SOLID STATE NMR STUDIES OF DNA AND RNA DYNAMICS

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It has been long accepted that detailed structural information is necessary in order to fully understand the means by which duplex DNA interacts with transcription factors, and in general performs the full range of its biological functions. Accordingly, our view of the structure of DNA has evolved through the years, as high resolution structural models of DNA obtained by X-ray diffraction and solution NMR display sequence specific variations from the canonical B form. It is reasonable to ask how an equally detailed picture of the internal dynamics of DNA would modify our view of these structural variations and thus our understanding of how DNA performs its biological functions. Solid state NMR is an ideal technique for probing the localized dynamics of biopolymers from near solution conditions to the crystalline environment. In addition, a number of novel solid state NMR techniques have been developed in recent years that enable the definition of biomolecular structure with high accuracy and precision over the same broad range of sample conditions, with no molecular weight limit. In the next five years we plan to use solid state NMR structural and dynamical techniques to define the relationship(s) between structure, dynamics and function in DNA and RNA. Specifically we propose the following five stage program: 1) Use solid state NMR to explore the sequence specificity of localized dynamics in DNA oligomers containing binding sites for restriction enzymes, transcription factors and methyltransferases; 2) Use solid state NMR to investigate the dynamic and structural impact of DNA methylation, especially in CpG-rich DNA sequences; 3) Use solid state NMR techniques to investigate the dynamics and structure of the catalytic sites of the hammerhead and hairpin ribozymes; 4) Continue to develop and optimize solid state NMR techniques for structure elucidation in high molecular weight nucleic acids. Extend these NMR techniques to 17.6 Tesla by completing construction of a multi-resonant CPMAS probe, thus aiding in the completion of specific goals 1-4; 5) Synthesize all isotopically labeled DNA and RNA phosphoramidites required to achieve specific goals 1-4.