SITE SPECIFICALLY MODIFIED RNAS

该项目的长期目标是了解 核糖核苷修饰的功能意义。尽管 大量的研究已经确定了90多个修改， 以及它们在tRNA和更大的RNA分子中的位置， 关于这些修饰对RNA的单独贡献， 结构和功能。我们假设修饰的核苷 它们通过改变RNA的稳定性或结构 在RNA中的特定位点。因此，本项目旨在确定 修饰的核苷对RNA结构的特定贡献， 稳定无法将修饰过的核苷整合到一个位点- 以特定的方式转化为大RNA， 这些分子的功能分析。具体目标是 建议是：（1）制定方法， 将修饰的核苷掺入到RNA中，和2）确定 单个改性碱基对两种结构稳定性的影响 核糖核苷酸片段（模型RNA）和全长RNA分子 具有特定的生物学功能。特定地点注册成立公司， 是通用的，并可适应各种修改。因此，我们认为， 提出了两种互补的方法：第一种方法涉及 亚磷酰胺化学，其中大量合成RNA 可以在一个常规的方式获得;第二个涉及更多的 一般来说，酶促掺入适用于所有 修饰的碱基和更大的生物学上有趣的RNA 分子。这些研究将使我们了解 对RNA结构和功能的影响。 在特定位点修饰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 改性基地的影响。RNA是人们关注的焦点， 它催化特定反应的能力。经修饰的RNA， 增强的催化活性也可能具有潜在的治疗效果， 应用.

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.