基于DNA自组装技术的microRNA传感新方法研究

发布时间：2018-08-16 08:36

【摘要】：Micro RNAs(mi RNAs)参与细胞生长的一系列重要进程,包括细胞增殖、分化、凋亡及死亡等过程。mi RNAs表达失调将导致细胞的过度生长、增殖及分化,从而导致肿瘤的发生发展,因此,其作为一种潜在的新型核酸类肿瘤标志物和潜在的治疗靶点具有广阔的临床应用前景。现有mi RNA检测方法主要有RT-PCR,微阵列芯片及Northern blot等,虽然这些方法都有一定的优势,但也存在一些不足,如操作繁琐,费时费力、灵敏度低等,这些都会在很大程度上影响其临床实用性。因此,发展简单、实用、快速、灵敏的mi RNAs检测技术对于阐明肿瘤发生发展的机制,寻找新的肿瘤标志物,肿瘤的早期诊断和治疗,探寻新的肿瘤治疗靶点具有重要价值;有助于实现mi RNAs标志物在肿瘤临床诊疗中的应用。本论文整合分子生物学、临床检验诊断学及生物分析化学等学科的最新研究成果,基于功能核酸、DNA自组装和等温扩增技术等,构建两种新颖、简单、快速的mi RNAs传感检测新方法,为肿瘤标志物的早期诊断与治疗提供潜在的检测平台。本研究主要包括以下两个部分:1.核酸信号放大策略用于mi RNA的比色传感整合链替代聚合反应和哑铃状密封探针介导的滚环扩增双重信号放大策略,本研究构建了一个快速、免标记的比色传感器用于mi RNA的高灵敏、高特异性检测。该传感器主要包括4个组成部分,分别是由trigger模板、C-rich DNA修饰的MB,哑铃状滚环模板,剪切酶以及聚合酶。靶mi RNA引发链替代聚合反应,释放滚环模板引物以启动基于toehold的滚环扩增反应,从而扩增出大量富G的DNA(GDNA)序列;GDNA序列与Hemin结合形成DNAzyme催化ABTS2-和H2O2进行比色反应,从而实现对mi RNAs的高灵敏、高特异性检测。该方法检测限低至10 a M,检测线性范围为10 a M~1 n M,能很好的应用于实际样本的检测。本章所构建的mi RNA超灵敏检测方法简单、实用,有望成为肿瘤相关核酸标志物的筛选、临床诊断应用的有力工具。2.新型DNA纳米镊的设计及其多组份miRNA的同时化学发光检测本方法构建了一种基于靶物质诱导纳米结构变化的新型DNA纳米镊,通过结合催化茎环自组装(CHA)和邻位触及形成辣根过氧化物酶模拟酶(DNAzyme),实现了AND逻辑门操作和对多组份mi RNA的同时检测。本研究构建的基于DX(DNA double crossover,DX)模块的DNA纳米镊的包含有信号输入和信号输出结构,两臂中间含弓形结构的DNA双股螺旋作为信号输入的识别域,用于同时特异性甄别两种不同的靶物质;两臂末端的拆分富G序列用于信号输出。利用CHA信号放大策略,少量的靶物质输入可以靶向诱导大量的DNA纳米镊有打开状态转向闭合状态,在Hemin存在时,镊两臂末端的拆分富G序列在邻位触及作用下形成大量DNAzyme,从而产生放大的传感输出信号。两种靶物质通过AND逻辑门诱导调控DNA纳米镊的开关,通过聚丙烯酰胺电泳(PAGE)、表面等离子体共振(SPR)表征了该调控机制,证实新型DNA纳米镊构建成功。构建的生物传感系统在输出的化学发光信号和输入mi RNA之间呈良好的线性关系,检测线达30 f M。该生物传感器可用于复杂的生物样本中mi RNA的检测分析。本方法构建了一个简单,可重复利用的生物传感平台,该传感平台可用于逻辑门的构建以及无酶的高灵敏高特异性的mi RNA检测。
[Abstract]:Micro RNAs (mi RNAs) are involved in a series of important processes of cell growth, including cell proliferation, differentiation, apoptosis and death. Misexpression of MI RNAs will lead to excessive cell growth, proliferation and differentiation, leading to the occurrence and development of tumors. Therefore, mi RNAs are a potential new type of tumor markers and potential therapeutic targets. The existing methods for detecting microRNA include RT-PCR, microarray chip and Northern blot. Although these methods have some advantages, they also have some disadvantages, such as complicated operation, time-consuming, laborious and low sensitivity, which will greatly affect their clinical practicability. Rapid and sensitive detection of MI RNAs is of great value in elucidating the mechanism of tumorigenesis and development, searching for new tumor markers, early diagnosis and treatment of tumors, and searching for new therapeutic targets for tumors. It is helpful to realize the application of MI RNAs markers in clinical diagnosis and treatment of tumors. Based on functional nucleic acids, DNA self-assembly and isothermal amplification techniques, two novel, simple and fast methods for detecting mi RNAs are proposed, which provide a potential platform for early diagnosis and treatment of tumor markers. This study mainly includes the following two parts: 1. Nucleic acid messenger A fast, label-free colorimetric sensor for high sensitivity and high specificity detection of MI RNA was constructed. The sensor consists of four components, namely, trigg. Er template, C-rich DNA modified MB, dumbbell-shaped ring template, shearing enzyme and polymerase. Target mi RNA initiates chain substitution polymerization, releases roller template primers to initiate toehold-based ring-rolling amplification reaction, thereby amplifying a large number of G-rich DNA (GDNA) sequences; GDNA sequence combines with hemin to form DNA zyme catalyzed ABTS2-and H2O2 for colorimetric inversion. The detection limit of this method is as low as 10 a M, and the detection linear range is 10 a M~1 n M. This method can be well applied to the detection of real samples. Force tool. 2. Design of novel DNA nanotweezers and simultaneous chemiluminescence detection of multi-component microRNAs. A novel DNA nanotweezers based on target-induced nanostructure changes were constructed. By combining catalytic stem-ring self-assembly (CHA) and ortho-contact formation of horseradish peroxidase mimetic enzyme (DNA zyme), the AND logic gate operation was realized. In this study, DNA nanotweezers based on DX (DNA double crossover, DX) module consisted of signal input and signal output structures, and DNA double helix containing bow-shaped structure between two arms was used as the recognition domain of signal input for simultaneous specific screening of two different target substances. Using the CHA signal amplification strategy, a small amount of target material input can be targeted to induce a large number of DNA nanotweezers to turn to the closed state. In the presence of hemin, the splitting rich G sequences at the ends of the tweezers arm form a large number of DNA zymes under the action of adjacent touch, thus generating amplified sensing output signals. The novel DNA nanotweezers were successfully constructed by inducing and controlling the switch of DNA nanotweezers through the logic gate of AND. The mechanism was characterized by polyacrylamide electrophoresis (PAGE) and surface plasmon resonance (SPR). The biosensor system was constructed with a good linear relationship between the output chemiluminescence signal and the input mi RNA. The biosensor can be used for the detection and analysis of MI RNA in complex biological samples. A simple and reusable biosensor platform is constructed. The biosensor platform can be used for the construction of logic gates and the detection of MI RNA with high sensitivity and specificity without enzyme.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730;TP212.3

【相似文献】