The central objectives of this research is to elucidate the conformations, configurations and interactions of nuclei acids and nucleotide analogs in biological macromolecular assemblies. The methodology focuses on the application of Raman equilibrium and dynamic probes of nucleic acid structure and hydrogen isotope exchange kinetics.
The specific aims of the proposed research are: (1) To establish new Raman spectra/structure correlations which facilitate DNA and RNA structure determinations in biological assemblies. (2) To further develop the Raman dynamic probe for discrimination and resolution of structure sensitive exchange processes in nucleic acids and their complexes. (3) To apply the Raman methodologies to biological assemblies which cannot readily be investigated by other structural techniques. Realization of these aims will significantly advance our understanding of DNA (and RNA) packaging, conformation and interactions in viruses and cell nuclei. Information will also be obtained to advance our understanding of DNA molecular sites and secondary structure which may be involved in transcriptional regulation and gene expression. In the proposed equilibrium studies, we shall investigate model nucleic acids of known three dimensional structure (oligomeric DNA crystals solved by x-ray methods) for the purpose of expanding our library of spectra/structure correlations to include conformation-sensitive cytosine, adenine and phosphodiester vibrational modes. These empirical studies will be augmented with normal mode analyses. In the proposed dynamics studies, we shall investigate purine 8CH exchanges, at the level of individual nucleotides in DNA of defined sequence, in order to characterize the effects of base sequence on helix groove dimensions as manifested by solvent accessibility. The Raman dynamic probe will be further developed to distinguish the more rapid imino exchanges of base- paired structures. We shall continue our development of Raman single channel and multi-channel instrumentation for more rapid and accurate data acquisition from nucleic acids and their complexes. Fourier deconvolution, curve fitting and related data reduction protocols will be employed to optimize data analysis, refinement and interpretation.
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