SITE SPECIFICALLY MODIFIED RNAS

拟议项目的长期目标是了解 核糖核苷修饰的功能意义。尽管 已经确定了90多项修改的大量研究 以及它们在tRNA和更大的RNA分子中的位置，人们知之甚少 关于这些修饰对RNA的个别贡献 结构和功能。我们假设修饰后的核苷会对 它们通过改变稳定性或结构对RNA功能的影响 在RNA中的特定位置。因此，这个项目就是要确定 修饰的核苷对RNA结构和核糖核酸的特异性贡献 稳定性。不能将修饰的核苷结合到一个部位- 进入大RNA的特定方式已经使结构和 这些分子的功能分析。这样做的具体目的是 建议是1)制定针对特定地点的方法 将修饰的核苷掺入RNA和2)测定 单层改性基层对两者结构稳定性的影响 核糖核苷酸片段(模型RNA)和全长RNA分子 具有特定的生物功能。特定地点的合并具有 既多才多艺，又能适应各种修改。因此， 提出了两种互补的方法：第一种涉及 亚磷酰胺化学中大量合成的RNA 可以通过常规方式获得；第二个涉及更多 一般的，酶促结合，适用于所有 修饰的碱基和更大的、具有生物学意义的RNA 分子。这些研究将使我们能够理解独特的影响 单个修饰核苷酸对RNA结构和功能的影响。 在特定位置修饰RNA的能力对于 确定RNA和蛋白质之间的结构-功能关系 或小分子，对折叠和动力学的影响，以及金属结合 改性碱的影响。RNA是备受关注的中心，因为 它催化特定反应的能力。修改的RNA使用 增强的催化活性也可能具有潜在的治疗作用 申请。

The long-term goal of the proposed project is to understand the functional significance of ribonucleoside modifications. In spite of the large number of studies which have identified over 90 modifications and their locations in tRNAs and larger RNA molecules, little is known regarding the individual contributions of these modifications to RNA structure and function. We hypothesize that modified nucleosides exert their effects on RNA function by altering the stability or structure at specific sites within the RNA. Thus, this project is to determine the specific contributions of modified nucleosides to RNA structure and stability. The inability to incorporate modified nucleosides in a site- specific manner into large RNAs has complicated both structural and functional analyses of these molecules. The specific aims of this proposal are 1) to develop methodologies for site-specific incorporation of modified nucleosides into RNA and 2) to determine the effects of individual modified bases on structural stability of both ribonucleotide fragments (model RNAs) and of full-length RNA molecules with specific biological functions. The site-specific incorporation has to be both versatile and adaptable to various modifications. Therefore, two complementary approaches are proposed: the first involves phosphoramidite chemistry in which large quantities of synthetic RNAs can be obtained in a routine manner; the second involves a more general, enzymatic incorporation that is applicable to all of the modified bases and to much larger, biologically interesting RNA molecules. These studies will allow us to understand the unique effects of the individual modified nucleotides on RNA structure and function. The ability to modify RNA at specific sites is important for determining structure-function relationships between RNA and proteins or small molecules, effects on folding and dynamics, and metal-binding effects of modified bases. RNA is the center of much attention because of its ability to catalyze specific reactions. Modified RNAs with enhanced catalytic activities may also have potential therapeutic applications.