions. For example, the region capable of hybridizing with the target miRNA may be one having at least 90%, preferably at least 95% complementary sequence with a portion of the target miRNA. The region that can hybridize with the target miRNA may be appropriately adjusted depending on the target miRNA and is not limited to a specific length.

다양한 질병 및 암 조절에 관련된 표적 miRNA의 서열은 http://www.mirbase.org/, http://genome.ucsc.edu/, http://www.bioinfo.rpi.edu/zukerm/rna/mfold-3.html, https://www.ncbi.nlm.nih.gov/ 등으로 이루어진 군으로부터 선택된 데이터베이스를 이용하여 확보할 수 있다.Sequences of target miRNAs involved in various disease and cancer regulation are http://www.mirbase.org/ , http://genome.ucsc.edu/ , http://www.bioinfo.rpi.edu/zukerm/rna/ It can be obtained using a database selected from the group consisting of mfold-3.html and https://www.ncbi.nlm.nih.gov/ .

DNA나 RNA와 같은 핵산 분자는 해당 분자를 구성하는 염기 서열에 따라서 상호간에 특이적으로 결합하는 성질이 있으며, 그 염기 서열을 적절하게 디자인함으로써 반응을 조절할 수 있다. Nucleic acid molecules such as DNA and RNA have specific properties of binding to each other depending on the nucleotide sequences constituting the molecule, and the reaction can be controlled by appropriately designing the nucleotide sequences.

본 발명에서 상기 제1프로브의 일 말단에 결합되어 있는 형광물질은 형광을 발할 수 있는 물질이라면 구체적인 종류는 특별히 한정되지 않으며, 이 분야에서 통상적으로 알려진 형광물질이라면 제한 없이 사용할 수 있다. 상기 형광물질은 예를 들어, Cy3, Cy5, Cy5.5, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, Alexa 680, 로다민(rhodamine), TAMRA, FAM, FITC, TRITC, Fluor X, DAB, ROX, Texas Red, Orange green 488X, Orange green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodify 630/650, Bodify 650/665, CAL Fluor orange 546, CAL Fluor red 610, Quasar 670 및 비오틴 등을 사용할 수 있으나, 이에 제한되는 것은 아니다. 바람직하게는, 본 발명에서 상기 형광물질은 FAM, Cy3 또는 Cy5일 수 있다.In the present invention, the fluorescent material bound to one end of the first probe is not particularly limited as long as it is a material capable of emitting fluorescence, and any fluorescent material commonly known in the art may be used without limitation. The fluorescent material is, for example, Cy3, Cy5, Cy5.5, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, Alexa 680, Rhodamine, TAMRA, FAM, FITC, TRITC , Fluor X, DAB, ROX, Texas Red, Orange green 488X, Orange green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodify 630/650, Bodify 650/665, CAL Fluor orange 546, CAL Fluor red 610 , Quasar 670, biotin, and the like can be used, but is not limited thereto. Preferably, the fluorescent material in the present invention may be FAM, Cy3 or Cy5.

본 발명에서 상기 제1프로브의 타 말단에 결합되어 있는 소광물질 또한 형광을 제어할 수 있는 물질이라면 구체적인 종류는 특별히 한정되지 않으며, 이 분야에서 통상적으로 알려진 소광물질이라면 제한 없이 사용할 수 있다. 상기 소광물질은 예를 들면, DABCYL(DAB), DABSYL, BHQ1, BHQ2, BHQ3 등의 BHQ(Biosearch Technologies, Inc.) 등을 사용할 수 있으나, 이에 제한되는 것은 아니다. 바람직하게는, 본 발명에서 상기 소광물질은 DAB 또는 BHQ2일 수 있다.In the present invention, if the matting material bonded to the other end of the first probe is also a material capable of controlling fluorescence, a specific kind is not particularly limited, and any kind of matting material commonly known in the art may be used without limitation. The matting material may include, for example, BHQ (Biosearch Technologies, Inc.) such as DABCYL (DAB), DABSYL, BHQ1, BHQ2, and BHQ3, but is not limited thereto. Preferably, the matting material in the present invention may be DAB or BHQ2.

본 발명에 따른 바이오 프로브는 세포 내 표적 miRNA의 존재에 따라 형광 신호의 온-오프가 조절되는 방식을 취하고 있다.The bioprobe according to the present invention takes a manner in which on-off of the fluorescence signal is controlled according to the presence of the target miRNA in the cell.

제1프로브, 제2프로브, 제3프로브 및 제4프로브를 포함하는 본 발명에 따른 바이오 프로브와 시료를 접촉시켰을 때 시료 내에 표적 miRNA가 존재하는 경우, 표적 miRNA의 일부분과 상보적인 서열을 가지는 제1프로브는 표적 miRNA와 혼성화되어 형광신호를 생성하게 된다. 그리고, 프로브 분자들 간의 경쟁적 혼성화 반응에 의해 상기 제1프로브의 적어도 3 내지 20개의 서열에 상보적으로 결합하는 염기서열을 포함하는 제2프로브가 제1프로브에 혼성화되고, 다시 상기 제2프로브의 적어도 3 내지 15개의 서열에 상보적으로 결합하는 염기서열을 포함하는 제3프로브가 제2프로브에 혼성화되면서 제1프로브와 제2프로브가 분리되고, 제3프로브와 제1프로브에 상보적으로 결합하는 6 내지 30개의 염기서열을 포함하는 제4프로브가 다시 제3프로브와 제1프로브에 결합하는 자가 순환 과정을 통해 형광 신호 증폭이 가능해진다(도 1).When the target miRNA is present in the sample when the sample is contacted with the bioprobe according to the present invention including the first probe, the second probe, the third probe, and the fourth probe, the first probe has a sequence complementary to a portion of the target miRNA. One probe hybridizes with the target miRNA to produce a fluorescence signal. In addition, a second probe including a base sequence complementarily binding to at least 3 to 20 sequences of the first probe by a hybrid hybridization reaction between probe molecules is hybridized to the first probe, and then, The first probe and the second probe are separated while the third probe including the base sequence complementarily binding to at least 3 to 15 sequences hybridizes to the second probe, and complementarily binds to the third probe and the first probe. A fourth probe including 6 to 30 nucleotide sequences may be amplified by a fluorescence signal through a self-cycling process in which the fourth probe binds to the third probe and the first probe again (FIG. 1).

더욱 상세하게는, 본 발명에서 제1프로브, 제2프로브, 제3프로브 및 제4프로브를 포함하는 바이오 프로브 세트는 표적 miRNA 처리 전 소정의 조건하에서 어닐링됨으로써 헤어핀 구조를 유지하게 된다. 그 후, 어닐링된 제1프로브, 제2프로브, 제3프로브 및 제4프로브를 포함하는 바이오 프로브 세트에 표적 miRNA를 처리하여 소정의 조건하에서 혼성화시킴으로써 형광신호를 생성하게 된다. More specifically, in the present invention, the bioprobe set including the first probe, the second probe, the third probe, and the fourth probe is annealed under predetermined conditions before the target miRNA treatment to maintain the hairpin structure. Thereafter, the biomimetic set comprising the annealed first probe, the second probe, the third probe and the fourth probe is treated with a target miRNA to hybridize under a predetermined condition to generate a fluorescent signal.

본 발명의 구체적인 실시예에서, 본 발명에 따른 제1프로브, 제2프로브, 제3프로브 및 제4프로브(올리고 1, 2, 3, 4)를 포함하는 바이오 프로브 세트를 이용하여 표적 miRNA를 검출한 결과, 제1프로브(올리고 1)만을 이용한 것에 비해 형광 신호가 현저하게 증폭됨을 확인할 수 있었다(도 3 내지 9). In a specific embodiment of the present invention, target miRNAs are detected using a bioprobe set comprising a first probe, a second probe, a third probe and a fourth probe (oligos 1, 2, 3, 4) according to the present invention. As a result, it was confirmed that the fluorescence signal was significantly amplified compared to using only the first probe (oligo 1) (Figs. 3 to 9).

상기 목적을 달성하기 위한 또 다른 실시양태로서, 본 발명은 상기 바이오 프로브 세트를 포함하는 miRNA 검출용 유전자 칩을 제공한다. 유전자 칩은 마이크로어레이와 상호 교환하여 사용할 수 있다. 상기 바이오 프로브 세트는 전술한 바와 같다.As another embodiment for achieving the above object, the present invention provides a gene chip for miRNA detection comprising the bio-probe set. Gene chips can be used interchangeably with microarrays. The bioprobe set is as described above.

본 발명의 miRNA 검출용 유전자 칩은 기판(substrate) 상에 miRNA 검출용 바이오 프로브 세트가 높은 밀도로 고정화되어 있는 형태로 구성될 수 있다. 이러한 구성은 당업계에 잘 알려져 있다. The gene chip for detecting miRNA of the present invention may be configured in a form in which a set of bio probes for detecting miRNA is fixed at a high density on a substrate. Such configurations are well known in the art.

상기 기판은 혼성화 특성을 보유하고, 혼성화의 배경 수준이 낮게 유지되는 조건하에 올리고뉴클레오티드 프로브가 커플링될 수 있는 임의의 기판일 수 있다. 통상적으로, 상기 기판은 미세역가 플레이트(microtiter plate), 막(예를 들어, 나일론 또는 니트로셀룰로오스) 또는 미세구(비드) 또는 칩일 수 있다.The substrate can be any substrate to which the oligonucleotide probe can be coupled under conditions that retain hybridization properties and keep the background level of hybridization low. Typically, the substrate may be a microtiter plate, membrane (eg nylon or nitrocellulose) or microspheres (beads) or chips.

상기 목적을 달성하기 위한 또 다른 실시양태로서, 본 발명은 상기 바이오 프로브 세트 및 혼성화 용액을 포함하는 miRNA 검출용 키트를 제공한다. 상기 바이오 프로브 세트는 전술한 바와 같다.As another embodiment for achieving the above object, the present invention provides a kit for detecting miRNA comprising the bioprobe set and hybridization solution. The bioprobe set is as described above.

상기 혼성화 용액은 프로브 및 핵산 시료가 혼성화할 수 있게 하는 완충액으로서 당업계에 공지된 용액을 이용할 수 있다. 본 발명의 키트는 최적의 반응 수행 조건을 기재한 사용자 설명서를 추가로 포함할 수 있다.The hybridization solution may use a solution known in the art as a buffer that allows the probe and the nucleic acid sample to hybridize. The kit of the present invention may further comprise a user manual describing the conditions for performing the optimal reaction.

상기 목적을 달성하기 위한 또 다른 실시양태로서, 본 발명은 본 발명에 따른 바이오 프로브 세트를 어닐링하는 단계; 대상으로부터 수득한 생물학적 시료와 상기 어닐링된 바이오 프로브 세트를 접촉시켜 혼성화시키는 단계; 및 상기 혼성화로부터 발생되는 형광 신호를 검출하는 단계;를 포함하는 miRNA를 검출하는 방법을 제공하는 것이다. 상기 바이오 프로브 세트는 전술한 바와 같다.As another embodiment for achieving the above object, the present invention comprises the steps of annealing the bioprobe set according to the present invention; Contacting and hybridizing the annealed bioprobe set with a biological sample obtained from a subject; And it provides a method for detecting miRNA comprising a; detecting a fluorescence signal generated from the hybridization. The bioprobe set is as described above.

상기 어닐링하는 단계는 본 발명에 따른 바이오 프로브 세트를 어닐링하여 프로브의 형광성을 억제시킴으로써 표적 miRNA와 혼성화될 때 형광 신호가 회복되어 형광 세기를 측정할 수 있게 해준다. 상기 어닐링 조건은 95℃에서 5분간 가열하고 12시간 이상 실온까지 서서히 식히는 것이 바람직하다.The annealing step anneals the bioprobe set according to the present invention to inhibit the fluorescence of the probe, thereby allowing the fluorescence signal to recover and to measure the fluorescence intensity when hybridized with the target miRNA. The annealing conditions are preferably heated at 95 ° C. for 5 minutes and slowly cooled to room temperature for at least 12 hours.

상기 혼성화시키는 단계는 실온 또는 37℃에서 이루어지는 것이 바람직하며, 온도가 높을수록 프로브의 헤어핀 구조가 오픈되어 miRNA가 존재하지 않을 경우에도 프로브들 간의 혼성화가 발생할 수 있으므로 바람직하지 않다.The hybridization is preferably performed at room temperature or 37 ° C., and the higher the temperature, the more open the hairpin structure of the probe, and thus, hybridization between probes may occur even when no miRNA is present.

본 발명에서 사용되는 용어인 “생물학적 시료”는 표적 miRNA를 포함하는 임의의 시료를 의미한다. 상기 생물학적 시료는 관심 질병을 가진 대상으로부터 수득한 임의의 조직 또는 체액일 수 있다. As used herein, the term "biological sample" refers to any sample that contains a target miRNA. The biological sample can be any tissue or body fluid obtained from a subject with a disease of interest.

상기 생물학적 시료는 대상의 가래, 혈액, 혈청, 혈장, 혈구(예를 들어, 백혈구), 조직, 생검 샘플, 도말 샘플, 세척 샘플, 면봉 샘플, 세포 함유 체액, 유동 핵산, 소변, 복막액 및 흉수, 뇌 척수액, 대변, 누액 또는 이로부터의 세포를 포함하나, 이에 제한되지 않는다. 생물학적 시료는 조직학적 목적 하에 취해진 조직 절편, 즉 동결 또는 고정 절편 또는 그의 미세해부 세포 또는 세포외 부분을 또한 포함할 수 있다. 상기 생물학적 시료는 대상에게 위해를 끼치지 않는 방법으로 얻어질 수 있다.The biological sample may be a sputum, blood, serum, plasma, blood cells (eg, white blood cells), tissue, biopsy sample, smear sample, wash sample, swab sample, cell-containing body fluid, flow nucleic acid, urine, peritoneal fluid, and pleural effusion. , Cerebrospinal fluid, feces, lacrimal fluid or cells therefrom. The biological sample may also include tissue sections taken for histological purposes, ie frozen or fixed sections or microdissected cells or extracellular parts thereof. The biological sample can be obtained in a manner that does not harm the subject.

본 발명에서 형광 신호의 검출은 형광 물질의 종류에 따라 다양한 방법이 이용될 수 있다. In the present invention, various methods may be used for detecting the fluorescent signal depending on the type of the fluorescent material.

본 발명의 구체적인 실시예에서, miRNA-34a가 저발현된 인간 유방암 세포 MDA-MB-231 세포에 본 발명에 따른 바이오 프로브 세트(올리고 1 또는 올리고 1 내지 4)를 형질주입하고 형광 신호를 확인한 결과, 올리고 1만을 사용한 것에 비하여 올리고 1 내지 4를 모두 사용한 것의 형광 신호가 증폭되는 것을 확인할 수 있었다(도 10).In a specific embodiment of the present invention, miRNA-34a-expressed human breast cancer cell MDA-MB-231 cells were transfected with a bioprobe set (oligo 1 or oligo 1 to 4) according to the present invention and confirmed by fluorescent signals. As compared with using only oligo 1, it was confirmed that the fluorescence signal of using all of oligo 1 to 4 was amplified (FIG. 10).

본 발명의 바이오 프로브 세트는 자가 형광 신호 증폭이 가능하여 세포내 미량으로 발현되는 마이크로 RNA 등을 초고감도로 검출할 수 있게 하므로, 질병의 진단이나 예후용 바이오마커 등에 유용할 것으로 기대된다.The bioprobe set of the present invention is capable of amplifying autofluorescence signals, and thus can detect microRNAs expressed in trace amounts in cells with high sensitivity, and thus are expected to be useful for diagnosing diseases and prognostic biomarkers.

도 1은 본 발명에 따른 바이오 프로브 세트에 의한 자가 형광 신호 증폭 과정을 모식화한 도면이다.1 is a view schematically illustrating a process of amplifying an autofluorescence signal by a bioprobe set according to the present invention.

도 2는 본 발명에 따른 바이오 프로브 세트의 어닐링 효과를 나타낸 그래프이다.2 is a graph showing the annealing effect of the bioprobe set according to the present invention.

도 3은 본 발명에 따른 바이오 프로브 세트의 miRNA(0.1X) 검출능 평가 결과를 나타낸 그래프이다.3 is a graph showing the results of evaluating miRNA (0.1X) detectability of the bioprobe set according to the present invention.

도 4는 본 발명에 따른 바이오 프로브 세트의 miRNA(0.2X) 검출능 평가 결과를 나타낸 그래프이다.4 is a graph showing the results of evaluating miRNA (0.2X) detectability of the bioprobe set according to the present invention.

도 5는 본 발명에 따른 바이오 프로브 세트의 miRNA(0.4X) 검출능 평가 결과를 나타낸 그래프이다.5 is a graph showing the results of evaluating miRNA (0.4X) detectability of the bioprobe set according to the present invention.

도 6은 본 발명에 따른 바이오 프로브 세트의 miRNA(0.6X) 검출능 평가 결과를 나타낸 그래프이다.6 is a graph showing the results of evaluating miRNA (0.6X) detectability of the bioprobe set according to the present invention.

도 7은 본 발명에 따른 바이오 프로브 세트의 miRNA(0.8X) 검출능 평가 결과를 나타낸 그래프이다.7 is a graph showing the results of evaluating miRNA (0.8X) detectability of the bioprobe set according to the present invention.

도 8은 본 발명에 따른 바이오 프로브 세트의 miRNA(1X) 검출능 평가 결과를 나타낸 그래프이다.8 is a graph showing the results of evaluating miRNA (1X) detectability of the bioprobe set according to the present invention.

도 9는 본 발명에 따른 바이오 프로브 세트의 miRNA 처리 전/후의 형광 신호 세기를 비교한 결과를 나타낸 그래프이다. 9 is a graph showing the result of comparing the fluorescence signal intensity before and after the miRNA treatment of the bioprobe set according to the present invention.

도 10은 miRNA-34a가 저발현된 MDA-MB-231 세포에서 본 발명에 따른 바이오 프로브 세트를 이용하여 표적 miRNA-34a를 검출한 결과를 나타낸 도면이다. 10 is a diagram showing the result of detecting a target miRNA-34a using a bioprobe set according to the present invention in MDA-MB-231 cells with low expression of miRNA-34a.

이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

실시예Example

실시예Example 1: 바이오  1: bio 프로브Probe 세트의 제작 Making of sets

다양한 miRNA를 표적으로 하여 바이오 프로브를 고안할 수 있으며, 본 실시예에서는 검출대상이 되는 표적 miRNA로 인간 유방암과 관련된 miRNA-34a 를 사용하였다. 상기 miRNA-34a의 염기서열(서열번호 1)은 하기와 같으며, 본 발명에서는 세포외 조건에서 올리고머의 합성을 용이하게 하기 위하여 DNA 염기서열로 치환한 것(서열번호 2)을 사용하였다.Bio probes can be designed by targeting various miRNAs, and in this example, miRNA-34a related to human breast cancer was used as a target miRNA to be detected. The base sequence (SEQ ID NO: 1) of the miRNA-34a is as follows, and in the present invention, a DNA base sequence substituted (SEQ ID NO: 2) was used to facilitate the synthesis of the oligomer under extracellular conditions.

[표 1]TABLE 1

Figure PCTKR2017002103-appb-I000001
Figure PCTKR2017002103-appb-I000001

상기 표적 miRNA-34a의 검출을 위하여 상기 miRNA와 상보적으로 결합할 수 있는 바이오 프로브 세트를 설계하였으며, 이하 제조된 바이오 프로브를 각각 올리고 1, 2, 3 및 4라 한다. 하기 표 2에 본 발명에 따른 바이오 프로브 세트의 서열번호(서열번호 3 내지 6) 및 염기서열을 나타내었다. In order to detect the target miRNA-34a, a bioprobe set capable of complementarily binding to the miRNA was designed. Hereinafter, the prepared bioprobes were referred to as oligo 1, 2, 3, and 4, respectively. Table 2 below shows the sequence numbers (SEQ ID NOS: 3 to 6) and nucleotide sequences of the bioprobe set according to the present invention.

[ 표 2 ]TABLE 2

Figure PCTKR2017002103-appb-I000002
Figure PCTKR2017002103-appb-I000002

상기 표 2에 나타낸 바와 같이, 본 발명의 바이오 프로브 세트는 각각 표적 miRNA 서열에 상보적인 염기서열을 포함하는 염기서열로 구성된다. 여기에서, 상기 올리고 1은 5’-말단에 형광물질로서 Cy5을 부착하고, 3’-말단에는 소광물질로서 BHQ2를 부착하였다.As shown in Table 2, the bioprobe set of the present invention consists of a base sequence each comprising a base sequence complementary to the target miRNA sequence. Here, the oligo 1 attached Cy5 as a fluorescent material at the 5'-end and BHQ2 as a quencher at the 3'-end.

실시예Example 2: 바이오  2: bio 프로브의Of probe 어닐링Annealing 효과 확인 Check the effect

상기 실시예 1에서 제조된 올리고 1의 어닐링 효과를 확인하기 위한 실험을 수행하였다. The experiment to confirm the annealing effect of the oligo 1 prepared in Example 1 was performed.

상기 올리고 1을 농도별(각각 12.5, 25, 50, 100 pmol)로 준비하여 가열기(heat block)에서 각각 95℃에서 5 분 가열한 후, 가열기의 작동을 끄고 12시간 이상 실온(RT)까지 서서히 식히면서 어닐링하였다. 그 후, 상기 올리고 1의 어닐링 전/후의 형광 세기를 측정함으로써 어닐링 여부를 확인하였다. The oligo 1 was prepared by concentration (12.5, 25, 50, 100 pmol, respectively) and heated at 95 ° C. for 5 minutes in a heater (heat block), and then the heater was turned off and then slowly turned to room temperature (RT) for at least 12 hours. Annealed while cooling. Then, the annealing was confirmed by measuring the fluorescence intensity before / after annealing of the oligo 1.

또한, 상기 올리고 1과 같은 농도별로 표적 miRNA 또는 대조군(5’-T GGCAGAATCGGAGCTGGTCCT 3’, 서열번호 7)을 37℃에서 2시간 반응시킨 후 형광 세기를 측정하였다. 형광은 여기(excitation) 파장 620 nm 및 방출(emission) 파장 668 nm에서 형광세기를 측정하였다. 그 결과를 도 2에 나타내었다.In addition, the fluorescence intensity was measured after reacting the target miRNA or the control (5'-T GGCAGAATCGGAGCTGGTCCT 3 ', SEQ ID NO: 7) for 2 hours at 37 ℃ for each concentration such as oligo 1. Fluorescence was measured for fluorescence intensity at excitation wavelength 620 nm and emission wavelength 668 nm. The results are shown in FIG.

도 2에 나타난 바와 같이, 올리고 1은 표적 miRNA와 특이적으로 결합하여 형광물질이 소광물질과의 거리가 멀어지면서 형광 신호가 증가됨을 확인할 수 있었다.As shown in Figure 2, oligo 1 specifically binds to the target miRNA was confirmed that the fluorescent signal is increased as the fluorescent material is far from the quencher.

실시예Example 3: 바이오  3: bio 프로브Probe 세트의  Set of miRNAmiRNA 검출능Detectability 평가 evaluation

상기 실시예 1에서 제조된 각각의 올리고 1, 2, 3 및 4를 25 pmol (1X)을 기준으로 95℃에서 5분 가열한 후 12시간 이상 실온(RT)까지 서서히 식히면서 어닐링하고, 농도별로 표적 miRNA의 서열(0.1X ~ 1X)을 처리하여 37℃ 조건에서 10분마다 형광 신호를 측정하였다. 그 결과를 도 3 내지 8 및 도 9에 나타내었다. Each oligo 1, 2, 3 and 4 prepared in Example 1 was heated for 5 minutes at 95 ℃ based on 25 pmol (1X) and then anneal while slowly cooling to room temperature (RT) for at least 12 hours, and targeted by concentration The fluorescence signal was measured every 10 minutes at 37 ° C. by processing the miRNA sequence (0.1 × -1 ×). The results are shown in FIGS. 3 to 8 and 9.

도 9는 도 3 내지 8에 나타낸 결과를 바탕으로 miRNA 처리 전/후(180분 후)의 형광 신호를 비교한 결과이다. 여기에서, 파란색 막대 그래프는 miRNA 처리 전의 형광 신호값(before kinetic)을 나타내고, 붉은색 막대 그래프는 miRNA 처리 180분 후의 형광 신호값(after kinetic)을 나타낸 것이다. 9 is a result of comparing the fluorescent signal before / after miRNA treatment (after 180 minutes) based on the results shown in FIGS. Here, the blue bar graph represents the fluorescence signal value (before kinetic) before the miRNA treatment, and the red bar graph represents the fluorescence signal value (after kinetic) after 180 minutes of the miRNA treatment.

도 3 내지 9에 나타난 바와 같이, 본 발명에 따른 바이오 프로브 세트가 표적 miRNA를 특이적으로 검출 가능함을 확인할 수 있었다. 또한, 특히 같은 농도의 표적 miRNA 존재시, 올리고 1 대비 올리고 1, 2, 3 및 4가 모두 존재하는 경우 높은 형광 신호를 나타냄을 확인할 수 있었다. 이는 본 발명에 따른 바이오 프로브 세트가 자가 형광 신호 증폭이 가능함을 확인시켜 주는 결과이다.As shown in Figures 3 to 9, it was confirmed that the bio probe set according to the present invention can specifically detect the target miRNA. In addition, in the presence of the same concentration of the target miRNA, it was confirmed that the high fluorescence signal when oligo 1, oligo 1, 2, 3 and 4 are present. This is a result confirming that the bioprobe set according to the present invention can amplify the autofluorescence signal.

실시예Example 4:  4: 세포내Intracellular 표적  Target miRNAmiRNA 검출 detection

종양 억제인자(tumor suppressor)로 잘 알려진 miRNA-34a가 저발현된 유방암 세포주인 MDA-MB-231 세포를 이용하여, 본 발명에 따른 바이오 프로브 세트의 표적 miRNA 검출능을 확인하기 위한 실험을 수행하였다.Experiments were performed to confirm the target miRNA detection ability of the bioprobe set according to the present invention using MDA-MB-231 cells, a low-expressing breast cancer cell line miRNA-34a, well known as a tumor suppressor. .

MDA-MB-231 세포를 105 cells/well로 분주(seeding)하였다. 다음날 95℃에서 5분 가열한 후 12시간 이상 실온(RT)까지 서서히 식히면서 어닐링한 올리고 1 (80 ng)과 1.5 μL 리포펙타민(lipofectamin) 2000 시약을 100 μL Opti-MEM 배지에 넣은 후 혼합하여 5분간 상온에서 배양하여 반응시켰다(조건 1). 상기와 동일한 조건하에서 어닐링한 올리고 1, 2, 3 및 4 (각각 80 ng)와 1.5 μL 리포펙타민 2000 시약을 상기와 같은 방법으로 반응시켰다(조건 2). 5분 후 준비해놓은 세포의 배지를 제거한 후, 각각 상기 준비해둔 조건들(조건 1 및 2)을 처리한 뒤, 4, 8, 16 및 24시간 동안 37℃에서 배양하였다. 대조군으로는 200 μL의 Opti-MEM 배지에 세포를 넣고 배양한 것을 사용하였다.MDA-MB-231 cells were seeded at 10 5 cells / well. The next day, after heating for 5 minutes at 95 ℃ and slowly cooled to room temperature (RT) for more than 12 hours, annealed oligo 1 (80 ng) and 1.5 μL lipofectamin 2000 reagent in 100 μL Opti-MEM medium and mixed The reaction was carried out by incubating at room temperature for 5 minutes (condition 1). Oligos 1, 2, 3, and 4 (80 ng each) and 1.5 μL Lipofectamine 2000 reagent annealed under the same conditions as above were reacted in the same manner (condition 2). After 5 minutes, the prepared medium was removed, and the above prepared conditions (conditions 1 and 2) were treated, and then cultured at 37 ° C. for 4, 8, 16, and 24 hours. As a control, cells cultured in 200 μL Opti-MEM medium were used.

배양된 세포를 고정시켜 DAPI 염색한 후 형광 신호가 발생되는 것을 형광 현미경으로 관찰하였으며, DAPI 및 Cy5 형광 이미지를 촬영하였다. 그 결과를 도 10에 나타내었다.After culturing the cultured cells and staining with DAPI, the fluorescence signal was observed under a fluorescence microscope, and DAPI and Cy5 fluorescence images were taken. The results are shown in FIG.

도 10에 나타난 바와 같이, 올리고 1만 사용하였을 때와 비교하여 올리고 1, 2, 3 및 4를 사용하였을 때 형광 신호가 증폭되었음을 알 수 있으며, 형질주입 시간에 따라 형광 신호가 더욱 증폭됨을 알 수 있었다.As shown in FIG. 10, it can be seen that the fluorescence signal was amplified when using oligos 1, 2, 3, and 4 compared to when only oligo 1 was used, and the amplification of the fluorescent signal was further amplified according to the transfection time. there was.

Claims (11)

형광물질 및 소광물질을 포함하는 마이크로 RNA 검출용 제1프로브, 상기 제1프로브의 적어도 3 내지 20개의 서열에 상보적으로 결합하는 염기서열을 포함하는 제2프로브, 상기 제2프로브의 적어도 3 내지 15개의 서열에 상보적으로 결합하는 염기서열을 포함하는 제3프로브, 및 제3프로브와 제1프로브에 상보적으로 결합하는 6 내지 30개의 염기서열을 포함하는 제4프로브를 포함하는, 자가 형광 신호 증폭이 가능한 마이크로 RNA 검출용 바이오 프로브 세트. A first probe for detecting a microRNA including a fluorescent material and a quencher, a second probe comprising a base sequence complementarily binding to at least 3 to 20 sequences of the first probe, and at least 3 to 3 of the second probe Self-fluorescence comprising a third probe comprising a base sequence that complementarily binds 15 sequences, and a fourth probe comprising 6 to 30 base sequences that complementarily binds the third probe and the first probe Bio probe set for micro RNA detection with signal amplification. 제1항에 있어서,The method of claim 1, 상기 마이크로 RNA는 위암, 유방암, 뇌암 및 바이러스 감염병으로 이루어진 군으로부터 선택된 질병과 관련된 것인, 자가 형광 신호 증폭이 가능한 마이크로 RNA 검출용 바이오 프로브 세트. The micro RNA is associated with a disease selected from the group consisting of gastric cancer, breast cancer, brain cancer and viral infections, bio probe set for detecting a micro RNA capable of amplifying autofluorescence. 제1항에 있어서,The method of claim 1, 상기 형광물질은 Cy3, Cy5, Cy5.5, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, Alexa 680, 로다민(rhodamine), TAMRA, FAM, FITC, , TRITC, Fluor X, DAB, ROX, Texas Red, Orange green 488X, Orange green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodify 630/650, Bodify 650/665, Calfluor orange 546, Calfluor red 610, Quasar 670 및 비오틴으로 이루어진 군으로부터 선택되는, 자가 형광 신호 증폭이 가능한 마이크로 RNA 검출용 바이오 프로브 세트. The fluorescent material is Cy3, Cy5, Cy5.5, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, Alexa 680, Rhodamine, TAMRA, FAM, FITC,, TRITC, Fluor X DAB, ROX, Texas Red, Orange green 488X, Orange green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodify 630/650, Bodify 650/665, Calfluor orange 546, Calfluor red 610, Quasar 670 and Biotin A bioprobe set for micro RNA detection capable of amplifying autofluorescence signals, selected from the group consisting of: 제3항에 있어서,The method of claim 3, 상기 형광물질은 FAM 또는 Cy5인, 자가 형광 신호 증폭이 가능한 마이크로 RNA 검출용 바이오 프로브 세트.The fluorescent material is FAM or Cy5, a bio-probe set for micro RNA detection capable of amplifying autofluorescence signal. 제1항에 있어서,The method of claim 1, 상기 소광물질은 DABCYL(DAB), DABSYL, BHQ1, BHQ2 및 BHQ3으로 이루어진 군으로부터 선택되는, 자가 형광 신호 증폭이 가능한 마이크로 RNA 검출용 바이오 프로브 세트. The matting material is selected from the group consisting of DABCYL (DAB), DABSYL, BHQ1, BHQ2 and BHQ3, a bio-probe set for micro RNA detection capable of autofluorescence signal amplification. 제5항에 있어서,The method of claim 5, 상기 소광물질은 DAB 또는 BHQ2인, 자가 형광 신호 증폭이 가능한 마이크로 RNA 검출용 바이오 프로브 세트. The quencher is DAB or BHQ2, a bio-probe set for micro RNA detection capable of amplifying autofluorescence signal. 제1항에 따른 바이오 프로브 세트를 포함하는 유전자 칩.Gene chip comprising the bio-probe set according to claim 1. 제1항에 따른 바이오 프로브 세트를 포함하는 마이크로 RNA 검출용 키트.Micro RNA detection kit comprising a bio-probe set according to claim 1. 제1항에 따른 바이오 프로브 세트를 어닐링하는 단계;Annealing the bioprobe set according to claim 1; 대상으로부터 수득한 생물학적 시료와 상기 어닐링된 바이오 프로브 세트를 접촉시켜 혼성화시키는 단계; 및 Contacting and hybridizing the annealed bioprobe set with a biological sample obtained from a subject; And 상기 혼성화로부터 발생되는 형광 신호를 검출하는 단계;Detecting a fluorescence signal generated from the hybridization; 를 포함하는 마이크로 RNA를 검출하는 방법.Micro RNA detection method comprising a. 제9항에 있어서,The method of claim 9, 상기 어닐링 조건은 95℃에서 5분간 가열하고 12시간 이상 실온까지 서서히 식히는 것인, 마이크로 RNA를 검출하는 방법.Wherein the annealing conditions are heated at 95 ° C. for 5 minutes and slowly cooled to room temperature for at least 12 hours. 제9항에 있어서,The method of claim 9, 상기 혼성화는 실온 또는 37℃에서 이루어지는 것인, 마이크로 RNA를 검출하는 방법.Wherein said hybridization takes place at room temperature or at 37 ° C.
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