DNA纳米机器在生物检测中的应用及G-四链体Toehold结构对DNA链交换反应的影响
发布时间：2018-10-19 16:50
【摘要】：随着DNA纳米技术的不断发展和进步,越来越多的方法出现并应用于生物检测,临床诊断,药物治疗,计算化学等领域。现代生物传感器需要高灵敏度,极大的信号增强和显著的选择性来达到检测和诊断的目的。传统的分子信标无法进行信号放大,这极大的限制了它的适用范围。利用酶,DNA酶和纳米粒子的信号放大的过程通常是相当复杂的,并限于特定的应用范围。在本文中,我们设计了自动的DNA扩增系统来构建基于传统分子信标结构的崭新的和简便的生物传感器。通过这种纳米机器的杂交扩增,这种经过改进的可扩增分子信标可以大大提高探测信号和检出限,并能应用于核酸检测,区分单碱基错配。通过引入适配体序列,这种可扩增分子信标也可以检测蛋白质和小分子,比如对β-肌动蛋白基因和凝血酶的检测。我们同时进一步证实了可扩增分子信标具有较高的灵敏度和稳定性,其扩增能力为在活细胞中检测miRNA提供了可能。可扩增分子信标的简单性和灵活性使它具有更广泛的应用前景。Toehold结构,作为链交换反应的核心,具有举足轻重的作用。研究人员针对toehold的长度,与链交换区域的距离,位置等要素做了许多工作。不过,在大多数情况下,toehold结构始终被默认为双链结构。在本论文中,我们改变了它的基本构型和链交换机理,设计了一个仅仅以DNA G-四链体为toehold结构诱导的DNA链交换反应。当反应环境中加入PEG时,这种分子拥挤试剂使得反应可以容易的发生,我们可以通过调节PEG的浓度和聚合度来控制反应,同时也可以通过改变G-四链体的结构来进行调节。同时我们还在迁移链中引入了错配的碱基,使链交换反应在水溶液中也可以顺利进行。紧接着我们还设计了基于这种特殊链交换的DNA链式扩增反应,通过G-四链体这一结构的特征实现无需荧光标记的检测体系,这种方法具有成本低,无需标记,灵敏度高和选择性强的特性,具有在DNA的纳米机器,生物传感和疾病诊断方面的应用潜力。
[Abstract]:With the development and progress of DNA nanotechnology, more and more methods have been developed and applied in the fields of biological detection, clinical diagnosis, drug therapy, computational chemistry and so on. Modern biosensors require high sensitivity, great signal enhancement and significant selectivity for detection and diagnosis. Traditional molecular beacons can not amplify signals, which greatly limits its scope of application. The process of signal amplification using enzymes, DNA enzymes and nanoparticles is usually complex and limited to specific applications. In this paper, we designed an automatic DNA amplification system to construct novel and simple biosensors based on traditional molecular beacon structures. The improved amplification molecular beacon can greatly improve the detection signal and detection limit, and can be used to detect nucleic acid and distinguish single base mismatch. By introducing aptamer sequences, the amplified molecular beacons can also detect proteins and small molecules, such as 尾 -actin genes and thrombin. We also confirmed the high sensitivity and stability of the amplified molecular beacons, which provided the possibility for the detection of miRNA in living cells. The simplicity and flexibility of augmentable molecular beacons make it more widely used. Toehold structure, as the core of chain exchange reaction, plays an important role. Researchers have done a lot of work on the length of the toehold, the distance and location of the chain-exchange region. However, in most cases, the toehold structure is always default to a double-stranded structure. In this thesis, we have changed its basic configuration and chain switching mechanism, and designed a DNA strand exchange reaction induced only by DNA G- quadruplex as toehold structure. When PEG is added to the reaction environment, this kind of molecular crowding reagent makes the reaction happen easily. We can control the reaction by adjusting the concentration and degree of polymerization of PEG, and we can also adjust the structure of G- quad by changing the structure of PEG. At the same time, we also introduced mismatched bases into the transport chain, so that the chain exchange reaction can be carried out smoothly in aqueous solution. Then we also design the DNA chain amplification reaction based on this special chain exchange, and realize the detection system without fluorescent labeling by using the structure characteristic of G- quadruplex. This method has the advantages of low cost and no labeling. With high sensitivity and selectivity, it has potential applications in DNA nanomachines, biosensors and disease diagnosis.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78
[Abstract]:With the development and progress of DNA nanotechnology, more and more methods have been developed and applied in the fields of biological detection, clinical diagnosis, drug therapy, computational chemistry and so on. Modern biosensors require high sensitivity, great signal enhancement and significant selectivity for detection and diagnosis. Traditional molecular beacons can not amplify signals, which greatly limits its scope of application. The process of signal amplification using enzymes, DNA enzymes and nanoparticles is usually complex and limited to specific applications. In this paper, we designed an automatic DNA amplification system to construct novel and simple biosensors based on traditional molecular beacon structures. The improved amplification molecular beacon can greatly improve the detection signal and detection limit, and can be used to detect nucleic acid and distinguish single base mismatch. By introducing aptamer sequences, the amplified molecular beacons can also detect proteins and small molecules, such as 尾 -actin genes and thrombin. We also confirmed the high sensitivity and stability of the amplified molecular beacons, which provided the possibility for the detection of miRNA in living cells. The simplicity and flexibility of augmentable molecular beacons make it more widely used. Toehold structure, as the core of chain exchange reaction, plays an important role. Researchers have done a lot of work on the length of the toehold, the distance and location of the chain-exchange region. However, in most cases, the toehold structure is always default to a double-stranded structure. In this thesis, we have changed its basic configuration and chain switching mechanism, and designed a DNA strand exchange reaction induced only by DNA G- quadruplex as toehold structure. When PEG is added to the reaction environment, this kind of molecular crowding reagent makes the reaction happen easily. We can control the reaction by adjusting the concentration and degree of polymerization of PEG, and we can also adjust the structure of G- quad by changing the structure of PEG. At the same time, we also introduced mismatched bases into the transport chain, so that the chain exchange reaction can be carried out smoothly in aqueous solution. Then we also design the DNA chain amplification reaction based on this special chain exchange, and realize the detection system without fluorescent labeling by using the structure characteristic of G- quadruplex. This method has the advantages of low cost and no labeling. With high sensitivity and selectivity, it has potential applications in DNA nanomachines, biosensors and disease diagnosis.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78
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