FACTORS STABILIZING RNA STRUCTURES

由于RNA携带高负电荷，它折叠成二级和二级结构， 三级结构对盐浓度非常敏感。 RNA三级结构的折叠特别受到 二价离子的浓度以及一价和二价离子的类型 礼物.研究离子与RNA的结合是一个困难的问题。 问题，因为样品分子中的结合可能完全不同， 与RNA静电场的离域相互作用， 在特定位点的部分脱水离子。如何看待 离子在指导RNA折叠中的作用取决于 这种非特异性和特异性结合模式。 本研究的目的是系统地研究离子RNA 在一系列已知的和越来越复杂的非- 规范结构和三级结构。将采用的方法有 量热和UV熔融实验（其报告了 折叠和未折叠状态之间的离子缔合程度，以及 提供离子诱导稳定化的直接测量），以及 检测单价或二价离子与所述化合物缔合的程度， 折叠的RNA结构（使用荧光的平衡透析或滴定 自由离子浓度的指示剂）。工作的具体目标包括 (i)研究二价离子在特定RNA位点的相互作用， 包括亲和力、结合的单价离子盐依赖性，以及 对不同碱土金属离子的选择性; (ii)确定哪些RNA结构产生选择性 单价离子（碱金属和铵），以及选择性是否可以 归属于一个或几个特定的协调点; (iii)初步尝试比较计算和实验测量 RNA中的静电势 这些实验应该能提供一幅离子分布的图像 以及不同种类的相关RNA 离子有助于RNA结构的热力学稳定性。这 信息将填补非规范和第三 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.