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| 基于释放ATP的核苷酸探针的新型核酸检测技术 的开发
摘要
可以释放ATP的核苷酸探针（ATP-Releasing Nucleotides，ARNs）能够替代天然的脱氧核苷酸进行DNA聚合酶催化下的引物延伸反应，而且在引物延伸过程中会产生ATP。将ARNs与已有的核酸检测信号放大技术相结合，通过检测ATP分子就能够实现对靶标物的超灵敏检测。其中酶辅助的检测信号放大策略和分子信标相结合可以实现对检测信号的级联放大。基于上述内容，本论文利用 “桥梁”ATP转化为荧光信号或者化学发光信号的原理，设计了两种生物传感系统，并且实现了对靶标物质的超灵敏检测。
（1）本文开发了一种新的合成ARNs的方法，该方法规避了通过原料引入ATP的缺点，并通过该方法合成了三种核苷酸分子探针分别是dG-P4-A、dA-P4-A、dT-P4-A。
（2）利用ATP是T4 DNA 连接酶的辅酶这一性质，将ARNs与分子信标、T4 DNA连接酶、聚合酶相结合构建了一种超灵敏检测ATP的方法。已有的方法，
T4 DNA连接酶利用ATP将两条短的DNA片段连接，打开了分子信标的发卡结构，产生荧光信号。这种方法理论上一分子ATP可以产生一分子的荧光信号。我们在上述反应体系中加入ARNs原料，在DNA聚合酶的作用下进行酶反应，同时释放出ATP，产生的ATP继续被用于T4 DNA 连接酶的反应，打开更多的分子信标。通过以上设计，实现了一个ATP的循环系统，通过聚合酶实现了对ATP数量的指数式扩增。理论上，该系统可以通过一个ATP分子打开全部的分子信标。实验结果表明：该方法检测灵敏度达到了2.29 pM，比已有方法提高了近两个数量级，实现了对ATP的超灵敏检测。
（3）将ARNs与链置换扩增技术、发卡结构相互结合，在DNA聚合酶和限制性核酸内切酶的催化作用下，构建了一个可以用于超灵敏检测核酸链的生物传感系统。该传感系统设计了发卡结构HP1和HP2两个探针，并且HP1与HP2的3´端能够碱基互补配对。当无目标链存在时，HP1和HP2处于发卡结构状态；当目标链存在时，HP1（或HP2）与之特异性结合并处于打开状态，打开的HP1（或HP2）与HP2（或HP1）杂交，形成一个三元复合体，该复合体在DNA聚合酶和核酸内切酶的催化下持续发生链置换反应；将ARNs作为上述反应的原料，反应结束后体系内产生ATP，随后利用ATP生物发光法（萤光素酶和萤光素）读取检测信号。上述传感系统与核酸适配体技术相结合，进一步实现了对黄曲霉毒素B1（AFB1）的灵敏检测。
Abstract
ATP-releasing nucleotides（ARNs) can replace natural deoxynucleotides to perform primer extensionreactions catalyzed by DNA polymerase, and ATP is generated during the primer extension process. Combining ARNs with the existing nucleic acid detection signal amplification technology can achieve ultra-sensitive detection of targets by detecting ATP molecules. The combination of enzyme-assisted detection signal amplification strategy and molecular beacon can realize the cascade amplification of detection signals. Based on the above content,this paper uses the principle of "bridged" ATP to convert it into a fluorescent signal or a bioluminescence signal, and designs two biosensing systems to achieve ultra-sensitive detection of target substances.
(1) The paper developed a new method of synthesizing ARNs,which avoids the shortcomings of introducing ATP throughraw materials, and synthesized three kinds of nucleotide molecular probes by this method, namely dG-P4-A and dA-P4-A, dT-P4-A.
(2) Taking advantage of the property that ATP is the coenzyme of T4DNA ligase, combining ARNs with molecular beacons, T4 DNA ligase,and polymerase to construct an ultra-sensitive method for detecting ATP. In the existing method, T4 DNA ligase uses ATP to connect two short DNA fragments toopen the hairpin structure of the molecular beacon and generate a fluorescent signal. In this way, in theory, one molecule of ATP can generate one molecule of fluorescent signal.We add ARNs raw materials to the above reaction system,perform enzymatic reaction under the action of DNA polymerase, and release ATP at the same time, and the generated ATP will continue to be used in the reaction of T4 DNA ligase to open more molecular beacons. Through the above design, an ATP circulation system is realized,and the exponential amplification of the ATP quantity is realized by polymerase. In theory, the system can open all molecular beacons with one ATP molecule.The experimental results show that the detection sensitivity of this method has reached 2.29 pM, which is nearly two orders of magnitude higher than that of the existing methods, and it has realized the ultra-sensitive detection of ATP.
ra-sensitive detection of ATP.
(3) Combining ARNs with strand displacement amplification technology and hairpin structure, under the catalysis of DNA polymerase and restriction endonuclease,a biosensing system that can be used for ultra-sensitive detection of nucleic acid strands is constructed. The sensor system is designed with two probes of hairpin structure HP1 andHP2, and the 3´ ends of HP1 and HP2 can be paired with complementary bases. When the target chain exists, HP1 (or HP2) specifically binds to it and is in an open state.The opened HP1 (or HP2) hybridizes with HP2 (or HP1) to form a ternary complex.Continuous strand displacement reaction catalyzed by DNA polymerase and endonuclease;using ARNs as the raw material for the above reaction,ATP is produced in the system after the reaction, and then used ATP bioluminescence method (luciferase and luciferin) reads the detection signal. The combination of the above-mentioned sensing system and nucleic acid aptamer technology further realizes the sensitive detection of aflatoxin B1 (AFB1).
Key words: Nucleotide probe; ATP;Molecular beacons；Signal amplification； Biosensing system;
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