基于DNA机器构建的荧光生物传感器用于端粒酶活性检测

发布时间：2018-04-01 06:07

本文选题：端粒酶活性检测　切入点：DNA机器　出处：《山东大学》2017年硕士论文

【摘要】：端粒酶是一种核糖核蛋白复合物,其可在染色体末端添加端粒重复序列以维持端粒DNA长度。端粒酶活性的增强可导致细胞持续分裂乃至永生化,与癌症等疾病的发生密切相关;端粒酶被认为是一种重要的癌症生物标志物,其活性的灵敏检测对于癌症的早期诊断具有重要意义。DNA机器是指能够在燃料物质的引发下行使分子动态机械功能的DNA装备,其由于动力学可控以及在分子相互作用研究上的优势而备受关注。以DNA机器在纳米水平的可控运动为基础,其应用派生出多个方向,如信号放大型DNA机器、分子开关DNA机器,其在构建灵敏、特异的生物传感平台方面具有广阔的应用前景。鉴于以上分析,本文利用两种DNA机器构建了荧光传感平台用于端粒酶活性的灵敏、准确检测。本文共分为三章:第一章为绪论,概述了端粒和端粒酶的结构和功能、端粒酶活性与细胞衰老和肿瘤发生的关系、目前端粒酶活性的主要检测方法及存在的不足。此外还介绍了 DNA机器的作用原理及其应用进展。第二章构建了基于免标记分子信标的级联扩增DNA机器用于端粒酶活性的灵敏检测。该策略经过级联扩增产生多个G-四倍体结构,端粒酶延伸产物废物利用,与扩增产生的G-四倍体共同用于信号输出,提高了端粒酶活性检测灵敏度。该方法在50 HeLa cells/mL到2000 HeLa cells/mL的范围内线性良好,能够检测到最低浓度为50 HeLa cells/mL中的端粒酶活性,优于或媲美于现存的端粒酶活性检测方法。此外,该方法被成功用于端粒酶活性抑制剂的抑制效应分析,在其抑制剂筛选方面具有应用潜能。第三章构建了一种基于DNA纳米镊子分子开关的荧光策略用于端粒酶活性的比率型检测。DNA-NT初始为打开状态,荧光供体和受体远离,荧光共振能量转移(fluorescence resonance energy transfer,FRET)效率低。端粒酶延伸产物能够将镊子闭合,使荧光供体和受体靠近,FRET效率增强。通过DNA镊子闭合前后的FRET响应变化,将其用于端粒酶活性的比率型检测。并通过置换链对镊子的重新打开以实现对端粒酶持续合成能力的研究。本章利用凝胶电泳和荧光法验证了该方法的可行性,并在最优条件下研究了其对端粒酶活性的检测性能。
[Abstract]:Telomerase is a ribonucleoprotein complex that adds telomere repeats at the end of chromosomes to maintain telomere DNA length.The enhancement of telomerase activity leads to cell division and even immortality, which is closely related to the occurrence of diseases such as cancer. Telomerase is considered to be an important biomarker of cancer.The sensitive detection of its activity is of great significance for the early diagnosis of cancer. DNA machines are DNA equipment capable of performing molecular dynamic mechanical functions under the initiation of fuel substances.It has attracted much attention because of its controllable kinetics and its advantages in molecular interaction.Based on the controllable motion of the DNA machine at the nanometer level, its applications derive from many directions, such as signal playing large DNA machine and molecular switch DNA machine. It has a broad application prospect in the construction of sensitive and specific biosensor platform.In view of the above analysis, a fluorescence sensing platform was constructed by using two kinds of DNA machines for the sensitive and accurate detection of telomerase activity.This paper is divided into three chapters: the first chapter is the introduction, which summarizes the structure and function of telomere and telomerase, the relationship between telomerase activity and cell senescence and tumorigenesis.In addition, the principle of DNA machine and its application progress are also introduced.In chapter 2, a cascaded amplified DNA machine based on marker free molecular beacons was constructed for the sensitive detection of telomerase activity.The strategy can produce a number of G- tetraploid structures by cascade amplification and waste utilization of telomerase extension products, which can be used in signal output together with the expanded G- tetraploid, which improves the sensitivity of telomerase activity detection.The method has a good linearity in the range of 50 HeLa cells/mL to 2000 HeLa cells/mL, and can detect telomerase activity in 50 HeLa cells/mL at the lowest concentration, which is superior to or comparable to that of the existing methods.In addition, the method has been successfully used to analyze the inhibitory effect of telomerase activity inhibitors, and has potential application in screening of telomerase activity inhibitors.In chapter 3, we construct a fluorescence strategy based on DNA nano-tweezers molecular switch to detect telomerase activity. DNA-NT is initially open, fluorescence donor and receptor are far away, fluorescence resonance energy transfer resonance energy transfer fret is low.Telomerase extension products can close tweezers and increase the efficiency of fluorescent donors and receptors near fret.The changes of FRET response before and after closure of DNA tweezers were used to detect telomerase activity.The ability of telomerase sustained synthesis was studied by reopening tweezers by replacement chain.In this chapter, the feasibility of this method was verified by gel electrophoresis and fluorescence method, and the detection performance of telomerase activity was studied under the optimal conditions.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3

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