SOLID STATE NMR OF DNA DYNAMICS AND STRUCTURE AND INSTRUMENT DESIGN

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My research group will be involved in four major projects. The first project is the design and construction of a console for a very high field NMR spectrometer. The design of an NMR console capable of operating at proton frequencies ranging from 750 MHz. to 1000 MHz. is currently funded by the DOE, with the ultimate objective of construction a 1000 MHz. NMR at the Battelle pacific Northwest Laboratory in Richland, WA. During the next year, designs and prototype versions of the high speed acquisition system and the pulse programmer will be completed, installed and tested on the old NIH Program Project 500 MHz. spectrometer. The microVax computer which controls that console's operation will be replaced with a SGI workstation and software for controlling console function via a Silicon Graphics workstation will also be written and tested. During the next several years we will use NIH funding to assemble a 750MHz. NMR console which will incorporate all of these design enhancements. As a second project, we will continue our study of the internal dynamics of DNA oligomers using deuterium (2H), 13C, and 15N solid state NMR spectroscopy as our principle tools. Various sites on the bases of dT and dA in TnAn-containing DNA oligomers will be enriched with 13C and 15N in an attempt to distinguish the orientation of motional axes for the averaging of the asymmetry and the anisotropy of specific CSA tensors. Ultimately, solid state NMR techniques will be extended to the study of dynamics in DNA triplexes and Holliday junctions. As a third project, we proposed the first application of solid state NMR structural techniques like REDOR, DRAMA, and SEDOR(RFDR) to the measurement of internuclear distances in AnTn-containing DNA oligomers. We are interested in exploring the nature of structural features which result in the formation of a B'helix, and specifically we intend to use solid state NMR as a prove of the mutual orientation of bases within base pairs and between adjacent bases. Our fourth project involves the synthesis of labeled DNA oligomers. Selectively deuterated DNA oligomers have been synthesized in 100-200 milligram quantities, and we have succeeded in synthesizing 150 milligrams of [d(CGCGAATTCGCG)]2 selectively labeled at the C4, N1, and N3 positions of T7 and T8. In the near future we intend to synthesize dT and dA with the sugar rings labeled at C1', C2', C3', and C5'.