In previous work the rate of isotopic hydrogen exchange of the purine ring was determined for a collection of mono-, oligo-, and polynucleotide structures using techniques of laser Raman spectroscopy. The exchange kinetics were correlated with the type of nucleotide, the molecular conformation, and the hydrogen bonding interactions of the purine ring. It was determined that the exchange rate can be employed to monitor the kind and amount of DNA or RNA secondary structure, including the number of strands in multi-stranded helices and the presence of Hoogsteen-type interactions. The solvent accessibility of the 7N ring site has emerged as a particularly influential factor in determining the kinetics of 8C-H exchange. In the proposed work the Raman dynamic probe will be exploited to determine the molecular environment of A and G residues of viral RNA and DNA in native plant and bacterial viruses, and in partially assembled and mutant virions. We shall also investigate several well characterized nucleoprotein complexes, such as the fd virus gene 5 protein-DNA complex, the phage lambda (cI and Cro) repressor-operator and E. coli lac repressor-operator systems. The kinetic studies will be augmented with equilibrium Raman studies to determine changes in DNA and RNA structure which may accompany protein binding. These investigations will provide new information on the nature of protein-nucleic acid interactions which are important for virus assembly and stability, and for gene regulation. The project will improve our understanding of the nature of protein/nucleic acid recognition. We shall employ the powerful techniques of optical multichannel analysis and Fourier deconvolution for rapid and accurate data acquisition and analysis. The proposed applications will advance the use of laser Raman spectroscopy both as a complement to other structure-determining methods and as a quantitative probe of nucleic acid structures and dynamics.
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