SYNTHESIS OF 15N-LABELED RNA FRAGMENTS

本提案的长期目标是开发和展示 15 N的制备和利用所需的合成策略 标记的RNA片段。 这种位点特异性15 N标记的分子 具有成为核酸结构的无价探针的潜力， 交互. 事实上，有一些类型的信息可能是 仅可从15 N标记分子的15 N NMR获得。 目前，MODEM 具有必要能力的高场光谱仪通常 现有的，但合成方法所需的制备的 15 N标记的RNA片段则不是。 本研究的第一个目标是开发15 N的合成路线 标记嘌呤核苷[1- 15 N]腺苷，[6- 15 N]-腺苷，[3- 15 N]- 腺苷，[7- 15 N]-腺苷，[1- 15 N]-鸟苷，[2- 15 N]-鸟苷，[3- 15 N]-鸟苷和[7- 15 N]-鸟苷。 第二个目标是发展 该方法将允许将这些标记的单体转化为 15 N标记的RNA片段，以足够的量和足够的 用于NMR研究的纯度。 这些合成将由 已成功用于合成的H-磷酸盐方法 15 N标记的DNA片段。 H-磷酸盐的一个特别优点是 方法是单体可以回收再利用。 本方法 然而，对于核糖核苷的保护， YR保护的单体的产率适用于有价值的标记的 核苷 因此，本提案的一个重要综合目标是： 开发新的合成程序，使其有可能获得 保护单体的高产率。 实现这一目标将是 也可用于较便宜的单体。 我们的程序将是 对于任何需要改进RNA合成方法的研究人员都是有用的， 并且在规模和经济性很重要的情况下将特别有价值。 最终目标是开始使用这些15 N标记的RNA片段， 探针RNA结构和相互作用，以便开发更好的 理解RNA折叠的原理，RNA的特异性， 蛋白质的相互作用，以及RNA作为酶的功能。 具体而言，将使用[1- 15 N]、[2- 15 N]和[6- 15 N]标签 主要用于监测碱基配对，而[7- 15 N]和[3- 15 N]标签 将被用来探测主要和次要沟槽中的水合作用， 就像其他在复杂RNA中发生的相互作用一样， 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.