The local structure of oligonucleotides can be exquisitely sensitive to the presence of multivalent cations in solution. This structural variability depends significantly on oligonucleotide sequence as well as cation type, and hence does not result from nonspecific cation associations. The goal of the proposed work is to clarify the origins of these structural effects. The dynamic behavior of bound cations is sensitive to the relative contributions of specific and nonspecific cation binding. These dynamics will be monitored by the nmr of the bound cations. The structural consequences of cation binding will in turn be determined by two-dimensional proton nmr. Multinuclear nmr and equilibrium binding studies are proposed in order to examine the roles of simple cations in stabilizing oligonucleotide conformations and in promoting oligonucleotide structural transitions. Circular dichroism will provide an initial characterization of cation-induced structural perturbations. Cation nmr relaxation and pulsed field gradient diffusion experiments will be used to monitor the mobility (translational, rotational and internal) of cations in oligonucleotide solution. Cation nmr experiments will also be used to examine the effect of DNA length and salt concentration on specific and nonspecific cation associations. These studies will be complemented by thermodynamic binding measurements of Donnan equilibria. Proton nmr, x-ray diffraction studies and molecular modelling will provide additional information regarding the nature of cation-induced structural perturbations, and the geometry of cation binding sites. The primary focus of the proposed work will be on double-helical oligonucleotide structures involving Watson-Crick base-pairing. However, exploratory work is also proposed to examine cation effects on unusual structures that may be important for homologous recombination (e.g. junction structures), regulation of transcription (e.g. triple helical structures) and for the stabilization of the ends of chromosomes (e.g. G-rich telomeric sequences). Specifically, experiments are proposed in order (1) To define cation effects on the overall geometry of selected oligonucleotides. (2) To characterize the local environment of specifically bound cations. (3) To correlate the dynamics of bound cations to cation-induced oligonucleotide structural transitions. (4) To determine experimentally the effect of DNA length on local and global cation binding to short oligonucleotides. (5) To explore cation binding to unusual oligonucleotide structures.
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