KR101249493B1 - Method for Detecting Nucleic Acid Using DNAzyme-Molecular Beacon - Google Patents
Method for Detecting Nucleic Acid Using DNAzyme-Molecular Beacon Download PDFInfo
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Abstract
본 발명은 핵산효소와 분자비콘의 복합체 및 핵산 신호증폭방법을 이용한 핵산 검출방법에 관한 것으로, 더욱 자세하게는 과산화효소 활성이 있는 핵산효소와 헤어핀 구조를 형성하는 분자비콘의 복합체와 블로커 핵산 및 핵산중합효소 반응을 통한 표적핵산을 검출하는 방법에 관한 것이다.
본 발명에 따른 핵산검출방법은 핵산중합효소의 가닥치환 활성을 이용하여 표적핵산이 발색반응을 통한 신호 생성 과정에 반복적으로 재사용될 수 있도록 하여 높은 민감도로 표적핵산을 검출할 수 있으며, 이는 핵산뿐 아니라 단백질 및 소분자의 검출에도 적용될 수도 있다.The present invention relates to a nucleic acid detection method using a complex of a nucleic acid enzyme and a molecular beacon and a nucleic acid signal amplification method, and more particularly, a complex, a blocker nucleic acid and a nucleic acid polymerization of a molecular beacon which forms a hairpin structure with a nucleic acid enzyme having peroxidase activity. The present invention relates to a method for detecting a target nucleic acid through an enzymatic reaction.
The nucleic acid detection method according to the present invention can detect target nucleic acids with high sensitivity by allowing the target nucleic acid to be repeatedly reused in the signal generation process through the color reaction using the strand replacement activity of the nucleic acid polymerase. It may also be applied to the detection of proteins and small molecules.
Description
본 발명은 핵산효소와 분자비콘의 복합체 및 핵산 신호증폭방법을 이용한 핵산 검출방법에 관한 것으로, 더욱 자세하게는 과산화효소 활성이 있는 핵산효소와 헤어핀 구조를 형성하는 분자비콘의 복합체와 블로커 핵산 및 핵산중합효소 반응을 통한 표적핵산을 검출하는 방법에 관한 것이다.
The present invention relates to a nucleic acid detection method using a complex of a nucleic acid enzyme and a molecular beacon and a nucleic acid signal amplification method, and more particularly, a complex, a blocker nucleic acid and a nucleic acid polymerization of a molecular beacon which forms a hairpin structure with a nucleic acid enzyme having peroxidase activity. The present invention relates to a method for detecting a target nucleic acid through an enzymatic reaction.
종래의 분자비콘 형태의 프로브를 이용하는 핵산 검출 바이오센서는 표적핵산의 서열에 특이적인 핵산 연쇄 중합반응을 통하여 생성된 중합 생성물이 분자비콘의 구조 변화를 유도하여 그에 따른 형광신호를 생성하게 된다 (Tyagi,S et al ., Nature biotechnology, 14:303-308, 1996). 일반적으로 분자비콘 프로브의 양 말단에 각각 형광체와 소광체(quencher)가 수식되어 있어, 헤어핀(hairpin) 구조상태로 있는 경우에는 형광을 띄지 않지만 분자비콘의 고리(loop) 부분이 표적핵산에 상보적으로 결합하여 헤어핀 구조가 열리면서 형광신호를 나타내게 된다. 이런 방법을 사용하게 되면 표적핵산과 분자비콘이 1:1로 정량적으로 결합하여 형광신호를 내게 된다. 즉, 한 분자의 표적핵산이 한 분자의 형광물질을 발생시키기 때문에 정확한 정량은 가능하지만, 낮은 농도의 표적핵산을 검출하는 데에는 한계가 있으며, 또한, 이 경우 분자비콘을 형광으로 수식해야하는데에 고비용이 든다.Nucleic acid detection biosensor using a conventional molecular beacon type probe is a polymerization product generated through a nucleic acid chain polymerization reaction specific to the sequence of the target nucleic acid induces a structural change of the molecular beacon to generate a fluorescent signal accordingly (Tyagi , S et al ., Nature biotechnology , 14: 303-308, 1996). In general, phosphors and quenchers are modified at both ends of the molecular beacon probe, so when the hairpin structure is not fluorescence, the loop portion of the molecular beacon is complementary to the target nucleic acid. Combining to open the hairpin structure will show a fluorescent signal. Using this method, the target nucleic acid and the molecular beacon are quantitatively combined 1: 1 to emit a fluorescent signal. In other words, since one molecule of target nucleic acid generates one molecule of fluorescent material, accurate quantification is possible, but there is a limit in detecting a low concentration of target nucleic acid, and in this case, the molecular beacon must be modified with fluorescence. This costs
최근, 핵산 검출에 있어 민감도를 높이기 위해서 핵산효소를 분자비콘에 결합한 바이오센서들이 제안되었다. 대표적인 예로, 분자비콘이 헤어핀 구조를 유지할 때는 기질과 결합하지 못하다가 표적핵산이 있는 경우에는 분자비콘의 헤어핀 구조가 열리면서 기질과 결합할 수 있게 되어 핵산분해활성의 핵산효소에 의해 기질이 절단되는 방법이다. 이때 기질의 양 말단이 각각 형광체와 소광체로 수식되어 있기 때문에 분해반응을 통해서 형광신호를 내게 된다 (Stojanovic, M. N. et al ,. ChemBioChem., 2:411-415, 2001). 또한, 한 가닥의 과산화효소 활성의 핵산효소(peroxidase DNAzyme)가 이용된 바이오센서를 들 수 있는데, 이 핵산효소는 표적핵산이 없는 경우 헤어핀 구조를 가지게 되어 불활성화 상태로 존재한다. 반면 표적핵산이 존재하는 경우에는 핵산효소의 헤어핀 구조가 열리면서 활성화되어 과산화효소 활성을 통한 발색 신호를 낸다 (Xiao,Y., et al ., J. Am . Chem . Soc ., 126:7430-7431, 2004). 그러나 종래의 분자비콘 핵산효소 기반 기술들의 분석 민감도는 전통적인 분자비콘을 이용한 바이오센서와 비슷하거나 오히려 낮은 수준인 것으로 나타났다.Recently, biosensors that bind nucleic acid enzymes to molecular beacons have been proposed to increase the sensitivity in nucleic acid detection. As a representative example, the molecular beacon does not bind to the substrate when maintaining the hairpin structure, but when the target nucleic acid is present, the hairpin structure of the molecular beacon is opened to bind to the substrate, thereby cutting the substrate by a nucleic acid enzyme having a nuclease activity. to be. At this time, since both ends of the substrate are modified with phosphor and quencher, a fluorescent signal is emitted through decomposition reaction (Stojanovic, MN et al ,. Chem Bio Chem., 2: 411-415, 2001). In addition, a biosensor using a peroxidase DNAzyme (peroxidase DNAzyme) of one strand is used, which has a hairpin structure in the absence of a target nucleic acid and exists in an inactive state. On the other hand, if the target nucleic acid is present, the hairpin structure of the nucleic acid enzyme is opened and activated to give a color signal through peroxidase activity (Xiao, Y., meat al ., J. Am . Chem . Soc ., 126: 7430-7431, 2004). However, the analysis sensitivity of the conventional molecular beacon nucleic acid enzyme-based techniques has been shown to be similar or rather low than the biosensor using the conventional molecular beacon.
이에, 본 발명자들은 핵산중합효소의 가닥치환 활성을 이용하여 표적핵산이 발색반응을 통한 신호 생성 과정에 반복적으로 재사용될 수 있도록 하여 높은 민감도로 표적핵산을 검출하기 위하여 예의 노력한 결과, 과산화효소 활성의 두 가닥 핵산 효소-분자비콘 복합체와 블로커 핵산으로 이루어진 복합체 프로브를 가닥 치환 활성을 가지는 핵산중합효소의 중합반응을 통하여 신호를 증폭시켜 매우 높은 민감도로 표적핵산을 검출할 수 있는 것을 확인하고 본 발명을 완성하였다.
Thus, the present inventors have made efforts to detect target nucleic acids with high sensitivity by using the strand replacement activity of nucleic acid polymerase so that the target nucleic acid can be repeatedly reused in the signal generation process through the color reaction. The present invention provides a complex probe consisting of a two-stranded nucleic acid enzyme-molecule beacon complex and a blocker nucleic acid and amplifies a signal through a polymerization reaction of a nucleic acid polymerase having strand substitution activity. Completed.
본 발명의 목적은 핵산효소-분자비콘 복합체와 블로커 핵산으로 이루어진 프로브를 가닥 치환 활성을 가지는 핵산중합효소의 중합반응을 통하여 높은 민감도로 표적핵산을 검출하는 방법을 제공하는데 있다.
An object of the present invention is to provide a method for detecting a target nucleic acid with high sensitivity through a polymerization reaction of a nucleic acid polymerase having strand substitution activity of a probe consisting of a nucleic acid enzyme-molecule beacon complex and a blocker nucleic acid.
상기 목적을 달성하기 위하여, 본 발명은 (a) i) 핵산효소-분자비콘 복합체;In order to achieve the above object, the present invention (a) i) nucleic acid enzyme-molecule beacon complex;
ii) 한쪽 말단에 상기 핵산효소-분자비콘 복합체의 루프부분과 상보적인 서열을 가지고, 다른 말단에 표적핵산의 일부와 상보적인 서열을 가지며, 어느 한 말단에 표적핵산 치환 프라이머와 상보적인 서열을 함유하는 블로커 핵산;ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, complementary to a portion of the target nucleic acid at the other end, and contains a sequence complementary to the target nucleic acid substitution primer at either end Blocker nucleic acid;
iii) 핵산중합효소 연쇄반응시 블로커 핵산에 붙어있는 표적핵산을 분리시키고, 대신에 블로커 핵산에 상보적인 서열을 가지는 핵산으로 치환시키는 표적핵산 치환 프라이머;iii) a target nucleic acid substitution primer that separates a target nucleic acid attached to a blocker nucleic acid during a nucleic acid polymerase chain reaction, and instead substitutes a nucleic acid having a sequence complementary to the blocker nucleic acid;
iv) 헤민;iv) hemin;
v) 핵산중합효소; 및v) nucleic acid polymerase; And
vi) dNTP가 함유된 핵산검출용액에 표적핵산을 함유하는 샘플을 혼합하고, 핵산중합효소 연쇄반응시켜 표적핵산을 블로커 핵산으로부터 분리시키는 단계; 및 (b) 상기 분리된 표적핵산에 의해 활성화된 핵산효소-분자비콘 복합체를 포함하는 용액에 과산화효소 기질을 첨가하여 표적핵산을 검출하는 단계를 포함하는 핵산의 검출방법을 제공한다.vi) mixing the sample containing the target nucleic acid with the nucleic acid detection solution containing dNTP, and nucleic acid polymerase chain reaction to separate the target nucleic acid from the blocker nucleic acid; And (b) adding a peroxidase substrate to a solution containing the nucleic acid enzyme-molecule beacon complex activated by the separated target nucleic acid to detect the target nucleic acid.
본 발명은 또한, 한쪽 말단에 핵산효소-분자비콘 복합체의 루프부분과 상보적인 서열을 가지고, 다른 말단에 표적핵산의 일부와 상보적인 서열을 가지며,어느 한 말단에 핵산중합효소 연쇄반응시 블로커 핵산에 붙어있는 표적핵산을 분리시키고, 대신에 블로커 핵산에 상보적인 서열을 가지는 핵산으로 치환시키는 표적핵산 치환 프라이머와 상보적인 서열을 함유하는 블로커 핵산을 제공한다.The present invention also has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, a sequence complementary to a portion of the target nucleic acid at the other end, and a blocker nucleic acid during the nucleic acid polymerase chain reaction at either end. A blocker nucleic acid is provided that contains a sequence complementary to a target nucleic acid substitution primer that isolates a target nucleic acid attached to the target nucleic acid and replaces the target nucleic acid with a nucleic acid having a sequence complementary to the blocker nucleic acid.
본 발명은 또한, i) 핵산효소-분자비콘 복합체; ii) 한쪽 말단에 핵산효소-분자비콘 복합체의 루프부분과 상보적인 서열을 가지고, 다른 말단에 표적핵산의 일부와 상보적인 서열을 가지며, 어느 한 말단에 표적핵산 치환 프라이머와 상보적인 서열을 함유하는 블로커 핵산; iii) 핵산중합효소 연쇄반응시 블로커 핵산에 붙어있는 표적핵산을 분리시키고, 대신에 블로커 핵산에 상보적인 서열을 가지는 핵산으로 치환시키는 표적핵산 치환 프라이머; iv) 헤민; v) 핵산중합효소; 및 vi) dNTP를 함유하는 핵산검출용액을 제공한다.The present invention is also directed to a process for preparing i) a nucleic acid enzyme-molecular beacon complex; ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, a sequence complementary to a portion of the target nucleic acid at the other end, and contains a sequence complementary to the target nucleic acid substitution primer at either end Blocker nucleic acid; iii) a target nucleic acid substitution primer that separates a target nucleic acid attached to a blocker nucleic acid during a nucleic acid polymerase chain reaction, and instead substitutes a nucleic acid having a sequence complementary to the blocker nucleic acid; iv) hemin; v) nucleic acid polymerase; And vi) a nucleic acid detection solution containing dNTP.
본 발명은 또한, (a) i) 핵산효소-분자비콘 복합체;The invention also provides a composition comprising (a) i) a nucleic acid enzyme-molecular beacon complex;
ii) 한쪽 말단에 상기 핵산효소-분자비콘 복합체의 루프부분과 상보적인 서열을 가지고, 다른 말단에 표적 소분자 또는 단백질과 결합능을 가지는 압타머 서열을 가지며, 어느 한 말단에 표적 소분자 또는 단백질 치환 프라이머와 상보적인 서열을 함유하는 블로커 핵산;ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, and has an aptamer sequence at the other end having a binding capacity with a target small molecule or protein, and at either end with a target small molecule or protein substitution primer Blocker nucleic acids containing complementary sequences;
iii) 핵산중합효소 연쇄반응시 블로커 핵산에 붙어있는 소분자 또는 단백질을 분리시키고, 대신에 블로커 핵산에 상보적인 서열을 가지는 핵산으로 치환시키는 표적 소분자 또는 단백질 치환 프라이머;iii) a target small molecule or protein substitution primer that separates the small molecule or protein attached to the blocker nucleic acid in the nucleic acid polymerase chain reaction and replaces it with a nucleic acid having a sequence complementary to the blocker nucleic acid;
iv) 헤민;iv) hemin;
v) 핵산중합효소; 및 v) nucleic acid polymerase; And
vi) dNTP가 함유된 소분자 또는 단백질검출용액에 표적 소분자 또는 단백질을 함유하는 샘플을 혼합하고, 핵산중합효소 연쇄반응시켜 표적 소분자 또는 단백질을 블로커 핵산으로부터 분리시키는 단계; 및 (b) 상기 분리된 표적 소분자 또는 단백질에 의해 활성화된 핵산효소-분자비콘 복합체를 포함하는 용액에 과산화효소 기질을 첨가하여 표적 소분자 또는 단백질을 검출하는 단계를 포함하는 소분자 또는 단백질의 검출방법을 제공한다.vi) mixing a sample containing a target small molecule or protein with a small molecule or protein detection solution containing dNTP, and separating the target small molecule or protein from the blocker nucleic acid by nucleic acid polymerase chain reaction; And (b) detecting the target small molecule or protein by adding a peroxide enzyme to a solution containing the isolated nucleic acid enzyme-molecule beacon complex activated by the target small molecule or protein. to provide.
본 발명은 또한, 한쪽 말단에 핵산효소-분자비콘 복합체의 루프부분과 상보적인 서열을 가지고, 다른 말단에 표적 소분자 또는 단백질과 결합능을 가지는 압타머 서열을 가지며, 어느 한 말단에 핵산중합효소 연쇄반응시 블로커 핵산에 붙어있는 소분자 또는 단백질을 분리시키고, 대신에 블로커 핵산에 상보적인 서열을 가지는 핵산으로 치환시키는 표적 소분자 또는 단백질 치환 프라이머와 상보적인 서열을 함유하는 블로커 핵산을 제공한다.The present invention also has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, and has an aptamer sequence having a binding capacity with a target small molecule or protein at the other end, and a nucleic acid polymerase chain reaction at either end. A blocker nucleic acid is provided that contains a sequence complementary to a target small molecule or protein substitution primer that separates the small molecule or protein attached to the time blocker nucleic acid and replaces it with a nucleic acid having a sequence complementary to the blocker nucleic acid.
본 발명은 또한, i) 핵산효소-분자비콘 복합체; ii) 한쪽 말단에 상기 핵산효소-분자비콘 복합체의 루프부분과 상보적인 서열을 가지고, 다른 말단에 표적 소분자 또는 단백질과 결합능을 가지는 압타머 서열을 가지며, 어느 한 말단에 표적 소분자 또는 단백질 치환 프라이머와 상보적인 서열을 함유하는 블로커 핵산; iii) 핵산중합효소 연쇄반응시 블로커 핵산에 붙어있는 소분자 또는 단백질을 분리시키고, 대신에 블로커 핵산에 상보적인 서열을 가지는 핵산으로 치환시키는 표적 소분자 또는 단백질 치환 프라이머; iv) 헤민; v) 핵산중합효소; 및 vi) dNTP가 함유된 소분자 또는 단백질검출용액을 제공한다.
The present invention is also directed to a process for preparing i) a nucleic acid enzyme-molecular beacon complex; ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, and has an aptamer sequence at the other end having a binding capacity with a target small molecule or protein, and at either end with a target small molecule or protein substitution primer Blocker nucleic acids containing complementary sequences; iii) a target small molecule or protein substitution primer that separates the small molecule or protein attached to the blocker nucleic acid in the nucleic acid polymerase chain reaction and replaces it with a nucleic acid having a sequence complementary to the blocker nucleic acid; iv) hemin; v) nucleic acid polymerase; And vi) provides a small molecule or protein detection solution containing dNTP.
본 발명에 따른 핵산검출방법은 핵산중합효소의 가닥치환 활성을 이용하여 표적핵산이 발색반응을 통한 신호 생성 과정에 반복적으로 재사용될 수 있도록 하여 높은 민감도로 표적핵산을 검출할 수 있으며, 이는 핵산뿐 아니라 단백질 및 소분자의 검출에도 적용될 수도 있다.
The nucleic acid detection method according to the present invention can detect target nucleic acids with high sensitivity by allowing the target nucleic acid to be repeatedly reused in the signal generation process through the color reaction using the strand replacement activity of the nucleic acid polymerase. It may also be applied to the detection of proteins and small molecules.
도 1은 표적물질에 의한 신호 증폭 원리와 과산화효소 활성의 핵산효소(DNAzyme)를 이용하여 표적핵산을 검출하는 원리를 나타낸 그림이다.
도 2는 신호증폭효과가 결합된 핵산효소-분자비콘 복합체를 이용하여 일정한 농도의 표적핵산을 분석할 때, 서로 다른 길이의 프라이머를 사용할 경우 얻어지는 흡광도 세기를 나타낸 그래프이다.
도 3은 핵산중합효소와 디옥시뉴클레오타이드(dNTP)를 첨가 유무에 따른 표적 핵산 농도에 따른 흡광도 세기 변화를 나타낸 그래프이다.
도 4는 상기 신호증폭 핵산효소-분자비콘 복합체를 이용하여 서로 다른 농도의 표적핵산을 분석할 때 얻어지는 흡광도 세기를 나타낸 그래프이다.
도 5는 표적 핵산이 없는 경우와 다른 병원균 핵산(Chlamydia trachomatis)이 들어있는 경우의 흡광도 세기를 나타낸 그래프이다.1 is a diagram showing the principle of detecting target nucleic acid using the signal amplification principle by the target material and the nucleic acid enzyme (DNAzyme) of the peroxidase activity.
Figure 2 is a graph showing the absorbance intensity obtained when using a different length of the primer when analyzing the target nucleic acid using a nucleic acid enzyme-molecule beacon complex coupled signal amplification effect.
Figure 3 is a graph showing the change in absorbance intensity according to the target nucleic acid concentration with or without the addition of nucleic acid polymerase and deoxynucleotide (dNTP).
4 is a graph showing absorbance intensities obtained when analyzing target nucleic acids at different concentrations using the signal amplifying nucleic acid enzyme-molecule beacon complex.
5 shows pathogen nucleic acids different from those without target nucleic acids (C hlamydia). trachomatis ) is a graph showing the absorbance intensity in the case of containing.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
일 관점에서 본 발명은, (a) i) 핵산효소-분자비콘 복합체;In one aspect, the present invention, (a) i) nucleic acid enzyme-molecule beacon complex;
ii) 한쪽 말단에 상기 핵산효소-분자비콘 복합체의 루프부분과 상보적인 서열을 가지고, 다른 말단에 표적핵산의 일부와 상보적인 서열을 가지며, 어느 한 말단에 표적핵산 치환 프라이머와 상보적인 서열을 함유하는 블로커 핵산;ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, complementary to a portion of the target nucleic acid at the other end, and contains a sequence complementary to the target nucleic acid substitution primer at either end Blocker nucleic acid;
iii) 핵산중합효소 연쇄반응시 블로커 핵산에 붙어있는 표적핵산을 분리시키고, 대신에 블로커 핵산에 상보적인 서열을 가지는 핵산으로 치환시키는 표적핵산 치환 프라이머;iii) a target nucleic acid substitution primer that separates a target nucleic acid attached to a blocker nucleic acid during a nucleic acid polymerase chain reaction, and instead substitutes a nucleic acid having a sequence complementary to the blocker nucleic acid;
iv) 헤민;iv) hemin;
v) 핵산중합효소; 및v) nucleic acid polymerase; And
vi) dNTP가 함유된 핵산검출용액에 표적핵산을 함유하는 샘플을 혼합하고, 핵산중합효소 연쇄반응시켜 표적핵산을 블로커 핵산으로부터 분리시키는 단계; 및 (b) 상기 분리된 표적핵산에 의해 활성화된 핵산효소-분자비콘 복합체를 포함하는 용액에 과산화효소 기질을 첨가하여 표적핵산을 검출하는 단계를 포함하는 핵산의 검출방법에 관한 것이다.vi) mixing the sample containing the target nucleic acid with the nucleic acid detection solution containing dNTP, and nucleic acid polymerase chain reaction to separate the target nucleic acid from the blocker nucleic acid; And (b) adding a peroxidase substrate to a solution containing the nucleic acid enzyme-molecule beacon complex activated by the isolated target nucleic acid to detect the target nucleic acid.
다른 관점에서 본 발명은 한쪽 말단에 핵산효소-분자비콘 복합체의 루프부분과 상보적인 서열을 가지고, 다른 말단에 표적핵산의 일부와 상보적인 서열을 가지며, 어느 한 말단에 핵산중합효소 연쇄반응시 블로커 핵산에 붙어있는 표적핵산을 분리시키고, 대신에 블로커 핵산에 상보적인 서열을 가지는 핵산으로 치환시키는 표적핵산 치환 프라이머와 상보적인 서열을 함유하는 블로커 핵산에 관한 것이다.In another aspect, the present invention has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, a sequence complementary to a portion of the target nucleic acid at the other end, and blocker during the nucleic acid polymerase chain reaction at either end. A blocker nucleic acid containing a sequence complementary to a target nucleic acid substitution primer isolating a target nucleic acid attached to the nucleic acid and instead replacing the target nucleic acid with a nucleic acid having a sequence complementary to the blocker nucleic acid.
또 다른 관점에서 본 발명은 i) 핵산효소-분자비콘 복합체; ii) 한쪽 말단에 핵산효소-분자비콘 복합체의 루프부분과 상보적인 서열을 가지고, 다른 말단에 표적핵산의 일부와 상보적인 서열을 가지며, 어느 한 말단에 표적핵산 치환 프라이머와 상보적인 서열을 함유하는 블로커 핵산; iii) 핵산중합효소 연쇄반응시 블로커 핵산에 붙어있는 표적핵산을 분리시키고, 대신에 블로커 핵산에 상보적인 서열을 가지는 핵산으로 치환시키는 표적핵산 치환 프라이머; iv) 헤민; v) 핵산중합효소; 및 vi) dNTP를 함유하는 핵산검출용액에 관한 것이다.
In another aspect, the present invention provides a kit comprising: i) a nucleic acid enzyme-molecular beacon complex; ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, a sequence complementary to a portion of the target nucleic acid at the other end, and contains a sequence complementary to the target nucleic acid substitution primer at either end Blocker nucleic acid; iii) a target nucleic acid substitution primer that separates a target nucleic acid attached to a blocker nucleic acid during a nucleic acid polymerase chain reaction, and instead substitutes a nucleic acid having a sequence complementary to the blocker nucleic acid; iv) hemin; v) nucleic acid polymerase; And vi) a nucleic acid detection solution containing dNTP.
또 다른 관점에서 본 발명은 (a) i) 핵산효소-분자비콘 복합체;In another aspect, the present invention provides a composition comprising: (a) i) a nucleic acid enzyme-molecular beacon complex;
ii) 한쪽 말단에 상기 핵산효소-분자비콘 복합체의 루프부분과 상보적인 서열을 가지고, 다른 말단에 표적 소분자 또는 단백질과 결합능을 가지는 압타머 서열을 가지며, 어느 한 말단에 표적 소분자 또는 단백질 치환 프라이머와 상보적인 서열을 함유하는 블로커 핵산;ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, and has an aptamer sequence at the other end having a binding capacity with a target small molecule or protein, and at either end with a target small molecule or protein substitution primer Blocker nucleic acids containing complementary sequences;
iii) 핵산중합효소 연쇄반응시 블로커 핵산에 붙어있는 소분자 또는 단백질을 분리시키고, 대신에 블로커 핵산에 상보적인 서열을 가지는 핵산으로 치환시키는 표적 소분자 또는 단백질 치환 프라이머;iii) a target small molecule or protein substitution primer that separates the small molecule or protein attached to the blocker nucleic acid in the nucleic acid polymerase chain reaction and replaces it with a nucleic acid having a sequence complementary to the blocker nucleic acid;
iv) 헤민;iv) hemin;
v) 핵산중합효소; 및 v) nucleic acid polymerase; And
vi) dNTP가 함유된 소분자 또는 단백질검출용액에 표적 소분자 또는 단백질을 함유하는 샘플을 혼합하고, 핵산중합효소 연쇄반응시켜 표적 소분자 또는 단백질을 블로커 핵산으로부터 분리시키는 단계; 및 (b) 상기 분리된 표적 소분자 또는 단백질에 의해 활성화된 핵산효소-분자비콘 복합체를 포함하는 용액에 과산화효소 기질을 첨가하여 표적 소분자 또는 단백질을 검출하는 단계를 포함하는 소분자 또는 단백질의 검출방법에 관한 것이다.vi) mixing a sample containing a target small molecule or protein with a small molecule or protein detection solution containing dNTP, and separating the target small molecule or protein from the blocker nucleic acid by nucleic acid polymerase chain reaction; And (b) detecting the target small molecule or protein by adding a peroxidase substrate to a solution containing the isolated nucleic acid enzyme-molecule beacon complex activated by the target small molecule or protein. It is about.
또 다른 관점에서 본 발명은 한쪽 말단에 핵산효소-분자비콘 복합체의 루프부분과 상보적인 서열을 가지고, 다른 말단에 표적 소분자 또는 단백질과 결합능을 가지는 압타머 서열을 가지며, 어느 한 말단에 핵산중합효소 연쇄반응시 블로커 핵산에 붙어있는 소분자 또는 단백질을 분리시키고, 대신에 블로커 핵산에 상보적인 서열을 가지는 핵산으로 치환시키는 표적 소분자 또는 단백질 치환 프라이머와 상보적인 서열을 함유하는 블로커 핵산에 관한 것이다.In another aspect, the present invention has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, an aptamer sequence having a binding capacity with a target small molecule or protein at the other end, and a nucleic acid polymerase at either end It relates to a blocker nucleic acid containing a sequence complementary to a target small molecule or protein substitution primer that separates the small molecule or protein attached to the blocker nucleic acid in the chain reaction, and instead replaces it with a nucleic acid having a sequence complementary to the blocker nucleic acid.
또 다른 관점에서 본 발명은 i) 핵산효소-분자비콘 복합체; ii) 한쪽 말단에 상기 핵산효소-분자비콘 복합체의 루프부분과 상보적인 서열을 가지고, 다른 말단에 표적 소분자 또는 단백질과 결합능을 가지는 압타머 서열을 가지며, 어느 한 말단에 표적 소분자 또는 단백질 치환 프라이머와 상보적인 서열을 함유하는 블로커 핵산; iii) 핵산중합효소 연쇄반응시 블로커 핵산에 붙어있는 소분자 또는 단백질을 분리시키고, 대신에 블로커 핵산에 상보적인 서열을 가지는 핵산으로 치환시키는 표적 소분자 또는 단백질 치환 프라이머; iv) 헤민; v) 핵산중합효소; 및 vi) dNTP가 함유된 소분자 또는 단백질검출용액에 관한 것이다.In another aspect, the present invention provides a kit comprising: i) a nucleic acid enzyme-molecular beacon complex; ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, and has an aptamer sequence at the other end having a binding capacity with a target small molecule or protein, and at either end with a target small molecule or protein substitution primer Blocker nucleic acids containing complementary sequences; iii) a target small molecule or protein substitution primer that separates the small molecule or protein attached to the blocker nucleic acid in the nucleic acid polymerase chain reaction and replaces it with a nucleic acid having a sequence complementary to the blocker nucleic acid; iv) hemin; v) nucleic acid polymerase; And vi) a small molecule or protein detection solution containing dNTP.
본 발명에 있어서, 상기 핵산효소-분자비콘 복합체의 염기서열은 서열번호 1, 서열번호 4, 서열번호 7, 서열번호 10 및 서열번호 13으로 구성된 군에서 선택되는 것을 특징으로 할 수 있다.In the present invention, the nucleotide sequence of the nucleic acid enzyme-molecule beacon complex may be selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 13.
본 발명에 있어서, 상기 과산화효소 기질은 ABTS[2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], TMB(3,3',5,5'-tetramethyl benzidine) 및 OPD(o-phenylenediamine)로 구성된 군에서 선택되는 것을 특징으로 할 수 있다.In the present invention, the peroxidase substrate is ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], TMB (3,3 ', 5,5'-tetramethyl benzidine) and OPD ( o-phenylenediamine) may be selected from the group consisting of.
"분자비콘(Molecular Beacon)"은 3' 말단을 quencher 물질로 태그한 헤어핀 형태의 2차 구조를 형성하는 올리고뉴클레오타이드로, 분자비콘 프로브는 어닐링(annealing)과정에서 주형 DNA와 상보적인 영역에서 특이적으로 혼성화하며, 형광물질과 quencher 물질과의 거리가 멀어져 quencher 물질에 의한 발광의 억제가 해제되고 형광을 발하게 되는 것을 말한다. 반면, 혼성화되지 않은 분자비콘은 2차 구조를 유지하고 있으므로 quencher 에 의해 억제되어 형광을 띄지 않는다."Molecular Beacon" is an oligonucleotide that forms a hairpin secondary structure tagged with the quencher material at the 3 'end, and the molecular beacon probe is specific in a region complementary to the template DNA during annealing. Hybridization, and the distance between the fluorescent material and the quencher material is far away and the suppression of light emission by the quencher material is released and fluoresce. On the other hand, unhybridized molecular beacons maintain secondary structure and are suppressed by the quencher and thus do not fluoresce.
본 발명에서의 핵산효소-분자비콘 복합체는 루프부분에 표적핵산 서열의 일부를 가지며, 스템 구조의 양 말단은 헤민이 존재하는 경우에만 스스로 스템 구조를 형성하여 과산화수소 활성을 가질 수 있는 특징이 있다. The nucleic acid enzyme-molecular beacon complex in the present invention has a part of the target nucleic acid sequence in the loop portion, and both ends of the stem structure have a feature of forming a stem structure by itself only when hemin is present, and having hydrogen peroxide activity.
"핵산효소"는 통상적으로 효소활성을 가지는 핵산분자를 뜻한다. "Nuclease" generally refers to a nucleic acid molecule having enzymatic activity.
"압타머(aptamer)"는 표적분자에 특이적으로 결합하는 핵산(DNA, RNA, 변형핵산)으로 구성된 고분자를 뜻하며, 기존의 항원항체 반응과 같은 개념의 진단 및 의약품으로 인공적으로 대량생산이 가능하다."Aptamer" refers to a polymer composed of nucleic acids (DNA, RNA, modified nucleic acid) that specifically binds to a target molecule, and can be artificially mass-produced as a diagnostic and pharmaceutical product with the same concept as an antigen-antibody reaction. Do.
본 발명에서의 핵산효소는 과산화효소 활성을 가지며, 분자비콘과 함께 복합체를 형성하고 블로커 핵산과 결합함으로써 핵산효소가 불활성화된다. 이후, 블로커 핵산과 표적핵산이 결합하여 핵산효소-분자비콘 복합체가 활성화되어 ABTS의 산화를 통하여 발색반응을 나타낸다. The nucleic acid enzyme in the present invention has peroxidase activity, and the nuclease is inactivated by forming a complex with the molecular beacon and binding with the blocker nucleic acid. Thereafter, the blocker nucleic acid and the target nucleic acid bind to each other to activate the nucleic acid enzyme-molecule beacon complex, thereby displaying a color reaction through oxidation of ABTS.
본 발명에서 헤민은 핵산효소-분자비콘의 복합체의 양 말단 부분에 존재하여 스템구조를 형성하여 핵산효소-분자비콘이 과산화효소 활성을 가지게 한다.
In the present invention, hemin is present at both terminal portions of the complex of the nucleic acid enzyme-molecule beacon to form a stem structure so that the nucleic acid enzyme-molecular beacon has peroxidase activity.
이하, 본 발명을 구체적으로 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 도 1에서는 과산화효소 활성의 핵산효소를 이용하여 표적핵산을 검출하는 원리에 관한 것으로, 도 1의 A는 핵산효소-분자비콘 복합체와 블로커 핵산을 결합시켜 불활성의 복합체 프로브를 나타낸 것이다. 핵산효소-분자비콘 복합체는 표적핵산 서열의 일부를 루프 부분에 가지고 있고, 양 말단의 스템 구조를 통해서 두 가닥 과산화효소 활성의 핵산 효소를 형성할 수 있는 서열이 있다. 이 양 말단 부분은 헤민이 있을 경우에만 스스로 스템 구조를 형성하여 과산화효소 활성을 가지게 된다. 블로커 핵산은 핵산효소-분자비콘의 루프부분과 상보적인 서열, 표적핵산의 일부와 상보적인 서열 및 표적핵산 치환 프라이머와 결합 부위인 서열을 일부분으로 가지고 있다. 핵산효소-분자비콘 복합체는 블로커 핵산과 결합함으로써 핵산 효소가 불활성화되며, 표적핵산의 결합 부분인 서열이 핵산효소-분자비콘의 루프부분과 상보적인 서열에 비해 상대적으로 길게 설계되어, 표적핵산은 블로커 핵산과 더 강하게 결합함으로써, 핵산효소-분자비콘 복합체를 블로커 핵산으로부터 분리시켜 원래의 효소활성을 갖게 할 수 있다. 1 of the present invention relates to the principle of detecting a target nucleic acid using a peroxidase-enzymatic nucleic acid enzyme, Figure 1A shows the inactive complex probe by binding the nucleic acid enzyme-molecule beacon complex and the blocker nucleic acid. The nuclease-molecule beacon complex has a portion of the target nucleic acid sequence in the loop portion and has a sequence capable of forming a two-stranded peroxidase-activated nucleic acid enzyme through the stem structure at both ends. Both terminal portions form a stem structure by themselves only in the presence of hemin and have peroxidase activity. The blocker nucleic acid has a sequence complementary to the loop portion of the nuclease-molecule beacon, a sequence complementary to a portion of the target nucleic acid, and a sequence that is a binding site to the target nucleic acid substitution primer. The nucleic acid enzyme-molecule beacon complex is designed to be inactivated by binding to a blocker nucleic acid, and the sequence that is the binding portion of the target nucleic acid is designed to be relatively longer than the sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon. By binding more strongly with the blocker nucleic acid, the nuclease-molecule beacon complex can be separated from the blocker nucleic acid and retain its original enzymatic activity.
도 1의 B의 [1 단계]에서는 블로커 핵산과 표적핵산의 결합, 핵산효소-분자비콘 복합체의 활성화, 활성화된 핵산효소-분자비콘 복합체의 과산화효소 활성에 의한 ABTS(2,2'-azino-is-(3-ethylbenzothiazoline)-6-sulfonic acid)의 산화를 통하여 발색반응이 나타나고, [2 단계]에서는 표적핵산이 블로커 핵산과 결합함으로써 표적핵산 치환 프라이머와 결합 부위인 서열이 드러남에 따라 프라이머가 결합할 수 있게 되어, 핵산중합효소에 의한 DNA 생성반응이 진행된다. [3 단계]에서는 상기 [2 단계]에 따라 블로커 핵산의 상보적인 서열이 합성되어 블로커와 상보적인 핵산 쌍이 형성되면서 가닥치환 활성을 지닌 핵산 중합효소에 의해 표적핵산이 분리되는 단계이다. [4 단계]에서는 분리되어 나온 표적핵산이 새로운 신호발생과정에 사용되어 또 다른 핵산효소-분자비콘 복합체와 블로커 핵산의 복합체 프로브를 활성화시키는 단계에 관한 것이다. In step [B] of FIG. 1B, ABTS (2,2'-azino-) by binding of a blocker nucleic acid and a target nucleic acid, activation of a nucleic acid enzyme-molecular beacon complex, and peroxidase activity of an activated nucleic acid enzyme-molecular beacon complex The color reaction occurs through the oxidation of is- (3-ethylbenzothiazoline) -6-sulfonic acid), and in step [2], the target nucleic acid binds to the blocker nucleic acid, so that the primer is released as the binding site of the target nucleic acid substitution primer is revealed. It becomes possible to bind, and the DNA production | generation reaction by a nucleic acid polymerase advances. In step [3], the complementary sequence of the blocker nucleic acid is synthesized according to the step [2] to form a nucleic acid pair complementary to the blocker, and the target nucleic acid is separated by the nucleic acid polymerase having strand substitution activity. In
본 발명의 블로커 핵산은 가닥치환 활성을 가진 핵산 중합효소에 의한 중합반응을 통하여 신호 증폭을 유도하기 위하여 표적핵산 치환 프라이머와 결합하는 서열을 포함하는 것이 바람직하여, 상기 표적핵산 치환 프라이머 서열은 블로커 핵산과 핵산효소-분자비콘 복합체의 서열에도 변화를 주어 결과적으로 증폭 효율뿐만 아니라 핵산효소-분자비콘 복합체의 효소활성에도 영향을 준다.The blocker nucleic acid of the present invention preferably comprises a sequence which binds to a target nucleic acid substitution primer to induce signal amplification through a polymerization reaction by a nucleic acid polymerase having strand substitution activity. In addition, the sequence of the nuclease-molecule beacon complex is also changed, and as a result, it affects not only the amplification efficiency but also the enzyme activity of the nuclease-molecule beacon complex.
본 발명의 일 양태에서는 서열번호 1, 4, 7, 10 및 13의 핵산효소-분자비콘 복합체, 서열번호 2, 5, 8, 11 및 14의 블로커 핵산, 서열번호 3, 6, 9, 12 및 15의 표적핵산 치환 프라이머, 헤민, 핵산중합효소 및 dNTP가 함유된 핵산검출용액과 표적핵산을 포함하는 샘플을 혼합하여 가닥치환 활성을 가진 핵산중합효소를 통해 블로커 핵산에 상보적인 서열을 중합됨에 따라 블로커 핵산에 붙어있던 표적핵산이 분리되고, 블로커 핵산으로부터 분리되어진 표적핵산은 다른 불활성 핵산효소-분자비콘 복합체를 활성화 시키는데 사용된다. 분리된 표적핵산에 의해 활성화된 핵산효소-분자비콘 복합체를 포함하는 용액에 과산화효소 기질을 첨가하여 표적핵산의 흡광도의 세기를 측정한 결과, 표적핵산 치환 프라이머의 염기서열 길이가 줄어듦에 따라 흡광도의 세기가 증가한 것을 확인할 수 있었다.In one aspect of the invention, the nucleic acid enzyme-molecular beacon complexes of SEQ ID NOs: 1, 4, 7, 10, and 13, the blocker nucleic acids of SEQ ID NOs: 2, 5, 8, 11, and 14, SEQ ID NOs: 3, 6, 9, 12, and The nucleic acid detection solution containing the target nucleic acid substitution primer, hemin, nucleic acid polymerase and dNTP of 15 and a sample containing the target nucleic acid were mixed to polymerize the sequence complementary to the blocker nucleic acid through the nucleic acid polymerase having strand substitution activity. The target nucleic acid attached to the blocker nucleic acid is isolated, and the target nucleic acid isolated from the blocker nucleic acid is used to activate other inactive nucleic acid enzyme-molecular beacon complexes. As a result of measuring the intensity of absorbance of target nucleic acid by adding peroxide substrate to the solution containing the nucleic acid enzyme-molecule beacon complex activated by the isolated target nucleic acid, the absorbance of the absorbance was decreased as the base sequence length of the target nucleic acid substitution primer was reduced. It could be seen that the intensity increased.
본 발명의 다른 양태에서는 흡광도가 가장 높게 나타난 핵산효소-분자비콘과 블로커 핵산 복합체 프로브를 이용하여 핵산중합효소와 dNTP를 포함하는 경우와, 핵산중합효소와 dNTP를 포함하지 않는 경우를 각각 표적핵산의 농도에 따라 흡광도의 세기 변화를 측정한 결과, 표적핵산의 농도가 증가함에 따라 불활성 복합체 프로브로부터 분리된 핵산효소-분자비콘의 증가로 인해 흡광도 세기가 증가하였으나, 핵산중합효소와 dNTP를 포함하고 있는 경우에는 표적 핵산의 재사용에 의한 신호 증폭효과로 인해서 상대적으로 높은 흡광도의 세기를 나타내어, 중합반응의 유무에 의하여 신호의 세기가 달라진다는 것을 확인하였다.In another embodiment of the present invention, the nucleic acid polymerase-molecule beacon and the blocker nucleic acid complex probe having the highest absorbance are used to include the nucleic acid polymerase and the dNTP, and the nucleic acid polymerase and the dNTP, respectively. As a result of measuring the intensity change of absorbance according to the concentration, the absorbance intensity increased due to the increase of the nucleic acid enzyme-molecule beacon isolated from the inactive complex probe as the concentration of target nucleic acid increased, but it contained nucleic acid polymerase and dNTP. In the case, the signal amplification effect by the reuse of the target nucleic acid shows a relatively high intensity of absorbance, and it was confirmed that the intensity of the signal varies depending on the presence or absence of the polymerization reaction.
본 발명의 또 다른 양태에서는 표적핵산 농도에 따른 분석한계의 결정하기 위하여, 중합생성용액을 분석버퍼용액과 혼합한 후, pM~10nM의 표적핵산의 농도에서 흡광도 세기를 측정하여 발색 분석 반응을 실시한 결과, 흡광도는 표적핵산의 농도에 따라 선형적으로 증가한 것을 확인할 수 있었다.In another embodiment of the present invention, in order to determine the analysis limit according to the target nucleic acid concentration, the polymerization solution was mixed with the assay buffer solution, and then the absorbance intensity was measured at the concentration of the target nucleic acid of pM ~ 10nM to perform a color analysis reaction. As a result, it was confirmed that the absorbance increased linearly with the concentration of the target nucleic acid.
본 발명의 또 다른 양태에서는 표적핵산에 대해 높은 특이성을 분석하기 위하여, 표적핵산이 없는 음성시료, 표적핵산을 포함하는 시료, 표적핵산이 아닌 다른 핵산을 포함하는 시료를 분석한 결과, 표적핵산을 포함한 시료와 비교하여, 표적핵산이 없거나 또는 표적핵산이 아닌 다른 핵산이 존재하는 경우에는 최종 흡광도 세기가 배경신호인 헤민의 과산화효소 활성에 의한 것과 비슷한 수준으로 매우 낮게 나타남으로써, 본 검출방법은 표적핵산에 대해 높은 특이성이 있음을 확인할 수 있었다.In another embodiment of the present invention, in order to analyze high specificity for the target nucleic acid, a negative sample without the target nucleic acid, a sample containing the target nucleic acid, a sample containing a nucleic acid other than the target nucleic acid, Compared with the sample included, if the target nucleic acid is absent or other nucleic acid other than the target nucleic acid is present, the final absorbance intensity is very low, similar to that caused by the hemin peroxidase activity of the background signal. It was confirmed that there is a high specificity for the nucleic acid.
본 발명은 핵산효소-분자비콘 복합체/블로커 핵산의 복합체 프로브와 핵산중합효소에 의한 중합 반응을 이용한 신호증폭방법을 이용하여, 핵산을 고감도로 발색 분석하는 것으로, 본 발명을 통하여 1pM의 매우 낮은 농도의 표적핵산도 검출할 수 있고, 신호증폭에 사용된 가닥 치환 중합반응은 등온 상태에서도 가능하기 때문에 열적 사이클링이 필요하지 않은 장점이 있다.The present invention uses a signal amplification method using a complex probe of a nucleic acid enzyme-molecule beacon complex / blocker nucleic acid and a polymerization reaction by a nucleic acid polymerase. The target nucleic acid can also be detected, and since the strand substitution polymerization reaction used in the signal amplification is possible even in an isothermal state, there is an advantage that thermal cycling is not required.
본 발명은 압타머(aptamer) 서열을 포함하는 블로커 핵산을 이용하여 소분자 또는 단백질을 민감하게 검출할 수 있다. 압타머는 항체와 같은 역할을 하는 핵산분자로 서열 특이적으로 소분자 또는 단백질에 붙을 수 있다. 따라서, 압타머 서열을 가진 블로커 핵산은 핵산효소-분자비콘 복합체보다 소분자에 더 높은 친화력으로 붙기 때문에, 소분자가 존재시에 핵산효소-분자비콘/블로커 핵산의 복합체 프로브에서 분리되어 나오게 된다. 따라서 핵산효소는 활성을 가지게 되어 발색 신호를 낸다. 또한 본 발명에서 설명한 신호증폭방법을 이용하여, 블로커 핵산이 포함하는 프라이머와 상보적인 서열에 표적핵산 치환 프라이머가 붙을 수 있다. 그리고 핵산중합효소에 의해 중합반응이 일어남에 따라 블로커 핵산의 압타머 서열에 붙어있던 소분자가 떨어져 나와 다른 핵산효소-분자비콘 복합체/블로커 핵산 복합체를 활성화 시킨다. 이와 같은 원리에 따라 본 발명은 핵산 뿐만이 아니라 소분자 또는 단백질을 검출할 수 있다.
The present invention can sensitively detect small molecules or proteins using a blocker nucleic acid comprising an aptamer sequence. Aptamers are nucleic acid molecules that act like antibodies and can attach to small molecules or proteins in sequence. Therefore, since the blocker nucleic acid having an aptamer sequence attaches to a small molecule with a higher affinity than the nuclease-molecule beacon complex, it is separated from the complex probe of the nuclease-molecule beacon / blocker nucleic acid in the presence of the small molecule. Thus, nucleic acid enzymes become active and give off color signals. In addition, using the signal amplification method described in the present invention, a target nucleic acid substitution primer may be attached to a sequence complementary to a primer included in a blocker nucleic acid. As the polymerization reaction occurs by the nucleic acid polymerase, small molecules attached to the aptamer sequence of the blocker nucleic acid are released to activate other nucleic acid enzyme-molecule beacon complexes / blocker nucleic acid complexes. According to this principle, the present invention can detect not only nucleic acids but also small molecules or proteins.
이하, 본 발명을 실시예에 의하여 더욱 상세하게 설명한다. 이들 실시예는 단지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail by way of examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.
프라이머primer 길이에 따른 신호증폭효율 및 핵산효소- Signal Amplification Efficiency and Nucleic Acid Enzyme According to Length 분자비콘Molecular Beacon 효소활성 Enzyme activity
핵산효소(서열번호 1, 4, 7, 10 및 13), 블로커 핵산(서열번호 2, 5, 8, 11 및 14) 및 표적핵산 치환 프라이머(서열번호 3, 6, 9, 12 및 15)를 준비하고, 각각의 프라이머 길이에 따른 최종표적 핵산의 검출을 측정하기 위하여, 50μM의 핵산효소-분자비콘 복합체/블로커 핵산의 복합체 프로브 1㎕, 각각의 50μM의 프라이머 1㎕, 표적핵산 용액 1㎕, 25μM 헤민 1㎕, 핵산중합효소(Klenow fragment exo-) 1.6㎕, 0.2mM의 dNTP 2㎕, 8.4㎕ 물 및 2㎕ NEB (New England Biolabs) 버퍼 2를 이용하여 핵산검출용액을 제조하였다. 비뇨생식기 감염 균인 Ureaplasma urealyticum 유전자 서열의 일부분을 표적핵산으로 사용하여, 상기 핵산검출용액과 혼합하여 37℃에서 2시간 동안 중합반응을 실시하여 중합생성용액을 수득하였다. Nucleic acid enzymes (SEQ ID NOs: 1, 4, 7, 10, and 13), blocker nucleic acids (SEQ ID NOs: 2, 5, 8, 11, and 14) and target nucleic acid substitution primers (SEQ ID NOs: 3, 6, 9, 12, and 15) To prepare and measure the detection of the final target nucleic acid according to each primer length, 1 μl of a complex probe of 50 μM of nuclease-molecule beacon complex / blocker nucleic acid, 1 μl of each 50 μM of primer, 1 μl of target nucleic acid solution, The nucleic acid detection solution was prepared using 1 μl of 25 μM hemin, 1.6 μl of Klenow fragment exo-, 2 μl of 0.2 mM dNTP, 8.4 μl water and 2 μl NEB (New England Biolabs)
수득한 중합생성용액을 분석버퍼용액(25mM HEPES, 20nM KCL, 200mM NaCl, 0.05% Triton X-100, 1% DMSO)과 혼합하고, 혼합 용액을 1시간 동안 실내온도에 보관한 뒤, ABTS/H2O2 기질 용액을 상기의 중합생성용액과 분석버퍼용액의 혼합액과 혼합하고, 10분의 반응 후에 분광광도계를 이용해 415㎚에서의 흡광도 세기를 측정하였다. 그 결과, 프라이머 염기 서열의 길이가 12개(서열번호 3)에서 9개(서열번호 12)로 줄어듦에 따라 흡광도 세기가 증가함을 알 수 있었으나, 8개의 염기로 이루어진 프라이머(서열번호 15)의 경우, 흡광도의 세기는 9개의 염기로 이루어진 프라이머에 비하여 오히려 감소한 것을 확인하였다 (도 2).
The resulting polymerization solution was mixed with analytical buffer solution (25 mM HEPES, 20 nM KCL, 200 mM NaCl, 0.05% Triton X-100, 1% DMSO), the mixed solution was stored at room temperature for 1 hour, and then ABTS / H The 2 O 2 substrate solution was mixed with the mixed solution of the polymerization production solution and the analysis buffer solution, and the absorbance intensity at 415 nm was measured using a spectrophotometer after 10 minutes of reaction. As a result, it was found that the absorbance increased as the length of the primer base sequence decreased from 12 (SEQ ID NO: 3) to 9 (SEQ ID NO: 12). In this case, it was confirmed that the intensity of absorbance decreased rather than the primer consisting of 9 bases (FIG. 2).
표적핵산의 농도 및 핵산중합효소와 Concentration of target nucleic acid and nucleic acid polymerase dNTPdNTP 유무에 따른 변화 Change with or without
실시예 1에서 가장 높은 흡광도를 가지는 서열번호 10의 핵산효소-분자비콘/블로커 핵산 복합체를 이용하여 핵산중합효소와 dNTP를 포함하는 경우(◆)와 포함하지 않는 경우(■)에 각각의 표적 핵산 농도 (10pM ~ 100nM)에 따른 흡광도 세기의 변화를 측정하였다.Each target nucleic acid in the case of containing (◆) and not containing (■) a nucleic acid polymerase and dNTP using the nucleic acid enzyme-molecule beacon / blocker nucleic acid complex of SEQ ID NO: 10 having the highest absorbance in Example 1 The change in absorbance intensity with concentration (10 pM-100 nM) was measured.
상기의 서열번호 10의 핵산효소, 서열번호 11의 블로커 및 서열번호 12의 프라이머를 사용하여 제조한 핵산효소-분자비콘/블로커 핵산 복합체 프로브를 상기 실시예 1에서 사용한 동일한 조성의 핵산검출용액에 혼합하였다. 이때 핵산검출용액을 각각의 표적핵산농도에 맞도록 표적핵산과 혼합한 뒤 37℃에서 2시간 동안 중합시켜 중합생성용액을 제조하였다. 수득한 중합생성용액과 분석버퍼용액(25mM HEPES, 20nM KCL, 200mM NaCl, 0.05% Triton X-100, 1% DMSO)을 혼합하여 1시간 동안 실내온도에서 보관한 후, 발색 반응 분석을 실시하였다. The nucleic acid enzyme-molecule beacon / blocker nucleic acid complex probe prepared using the nucleic acid enzyme of SEQ ID NO: 10, the blocker of SEQ ID NO: 11 and the primer of SEQ ID NO: 12 was mixed with the nucleic acid detection solution of the same composition used in Example 1 above. It was. At this time, the nucleic acid detection solution was mixed with the target nucleic acid to meet the respective target nucleic acid concentration, and then polymerized at 37 ° C. for 2 hours to prepare a polymerization solution. The obtained polymerization product solution and the assay buffer solution (25 mM HEPES, 20 nM KCL, 200 mM NaCl, 0.05% Triton X-100, 1% DMSO) were mixed and stored at room temperature for 1 hour, followed by color reaction analysis.
그 결과, 핵산 농도가 증가함에 따라 표적핵산에 의해 불활성 복합체 프로브로부터 분리된 핵산효소-분자비콘의 증가로 인해 흡광도 세기가 증가하였다 (도 3). 그러나 핵산중합효소와 dNTP를 포함하고 있는 경우에는 표적 핵산의 재사용에 의한 신호 증폭효과로 인해서 상대적으로 매우 높은 흡광도를 나타냈으므로, 본 결과는 가닥 치환 활성을 가지는 핵산 중합효소에 의한 핵산중합반응을 통해서 표적핵산이 블로커 핵산으로부터 분리되고 새로운 핵산효소 활성에 재사용됨으로써 신호를 증폭시킨 것을 확인할 수 있었다.
As a result, the absorbance intensity increased due to the increase of the nucleic acid enzyme-molecular beacon isolated from the inactive complex probe by the target nucleic acid as the nucleic acid concentration increased (FIG. 3). However, in the case of containing nucleic acid polymerase and dNTP, the absorbance of the target nucleic acid was relatively high due to the signal amplification effect by the reuse of the target nucleic acid. Through this, it was confirmed that the target nucleic acid was separated from the blocker nucleic acid and reused for new nucleic acid enzyme activity, thereby amplifying the signal.
표적핵산 농도에 따른 분석한계의 결정Determination of Analysis Limits According to Target Nucleic Acid Concentration
상기 실시예 1에서 사용한 동일한 조성의 핵산검출용액과 각각의 농도에 해당하는 표적핵산용액을 혼합한 뒤 37℃에서 2 시간 동안 중합하였다. 이렇게 얻어진 중합생성용액을 분석버퍼용액(25mM HEPES, 20nM KCL, 200mM NaCl, 0.05% Triton X-100, 1% DMSO)과 혼합하여 실내온도에서 1시간 보관한 후, pM~10nM의 표적핵산의 농도에서 흡광도 세기를 측정하여 발색 분석 반응을 실시하였다.The nucleic acid detection solution of the same composition used in Example 1 and the target nucleic acid solution corresponding to each concentration were mixed and then polymerized at 37 ° C. for 2 hours. The polymerization solution thus obtained was mixed with analytical buffer solution (25 mM HEPES, 20 nM KCL, 200 mM NaCl, 0.05% Triton X-100, 1% DMSO), stored at room temperature for 1 hour, and then the concentration of target nucleic acid of pM ~ 10nM Absorbance intensity was measured at and colorimetric analysis was performed.
그 결과, 흡광도는 표적핵산의 농도에 따라 선형적으로 증가함을 확인하였으며, 본 발명에서는 다른 분자비콘을 이용한 이전 핵산 검출방법에 비해 현저히 낮은 값인 1pM에서 분석한계의 값을 갖는 것을 확인할 수 있었다.
As a result, the absorbance was confirmed to increase linearly with the concentration of the target nucleic acid, the present invention was confirmed to have a value of the analysis limit at 1pM, which is significantly lower than the previous nucleic acid detection method using other molecular beacons.
표적핵산에 대한 특이성 분석Specificity Analysis for Target Nucleic Acids
본 발명의 특이성을 측정하기 위하여, 표적핵산이 없는 음성시료, 표적핵산인 Urea 핵산(100nM)을 포함하는 시료, 표적핵산이 아닌 다른 핵산(100nM)을 포함하는 시료를 분석한 결과 얻어지는 흡광도를 측정하였다. 이때, 표적핵산이 아닌 핵산으로 Chlamydia trachomatis 유전자 서열의 일부가 사용되었다. 상기 실시예 1과 동일한 방법으로, 핵산검출용액에 Urea 핵산을 넣은 시료와 Chlamydia 핵산을 넣은 시료, 표적핵산을 넣지 않은 시료를 37℃에서 2시간 동안 중합한 뒤, 분석버퍼용액에 혼합하여 분광광도계를 이용해 415㎚에서의 흡광도를 측정하였다.In order to measure the specificity of the present invention, the absorbance obtained by analyzing a negative sample without a target nucleic acid, a sample containing a target nucleic acid Urea nucleic acid (100 nM), a sample containing a nucleic acid other than the target nucleic acid (100 nM) is measured It was. At this time, C hlamydia as a nucleic acid rather than a target nucleic acid Part of the trachomatis gene sequence was used. In the same manner as in Example 1, a sample containing Urea nucleic acid, a sample containing Chlamydia nucleic acid, and a sample without target nucleic acid were polymerized at 37 ° C. for 2 hours in a nucleic acid detection solution, and then mixed in an assay buffer solution. Absorbance at 415 nm was measured using.
그 결과, Urea 표적핵산이 없거나 또는 표적핵산이 아닌 다른 핵산이 존재하는 경우에는 최종 흡광도 세기가 배경신호인 헤민의 과산화효소 활성에 의한 것과 비슷한 수준으로 매우 낮게 나타났고, 표적핵산인 Urea 분석결과와는 명확히 구분됨을 알 수 있었다. 따라서, 본 발명의 검출방법은 표적핵산에 대해 높은 특이성을 가지고 있음을 확인할 수 있었다 (도 5).
As a result, in the absence of Urea target nucleic acid or in the presence of a nucleic acid other than the target nucleic acid, the final absorbance intensity was very low, similar to that caused by the hemin peroxidase activity of the background signal. Was clearly distinguished. Therefore, the detection method of the present invention was confirmed to have a high specificity for the target nucleic acid (Fig. 5).
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
<110> Korea Advanced Institute of Science and Technology <120> Method for Detecting Nucleic Acid Using DNAzyme Molecular Beacon <130> P10-B278 <160> 15 <170> KopatentIn 1.71 <210> 1 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> DNAzymeMB <400> 1 agggacggga gacacagaag taatggactt agttattagt ggagggt 47 <210> 2 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> blocker <400> 2 taatgcgttt gtaataacta agtccattac ttctgtgtct cccgt 45 <210> 3 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 acgggagaca ca 12 <210> 4 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> DNAzymeMB <400> 4 agggacggga gacacgaagt aatggactta gttattagtg gagggt 46 <210> 5 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> blocker <400> 5 taatgcgttt gtaataacta agtccattac ttcgtgtctc ccgt 44 <210> 6 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 acgggagaca c 11 <210> 7 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> DNAzymeMB <400> 7 agggacggga gacagaagta atggacttag ttattagtgg agggt 45 <210> 8 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> blocker <400> 8 taatgcgttt gtaataacta agtccattac ttctgtctcc cgt 43 <210> 9 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 acgggagaca 10 <210> 10 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> DNAzymeMB <400> 10 agggacggga gacgaagtaa tggacttagt tattagtgga gggt 44 <210> 11 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> blocker <400> 11 taatgcgttt gtaataacta agtccattac ttcgtctccc gt 42 <210> 12 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 acgggagac 9 <210> 13 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> DNAzymeMB <400> 13 agggacggga gagaagtaat ggacttagtt attagtggag ggt 43 <210> 14 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> blocker <400> 14 taatgcgttt gtaataacta agtccattac ttctctcccg t 41 <210> 15 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 acgggaga 8 <110> Korea Advanced Institute of Science and Technology <120> Method for Detecting Nucleic Acid Using DNAzyme Molecular Beacon <130> P10-B278 <160> 15 <170> Kopatentin 1.71 <210> 1 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> DNAzyme MB <400> 1 agggacggga gacacagaag taatggactt agttattagt ggagggt 47 <210> 2 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> blocker <400> 2 taatgcgttt gtaataacta agtccattac ttctgtgtct cccgt 45 <210> 3 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 acgggagaca ca 12 <210> 4 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> DNAzyme MB <400> 4 agggacggga gacacgaagt aatggactta gttattagtg gagggt 46 <210> 5 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> blocker <400> 5 taatgcgttt gtaataacta agtccattac ttcgtgtctc ccgt 44 <210> 6 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 acgggagaca c 11 <210> 7 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> DNAzyme MB <400> 7 agggacggga gacagaagta atggacttag ttattagtgg agggt 45 <210> 8 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> blocker <400> 8 taatgcgttt gtaataacta agtccattac ttctgtctcc cgt 43 <210> 9 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 acgggagaca 10 <210> 10 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> DNAzyme MB <400> 10 agggacggga gacgaagtaa tggacttagt tattagtgga gggt 44 <210> 11 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> blocker <400> 11 taatgcgttt gtaataacta agtccattac ttcgtctccc gt 42 <210> 12 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 acgggagac 9 <210> 13 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> DNAzyme MB <400> 13 agggacggga gagaagtaat ggacttagtt attagtggag ggt 43 <210> 14 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> blocker <400> 14 taatgcgttt gtaataacta agtccattac ttctctcccg t 41 <210> 15 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 acgggaga 8
Claims (14)
(a) i) 핵산효소-분자비콘 복합체;
ii) 한쪽 말단에 상기 핵산효소-분자비콘 복합체의 루프부분과 상보적인 서열을 가지고, 다른 말단에 표적핵산의 일부와 상보적인 서열을 가지며, 어느 한 말단에 표적핵산 치환 프라이머와 상보적인 서열을 함유하는 블로커 핵산;
iii) 핵산중합효소 연쇄반응시 블로커 핵산에 붙어있는 표적핵산을 분리시키고, 대신에 블로커 핵산에 상보적인 서열을 가지는 핵산으로 치환시키는 표적핵산 치환 프라이머;
iv) 헤민;
v) 핵산중합효소; 및
vi) dNTP가 함유된 핵산검출용액에 표적핵산을 함유하는 샘플을 혼합하고, 핵산중합효소 연쇄반응시켜 표적핵산을 블로커 핵산으로부터 분리시키는 단계; 및
(b) 상기 분리된 표적핵산에 의해 활성화된 핵산효소-분자비콘 복합체를 포함하는 용액에 과산화효소 기질을 첨가하여 표적핵산을 검출하는 단계.
A nucleic acid detection method comprising the following steps:
(a) i) a nucleic acid enzyme-molecular beacon complex;
ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, complementary to a portion of the target nucleic acid at the other end, and contains a sequence complementary to the target nucleic acid substitution primer at either end Blocker nucleic acid;
iii) a target nucleic acid substitution primer that separates a target nucleic acid attached to a blocker nucleic acid during a nucleic acid polymerase chain reaction, and instead substitutes a nucleic acid having a sequence complementary to the blocker nucleic acid;
iv) hemin;
v) nucleic acid polymerase; And
vi) mixing the sample containing the target nucleic acid with the nucleic acid detection solution containing dNTP, and nucleic acid polymerase chain reaction to separate the target nucleic acid from the blocker nucleic acid; And
(b) detecting the target nucleic acid by adding a peroxide enzyme substrate to a solution containing the nucleic acid enzyme-molecule beacon complex activated by the separated target nucleic acid.
The method of claim 1, wherein the nucleotide sequence of the nucleic acid enzyme-molecule beacon complex is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 13 .
According to claim 1, wherein the peroxidase substrate is ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], TMB (3,3 ', 5,5'-tetramethyl benzidine) and OPD (o-phenylenediamine) A method for detecting a nucleic acid, characterized in that selected from the group consisting of.
A target nucleic acid that has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, and a sequence complementary to a portion of the target nucleic acid at the other end, and is attached to the blocker nucleic acid in the nucleic acid polymerase chain reaction at either end. A blocker nucleic acid containing a sequence complementary to a target nucleic acid substitution primer that isolates and replaces with a nucleic acid having a sequence complementary to the blocker nucleic acid.
The blocker nucleic acid according to claim 4, wherein the nucleotide sequence of the nucleic acid enzyme-molecule beacon complex is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 13.
i) nucleic acid enzyme-molecular beacon complexes; ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, a sequence complementary to a portion of the target nucleic acid at the other end, and contains a sequence complementary to the target nucleic acid substitution primer at either end Blocker nucleic acid; iii) a target nucleic acid substitution primer that separates a target nucleic acid attached to a blocker nucleic acid during a nucleic acid polymerase chain reaction, and instead substitutes a nucleic acid having a sequence complementary to the blocker nucleic acid; iv) hemin; v) nucleic acid polymerase; And vi) a nucleic acid detection solution containing dNTP.
The nucleic acid detection solution according to claim 6, wherein the nucleotide sequence of the nucleic acid enzyme-molecule beacon complex is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 13.
(a) i) 핵산효소-분자비콘 복합체;
ii) 한쪽 말단에 상기 핵산효소-분자비콘 복합체의 루프부분과 상보적인 서열을 가지고, 다른 말단에 표적 소분자 또는 단백질과 결합능을 가지는 압타머 서열을 가지며, 어느 한 말단에 표적 소분자 또는 단백질 치환 프라이머와 상보적인 서열을 함유하는 블로커 핵산;
iii) 핵산중합효소 연쇄반응시 블로커 핵산에 붙어있는 소분자 또는 단백질을 분리시키고, 대신에 블로커 핵산에 상보적인 서열을 가지는 핵산으로 치환시키는 표적 소분자 또는 단백질 치환 프라이머;
iv) 헤민;
v) 핵산중합효소; 및
vi) dNTP가 함유된 소분자 또는 단백질검출용액에 표적 소분자 또는 단백질을 함유하는 샘플을 혼합하고, 핵산중합효소 연쇄반응시켜 표적 소분자 또는 단백질을 블로커 핵산으로부터 분리시키는 단계; 및
(b) 상기 분리된 표적 소분자 또는 단백질에 의해 활성화된 핵산효소-분자비콘 복합체를 포함하는 용액에 과산화효소 기질을 첨가하여 표적 소분자 또는 단백질을 검출하는 단계.
Method of detecting small molecule or protein comprising the following steps:
(a) i) a nucleic acid enzyme-molecular beacon complex;
ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, and has an aptamer sequence at the other end having a binding capacity with a target small molecule or protein, and at either end with a target small molecule or protein substitution primer Blocker nucleic acids containing complementary sequences;
iii) a target small molecule or protein substitution primer that separates the small molecule or protein attached to the blocker nucleic acid in the nucleic acid polymerase chain reaction and replaces it with a nucleic acid having a sequence complementary to the blocker nucleic acid;
iv) hemin;
v) nucleic acid polymerase; And
vi) mixing a sample containing a target small molecule or protein with a small molecule or protein detection solution containing dNTP, and separating the target small molecule or protein from the blocker nucleic acid by nucleic acid polymerase chain reaction; And
(b) detecting the target small molecule or protein by adding a peroxide enzyme substrate to the solution containing the nucleic acid enzyme-molecule beacon complex activated by the separated target small molecule or protein.
The small molecule or protein of claim 8, wherein the nucleotide sequence of the nucleic acid enzyme-molecule beacon complex is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 13 Detection method.
The method of claim 8, wherein the peroxidase substrate is ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], TMB (3,3 ', 5,5'-tetramethyl benzidine) and OPD A method for detecting a small molecule or protein, characterized in that selected from the group consisting of (o-phenylenediamine).
It has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, and has an aptamer sequence at the other end that has a binding capacity with a target small molecule or protein, and attaches to the blocker nucleic acid during the nucleic acid polymerase chain reaction at either end. A blocker nucleic acid containing a sequence complementary to a target small molecule or protein substitution primer that isolates a small molecule or protein that is present and instead replaces it with a nucleic acid having a sequence complementary to the blocker nucleic acid.
The blocker nucleic acid according to claim 11, wherein the nucleotide sequence of the nucleic acid enzyme-molecule beacon complex is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 13.
i) nucleic acid enzyme-molecular beacon complexes; ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, and has an aptamer sequence at the other end having a binding capacity with a target small molecule or protein, and at either end with a target small molecule or protein substitution primer Blocker nucleic acids containing complementary sequences; iii) a target small molecule or protein substitution primer that separates the small molecule or protein attached to the blocker nucleic acid in the nucleic acid polymerase chain reaction and replaces it with a nucleic acid having a sequence complementary to the blocker nucleic acid; iv) hemin; v) nucleic acid polymerase; And vi) a small molecule or protein detection solution containing dNTP.
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