DNA过氧化物酶的活性及在生物检测中应用的研究
摘要
DNA是生命体内十分重要的功能大分子,是绝大多数生物物种的遗传信息的载体。DNA具有不同的二级结构,包括三螺旋DNA、G-四链体和i-motif序列等。探讨DNA的结构与功能的关系具有十分重要的意义,可以加深对生命本质的认识以及拓展DNA在各个领域的应用。
DNA过氧化物酶是由G-四链体DNA与氯高铁血红素形成的复合物,具有过氧化物酶的活性,能够催化双氧水(H2O2)氧化2,2'-联氮-双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)发生显色反应。本论文研究了DNA过氧化物酶的结构与功能的关系,并设计了一种基于DNA过氧化物酶的检测腺苷的生物传感器。
1.通过对DNA过氧化物酶的DNA序列的末端进行突变的策略,利用紫外光谱,圆二色谱等实验手段,本文较系统地研究了DNA过氧化物酶的结构与酶活力的关系。我们发现在DNA过氧化物酶的DNA序列的末端增加核苷dTn会对其酶的活性发生较大的影响。当组装成的DNA过氧化物酶的结构越稳定,越有利于酶与底物(H2O2)间的结合,DNA过氧化物酶表现出的活性就越高,反之则越低。
2.进而我们设计了一种基于DNA过氧化物酶的生物传感器,它是以核酸适配子为识别分子,以Fok I限制性内切酶为信号控制器,以DNA过氧化物酶为信号输出装置。该DNA生物传感器具有很好的灵敏度和选择性,只对腺苷有识别作用,且其最低检测下限可达500nM,对于其他核苷则无响应。此生物传感器能通过酶促反应的显色效果实现可视化检测,而无需依赖于仪器。
DNA is an important functional macromolecule in the life, as the carrier of genetic information in the vast majority of biological species. There are different secondary structures of DNA, including triple helix DNA, G-quadruplex and i-motif sequences, etc.. It is of great significance to investigate the relationship between DNA structure and function, which could help us to deepen the understanding on the essence of life and expand the applications of DNA in various fields.
DNAzyme, formed by hemin and G-quadruplex DNA, has demonstrated peroxidase-like activities, and it can catalyze the oxidation of 2,2'-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid (ABTS) by H2O2 as color-changed reaction. This dissertation studied the relationship between the structure and function of DNAzyme, and designed a DNAzyme-based biosensor for the detection of adenosine.
1. By the strategy that mutated the end of the DNA sequence of DNAzyme, using ultraviolet spectroscopy, circular dichroism and other experimental methods, this paper systematically studied the relationship between the structure of DNAzyme and its peroxidase activity. We found that adding dTn to the end of the DNA sequence of the enzyme would make great influence on the peroxidase activity. And the more stable the assembled DNAzyme was, the more conducive the binding of enzyme and substrate (H2O2) could be, which showed a higher peroxidase activity, and vice versa, the lower.
2. And then we designed a DNAzyme-based biosensor that employed the aptamer as the receptor, restriction enzyme Fok I as the signal controller, DNAzyme as the resultant output device. This DNA biosensor had good sensitivity and selectivity, only to have identified the present of adenosine with the limit of detection (LOD) of 0.5μM, and there was no response to other nucleosides. This biosensor can achieve the optical detection through the color change of the enzymatic reaction, instead of relying on the instrument.
DNA过氧化物酶是由G-四链体DNA与氯高铁血红素形成的复合物,具有过氧化物酶的活性,能够催化双氧水(H2O2)氧化2,2'-联氮-双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)发生显色反应。本论文研究了DNA过氧化物酶的结构与功能的关系,并设计了一种基于DNA过氧化物酶的检测腺苷的生物传感器。
1.通过对DNA过氧化物酶的DNA序列的末端进行突变的策略,利用紫外光谱,圆二色谱等实验手段,本文较系统地研究了DNA过氧化物酶的结构与酶活力的关系。我们发现在DNA过氧化物酶的DNA序列的末端增加核苷dTn会对其酶的活性发生较大的影响。当组装成的DNA过氧化物酶的结构越稳定,越有利于酶与底物(H2O2)间的结合,DNA过氧化物酶表现出的活性就越高,反之则越低。
2.进而我们设计了一种基于DNA过氧化物酶的生物传感器,它是以核酸适配子为识别分子,以Fok I限制性内切酶为信号控制器,以DNA过氧化物酶为信号输出装置。该DNA生物传感器具有很好的灵敏度和选择性,只对腺苷有识别作用,且其最低检测下限可达500nM,对于其他核苷则无响应。此生物传感器能通过酶促反应的显色效果实现可视化检测,而无需依赖于仪器。
DNA is an important functional macromolecule in the life, as the carrier of genetic information in the vast majority of biological species. There are different secondary structures of DNA, including triple helix DNA, G-quadruplex and i-motif sequences, etc.. It is of great significance to investigate the relationship between DNA structure and function, which could help us to deepen the understanding on the essence of life and expand the applications of DNA in various fields.
DNAzyme, formed by hemin and G-quadruplex DNA, has demonstrated peroxidase-like activities, and it can catalyze the oxidation of 2,2'-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid (ABTS) by H2O2 as color-changed reaction. This dissertation studied the relationship between the structure and function of DNAzyme, and designed a DNAzyme-based biosensor for the detection of adenosine.
1. By the strategy that mutated the end of the DNA sequence of DNAzyme, using ultraviolet spectroscopy, circular dichroism and other experimental methods, this paper systematically studied the relationship between the structure of DNAzyme and its peroxidase activity. We found that adding dTn to the end of the DNA sequence of the enzyme would make great influence on the peroxidase activity. And the more stable the assembled DNAzyme was, the more conducive the binding of enzyme and substrate (H2O2) could be, which showed a higher peroxidase activity, and vice versa, the lower.
2. And then we designed a DNAzyme-based biosensor that employed the aptamer as the receptor, restriction enzyme Fok I as the signal controller, DNAzyme as the resultant output device. This DNA biosensor had good sensitivity and selectivity, only to have identified the present of adenosine with the limit of detection (LOD) of 0.5μM, and there was no response to other nucleosides. This biosensor can achieve the optical detection through the color change of the enzymatic reaction, instead of relying on the instrument.
引文
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