DNA/RNA Quadruplexes: Structure, Stability, Interactions

描述(由申请人提供)：本项目重点研究以鸟嘌呤四元组为基础的四链构象。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的应用，以及作为抗病毒和抗癌化疗药物的诊断试剂。基于鸟嘌呤四元组的结构在核酸中是独一无二的，因为它们依赖于某些稳定阳离子的特定结合。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。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。

DESCRIPTION (provided by applicant): 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, and as diagnostic agents, in addition to antiviral and anticancer chemotherapeutic agents. Structures based on guanine quartets are unique among nucleic acids in their dependence on specific binding of certain stabilizing cations. 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. This combination of approaches will afford a detailed examination of the conformation and interaction properties of the very important nucleic acid species.