SYNTHESIS OF 15N-LABELED RNA FRAGMENTS

这项提案的长期目标是开发和展示 制备和利用15N所需的合成策略 标记的RNA片段。这种位点特异性15N标记分子 有可能成为无价的核酸结构和 互动。事实上，有一些类型的信息可能是 仅可从15N标记分子的15N核磁共振中获得。目前，调制解调器 具备必要能力的高场光谱仪一般 可用，但合成方法学是必要的 15N标记的RNA片段不是。 这项研究的第一个目标是开发通向15N的合成路线 标记嘌呤核苷[1-15N]腺苷，[6-15N]-腺苷，[3-15N]- 腺苷，[7-15N]-腺苷，[1-15N]-鸟苷，[2-15N]-鸟苷，[3- 15N]-鸟苷和[7-15N]-鸟苷。第二个目标是发展 方法将允许将这些标记的单体转化为 15N标记的RNA片段，数量充足且足够 用于核磁共振研究的纯度。这些合成将通过以下方式进行 一种已成功用于合成苯并三甲胺的氢磷酸盐方法 15N标记的DNA片段。氢磷酸盐的一个特殊优势 方法是单体可以回收和重复使用。目前的方法 然而，为了保护核糖核苷，不要给予足够高的 YR保护单体的产率适用于有价值的标签 核苷。因此，这项建议的一个重要综合目标是 开发新的合成程序，使获得 受保护单体的高产率。这一目标的实现将是 对于价格较低的单体也很有用。我们的程序将是 对任何需要改进RNA合成方法的研究人员都很有用， 在规模和经济很重要的地方，它将特别有价值。 最终目标是开始使用这些15N标记的RNA片段来 探测RNA的结构和相互作用，以开发更好的 了解RNA折叠的原理，RNA的特异性- 蛋白质的相互作用，以及RNA作为酶发挥作用的能力。 具体地，将使用[1-15N]、[2-15N]和[6-15N]标签 主要用于监测碱基配对，而[7-15N]和[3-15N]标签 也将用于探测主槽和次槽中的水合情况 与其他在复杂RNA中发生的鲜为人知的相互作用一样 结构。

The long-term goals of this proposal are to develop and demonstrate the synthetic strategies necessary for the preparation and utilization of 15N labeled RNA fragments. Such site-specifically 15 N labeled molecules have the potential to be invaluable probes of nucleic acid structure and interactions. In fact, there are some types of information which may be available only from 15N NMR of 15 N labeled molecules. At present, modem high-field spectrometers with the necessary capabilities are generally available, but the synthetic methodology necessary for preparation of the 15 N labeled RNA fragments is not. The first goal of this research is to develop synthetic routes to the 15N labeled purine nucleosides [1-15N] adenosine, [6-15N]-adenosine,[3-15N]- adenosine, [7-15N]-adenosine,[1-15N]-guanosine, [2-15N]-guanosine, [3- 15N]-guanosine, and [7-15N]-guanosine. The second goal is to develop methodology which will allow conversion of these labeled monomers into 15N labeled RNA fragments, in sufficient quantity and of sufficient purity for use in NMR studies. These syntheses will be carried out by an H-phosphorate method that has been used successfully for synthesis of 15N labeled DNA fragments. A particular advantage of the H-phosphorate method is that the monomers can be recovered and reused. Present methods for protection of ribonucleosides, however, do not give high enough yields of yr protected monomers to be applicable to valuable labeled nucleosides. Thus, an important synthetic aim of this proposal is to develop new synthetic procedures which will make it possible to obtain high yields of protected monomers. Achievement of this goal will be useful for less expensive monomers as well. Our procedures will be useful for any researchers needing improved methods for RNA synthesis, and will be particularly valuable where scale and economy are important. The final goal is to begin to use these 15N labeled RNA fragments to probe RNA structure and interactions in order to develop a better understanding of the principles of RNA folding, the specificity of RNA- protein interactions, and the ability of RNA to function as an enzyme. Specifically, the [1-15N], [2-15N], and [6-15N] labels will be used primarily to monitor base-pairing, while the [7-15N] and [3-15N] labels will be used to probes hydration in the major and minor grooves, as well as other poorly understand interactions that occur in complex RNA structures.