FACTORS STABILIZING RNA STRUCTURES

因为RNA带有很高的负电荷，所以它折叠成次要的和 三级结构对盐浓度非常敏感。 RNA三级结构的折叠特别受 二价离子浓度与一价离子和二价离子的类型 礼物。研究与RNA的离子关联是一个困难的问题 问题，因为样品分子中的结合可能与完全不同 与RNA静电场的离域相互作用导致络合作用 特定部位的部分脱水离子。如何看待…的作用 离子在引导RNA折叠中的作用取决于所分配的相对重要性 到这种非特定和特定的绑定模式。 拟议工作的目的是系统地检查ion-rna。 一系列已知且日益复杂的非核糖核酸的相互作用 规范结构和三级结构。将使用的方法包括 量热和紫外线熔融实验(它们报告了 折叠和未折叠状态之间的离子缔合程度，以及 提供离子诱导的稳定性的直接测量)，以及 检测单价或二价离子与 折叠RNA结构(平衡透析法或荧光法滴定 自由离子浓度指示器)。工作的具体目标包括 (1)研究特定核糖核酸位点上的二价离子相互作用， 包括结合的亲和力、单价离子盐依赖性，以及 对不同碱土离子的选择性； (Ii)确定对什么RNA结构产生选择性 一价离子(碱金属和铵)，以及选择性是否可以 归因于一个或几个具体的协调地点； (3)初步尝试比较计算和实验测量的结果 RNA中的静电势。 这些实验应该提供离子如何分布的图景。 围绕每个RNA以及不同类别的关联程度 离子对RNA结构的热力学稳定性有贡献。这 信息将填补非规范和第三级的一个关键方面 RNA折叠能量学。

Because RNA carries a high negative charge, its folding into secondary and tertiary structures can be exquisitely sensitive to salt concentration. Folding of RNA tertiary structures is particularly affected by the concentration of divalent ions and the types of mono- and divalent ions presents. Investigation ion association with an RNA is a difficult problem, as binding in the sample molecule may vary from entirely delocalized interactions with the RNA electrostatic field to chelation of partially dehydrated ions at specific sites. How one views the role of ions in directing RNA folding depends on the relative importance assigned to such non-specific and specific binding modes. The purpose of the proposed work is to systematically examine ion-RNA interactions in a series of RNAs of known and increasingly complex non- canonical and tertiary structures. Approaches that will be used are calorimetric and UV melting experiments (which report the differences in the extent of ion association between folded and unfolded states, and provide a direct measure of ion-induced stabilization), and methods that detect the extent of monovalent or divalent ion association with the folded RNA structure (equilibrium dialysis or titrations using fluorescent indicators of free ion concentrations). Specific aims of the work include (i) Investigation of divalent ion interactions at specific RNA sites, including the affinity, monovalent ion salt dependence of binding, and selectivity for different alkaline earth ions; (ii) Determination of what RNA structures develop selectivity for monovalent ions (alkali metal and ammonium), and whether selectivity can be attributed to one or a few specific coordination sites; (iii) Initial attempts to compare calculated and experimentally measured electrostatic potentials in an RNA. These experiments should provide a picture of how ions are distributed around each RNA and the degree to which different classes of associated ions contribute to thermodynamic stability of RNA structures. This information will fill in a crucial aspect of non-canonical and tertiary RNA folding energetics.