EPR STUDIES OF METAL/NUCLEI ACID INTERACTIONS

二价金属离子通常是核酸结构和功能的关键组分，特别是催化RNA分子（核酶）。 然而，目前的生物物理方法不足以提供金属离子-RNA相互作用的完整描述。 因此，我们建议开发脉冲EPR作为一种技术，用于研究溶液中的RNA金属离子位点。使用单体系统，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，用于分析各种RNA官能团与顺磁性金属离子的连接。 在这项工作中的一个关键要素是一种新型的Kalpha波段（31 GHz）的ESE-ENDOR光谱仪使用固态微波源和功率放大器的设计和建设。 我们将这些方法应用于RNA金属生物化学中的一些突出问题，包括在促进大RNA的折叠中的“离子核心”的效用，各种核酶的基态和过渡态中的催化金属离子位点的结构，以及通过碱基修饰、序列和其他因素对离子结合模式的调制。这些研究有很大的潜力，以提高RNA和核糖核蛋白酶的基本理解，以及一般的生物催化。 此外，由于生物活性的核酶衍生物的药物应用的设计依赖于催化机制的充分理解，这项研究可能会显着推进核酶对他们的潜力，作为一个毁灭性的疾病，包括艾滋病，癌症和丙型肝炎的治疗。

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.