【作者】 刘凌凤；

【导师】 王柯敏；

【作者基本信息】 湖南大学 ， 分析化学， 2003， 硕士

【摘要】 核酸是生命体系中的关键物质，它承载着传递遗传信息、编码其它相关生物分子的重要任务，在分子生物学和生物技术领域受到广泛关注，是发展非常迅速的研究重点。核酸相关生命功能的完成需要蛋白质(酶)的协同参与。对核酸和相关蛋白的相互作用研究正是以功能基因组和蛋白质组的研究为标志的后基因组时代所关注的热点之一。 核酸的转录、修复和重组是细胞生命周期中的重要过程，由核酸连接酶催化的核酸连接反应是这些过程中不可或缺的步骤，传统的核酸连接分析的研究手段主要是凝胶电泳技术，无法满足实时监测核酸连接过程的要求，限制了人们对于这一作用过程更为具体和根本的机理的深入了解。有关核酸与连接酶作用的实时过程信息的获得将不仅有助于对一些生命过程机理的研究，还将为新药的设计提供新的技术手段，为疾病的治疗找到新的方法。 病毒基因组RNA的快速检测对于由RNA病毒感染的疾病诊断与防治具有重要意义。目前普遍运用的RT-PCR方法对于模板的提纯有较高的要求，于是，发展简便、快速而灵敏的技术用于病毒RNA的检出变得尤为重要。 1996年，Tyagi和Krammer研究出一种新型荧光探针—分子信标。这种探针是在寡聚核苷酸探针的基础上发展起来的一类具有茎—环结构的寡聚核苷酸单链探针，具有高灵敏度、高特异性和背景值极低，无需分离未杂交探针等优点，不仅适用于核酸分析，而且同样也适用于蛋白质(酶)等能与核酸发生相互作用的物质分析，为实时监测蛋白质(酶)与核酸作用的过程提供了极好的技术手段。 本实验室唐志文博士利用分子信标核酸探针，发挥其研究核酸及相关蛋白质(酶)的优势，创新性地开展对核酸连接、核酸磷酸化以及核酸酶切过程的实时监测研究，本论文作为唐志文博士论文工作的一部分，从分子信标实时监测核酸与蛋白质(酶)的作用过程和分子信标快捷地检测病毒RNA两个角度开展研究，发展了基于分子信标的新的检测技术和应用研究体系，可从分子水平上获取生物大分子之间相互作用的动态信息： (1)利用分子信标实时监测了T4 DNA连接酶催化的DNA连接过程，并建立与实现反应体系相应的动力学模型。 建立一了一种新的核酸连接实时监测方法，利用分子信标作为核酸连接反应的DNA模板和检测分子，在核酸分子连接的同时检测荧光信号。首次实时、准确地获取核酸连接过程信息，为核酸连接酶分析、核酸连接动力学过程研究提供全新、有效的手段，也为深入研究核酸连接酶与核酸分子间的相互作用提供了新的思路与技术。一与传统的研究方法相比，该方法避免了放射性同位素标记、变性凝胶电泳、放射性自显影等复杂的操作，为核酸连接过程研究提供一种新的非同位素分析方法，并建立了该方法的动力学模型，将有助于我们进一步揭示核酸与蛋白质这两种生命中最关键物质之间的相互作用与关系。在此基础上建立了快速、、准确检测T4 DNA连接酶的分析方法。其检测下限为2 .3xlo礴U/mL。 (2)基于分子信标实时监测大肠杆菌DNA连接酶催化DNA连接的活性，并建立了高灵敏地分析连接酶活性的方法。 核酸连接酶除了一类与T4 DNA连接酶相似，以戌1，P为辅酶的之外，另一类以NAD十为辅酶的连接酶也具有同要重要的研究价值。因此，在上一章建立的研究T41〕NA连接酶的技术的基础之上，本章进一步发展了对以NAD+为辅酶的E. coliDNA连接酶的活性及其催化的DNA连接过程的实时监测方法。 该方法灵敏、实时地监测了E coli DNA连接酶催化的DNA连接反应的进程。建立了快速、准确检测E。011 DNA连接酶的分析方法，检测下限为4.0xl了U/ml。与传统的研究方法相比，该方法既避免了同位素标记、变性凝胶电泳、放射自显影等复杂的操作，又为深入研究核酸连接酶与核酸的相互作用提供了实时、丰富的动力学数据，为蛋白质(酶)与核酸的相互作用研究提供了新的思路、方法和有效的手段，对于推动其动力学过程的深入研究具有重要的意义。 (3)基于分子信标荧光探针快速检测烟草花叶病毒的新方法研究 根据常见的烟草花叶病毒(tobacco mosaic vios，TMV)的遗传物质RNA序列，设计一了一种新型分子信标荧光探针，发展了一种直接检测病毒基因组RNA的新方法，由于分子信标具有高特异性、对病毒RNA的提取不需要严格的纯化，就可以用于植物病毒的检测，克服了传统的RFPCR过程要求制备高质量核酸模板的限制，实现了病毒RNA快速、准确的检测。更多还原

【Abstract】 Nucleic acid is one of the most important biomolecules in life that is in charge of transferring genetic information and coding other biomolecules. The investigation of nucleic acids has been one of the most important areas and currently one of the most rapidly growing fields in contemporary molecular biology and biotechnology.Nucleic acids ligation catalyzed by DNA ligase plays an indispensable role in cell life cycle, which includes many essential life processes such as replication, repair and recombination of nucleic acids. Unfortunately, up to now, our knowledge of interactions between ligase and nucleic acids has been limited because of the lack of adequate methodologies for real-time detection. The detailed and fundamental nature of these interactions as well as their contribution to the total organization of biological systems is yet to be discovered. Better understanding of these interactions not only will benefit the understanding of many biological mechanisms but also is expected to be of great medical use in designing new drugs and in developing new disease treatment strategies.Detection of virus genome RNA is of great importance for diagnosis of some diseases infected by RNA virus. RT-PCR, commonly used for RNA virus determination, requires template of high quality, which has restricted application of the method. Therefore, it is necessary to develop technology for quick and sensitive detection of virus RNA.Molecular beacons are a class of novel fluorescence probes, since first developed in 1996, they have been widely used in DNA/RNA and some protein studies. Molecular beacon has shown to be a useful tool in monitoring interactions between nucleic acids and proteins (enzymes), which makes it a pretty good tool for monitoring interactions between proteins and nucleic acids in real time.Furthermore, since molecular beacon is a kind of probe with high sensitivity, high selectivity, low background and no need of remove from the redundant probes from detecting mixture, it could be used to determine mRNA and genome RNA of somevirus without amplification by PCR.Based on molecular beacon probes, Dr. Zhiwen Tang in our lab has developed novel methods to research on ligation, phosphorylation and enzymatic cleavage of nucleic acids. As a part of his systematic research, the thesis has developed application systems based on molecular beacon for two goals: one is to monitor reactions between nucleic acids and proteins (enzymes) in real-time, the other is to detect virus RNA quickly and directly. The thesis is composed of three major projects:(1) Real time monitoring of nucleic acids ligation catalyzed by T4 DNA ligaseand foundation of kinetics model of the methodA new approach has been developed for monitoring DNA ligation using molecular beacon (MB) DNA probes. Molecular beacon is a hairpin-shaped oligonucleotide probe with high sensitivity and specificity. It has been employed as a DNA probe to monitor the ligation process. The sequence of the MB was designed to be complimentary with the product of the ligation, therefore, the ligation process can be monitored in a homogeneous solution in real-time. It could be realized based on this method to study nucleic acids ligation kinetics with convenience and to determine the activity of DNA ligase accurately. The result of kinetics experiment indicates that interaction in the detecting system is a single-substrate enzyme-catalyzed reaction and molecular beacon acts as not only the probe for monitoring ligation but also the template for DNA substrate formation. Effects of some external factors and base mismatches at the nick on DNA ligation have been studied using the novel approach. Based on this principle, a sensitive, precise and quick analytical method to determine the activity of T4 DNA ligase has also been developed.The major advantages of our method are its ultrasensitivity, excellent specificity, convenience, real-time monitoring of homogeneous solution. The now easily obtainable MBs would found wide application in the investigation o更多还原