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.
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