EPR STUDIES OF METAL/NUCLEI ACID INTERACTIONS

二价金属离子是核酸，特别是催化核酶分子的结构和功能的关键组成部分。然而，目前的生物物理方法不足以提供对金属离子-RNA相互作用的完整描述。因此，我们建议发展脉冲EPR作为一种研究溶液中RNA金属离子中心的技术。利用单体系统，我们将开发和优化基于电子自旋回波包络调制(ESEEM)和电子自旋回波-电子核双共振(ESE-Endor)的方法来分析不同RNA官能团与顺磁性金属离子的连接。这项工作的一个关键部分是设计和建造一种新型的卡尔法波段(31 GHz)ESE-Endor光谱仪，它使用了固态微波源和功率放大器。我们将把这些方法应用于RNA矿物生物化学中的一些突出问题，包括离子核在促进大RNA折叠中的作用，各种核酶的基态和过渡态中催化金属离子中心的结构，以及碱基修饰、序列等因素对离子结合模式的调节。这些研究对提高对RNA和核糖核蛋白酶的基本了解以及对生物催化的总体了解具有巨大的潜力。此外，由于设计用于药物应用的生物活性核酶衍生物依赖于对催化机制的充分了解，这项研究可能会极大地推动核酶作为治疗一些毁灭性疾病的潜力，包括艾滋病、癌症和丙型肝炎。

Divalent metal ions are a key component in the structure and function of nucleic acids in general and catalytic RNA molecules (ribozymes) in particular. Current biophysical methods, however, are inadequate to provide a full description of metal ion-RNA interactions. We therefore propose to develop pulsed EPR as a technology for the study of RNA metal ion sites in solution. Using monomeric systems, we will develop and optimize methodologies based on electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) for analyzing ligation by various RNA functional groups to paramagnetic metal ions. A key element in this work is the design and construction of a novel Kalpha-band (31 GHz) ESE-ENDOR spectrometer using a solid-state microwave source and power amplifier. We will apply these methods to a number of outstanding problems in RNA metallobiochemistry, including the utility of "ion cores" in promoting the folding of large RNAs, the structure of catalytic metal ion sites in the ground and transition states of various ribozymes, and the modulation of ion binding modes by base modification, sequence, and other factors. These studies have great potential to improve the fundamental understanding of RNA and ribonucleoprotein enzymes, as well as of biological catalysis in general. In addition, since the design of bioactive ribozyme derivatives for pharmaceutical applications depends on an adequate understanding of catalytic mechanism, this research may significantly advance ribozymes toward their potential as therapeutics for a number of devastating diseases, including AIDS, cancer, and hepatitis C.