DNA/RNA QUADRUPLEXES: STRUCTURE, STABILITY AND INTERACTIONS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。这个项目的重点是四链的不寻常的核酸构象，建立在鸟嘌呤四元组的基础上。在过去的五年里，越来越多的证据表明这些结构在生物系统中的存在及其功能作用：例如，四链结构被认为在调节端粒酶活性、启动转录、翻译抑制以及与大量蛋白质的特异性结合方面发挥作用。此外，已经描述了四链作为蛋白质识别的折叠基序、作为抗病毒和抗癌化疗药物之外的诊断剂的应用。我们的长期目标是加深对这类核酸的热力学、结构和相互作用的了解，特别是关于阳离子结合的特殊性质、折叠的RNA四链的性质及其与DNA四链的差异，以及它们与其他生物分子的相互作用。特别是，我们建议对一系列具有生物意义的DNA和RNA形式的富鸟嘌呤序列进行热力学分析，对这些序列进行结构分析，开发评估四链形式分子的方法，并详细研究催化DNA结构及其与氯化血红素的相互作用。用于这一目标的实验技术包括UV/CD光谱、等温滴定量热法、核磁共振、荧光和平衡沉淀。此外，还将使用质谱学在实验中确认连接产物，这些实验的目的是在生物学条件下表征人类端粒结构。这种方法的结合将提供对非常重要的核酸物种的构象和相互作用性质的详细检查。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project focuses on the unusual nucleic acid conformation of quadruplexes, built upon the guanine quartet. Evidence for the presence of these structures in biological systems, and their functional roles, has been mounting over the past five years: for example, quadruplex structures have been proposed to play a role in regulating telomerase activity, in initiation of transcription, in translational repression, and in binding specifically to a substantial number of proteins. Furthermore, applications have been described for quadruplexes as a folding motif for protein recognition, as diagnostic agents in addition to antiviral and anticancer chemotherapeutic agents. Our long term goal is an enhanced understanding of the thermodynamics, structure and interactions of this class of nucleic acids, in particular in terms of the specific nature of cation binding, the properties of folded RNA quadruplexes and their differences with respect to DNA quadruplexes, and their interactions with other molecules of biological interest. In particular, we propose thermodynamic analyses of a series of biologically significant guanine-rich sequences, both in DNA and RNA forms, structural analyses of these sequences, development of methods to assess the molecularity of quadruplex forms and a detailed investigation of a catalytic DNA construct and its interactions with hemin. Experimental techniques to be employed in this goal include UV/CD spectroscopy, isothermal titration calorimetry, NMR, and fluorescence, and equilibrium sedimentation. Additionally, mass spectrometry will be used to confirm ligation products in experiments aimed at characterizing the human telomere structure under biological conditions. This combination of approaches will afford a detailed examination of the conformation and interaction properties of the very important nucleic acid species.