Project Summary DNA and RNA exhibit an amazing degree of conformational heterogeneity as required for their many biological functions, including replication and gene regulation. The importance of this heterogeneity in oligonucleotides (ODN) is becoming more evident as more non-canonical structures that play essential roles in both eukaryotic and prokaryotic organisms are identified. The variety of conformations assumed by ODNs, be they either canonical or non-canonical, is dictated by the balance between their intrinsic conformational properties and interactions with their environment, including interactions with ions, other nucleic acids, proteins and small molecules. In the proposed study this balance will be investigated at an atomic level of detail using both quantum mechanical (QM) and molecular dynamics (MD) based theoretical calculations. Towards this goal Aim 1 will involve further development of an empirical force field for nucleic acids that explicitly treats electronic polarizability based on the classical Drude oscillator. Emphasis will be on completion of the 1st generation RNA force field (FF), extension to modified nucleotides, improved base stacking, balancing the nonbond interactions, evaluation of the DNA and RNA FF, and subsequent optimization yielding a 2nd generation Drude FF. Intrinsic contributions to conformational heterogeneity to be studied in Aim 2 will include QM analysis of base stacking, including a wide range of non-canonical base stacked conformations, and MD-based studies on the contribution of the 2'OH moiety to the conformational heterogeneity of RNA. Aim 3 will investigate environmental contributions to ODN conformational heterogeneity including the impact of Mg+2 and the protein Hfq on RNA folding landscapes, of ions on the electrostatic environment of DNA using experimental and theoretical analyses of solvochromatic shifts and the impact of ions on base flipping, a structural change that may be considered a surrogate of unfolding. Systems to be studied include a range of DNAs of various sequences and RNA hairpins, pseudoknots, and riboswitches for which extensive experimental data are available, as well as the small non-coding RNAs PrrF1 and 2, which are potential targets for novel antibiotics. To facilitate both force field development and the intrinsic and environmental impact studies, a novel multidimensional Hamiltonian Replica Exchange Method (HREMD) that includes both long-range and local perturbations of the Hamiltonian will be developed. Studies of the impact of Mg+2 will be undertaken using a novel Grand-Canonical Monte Carlo (GCMC) method that allows sampling of the spatial distribution of ions in and around RNA. In combination, the proposed work will yield improved theoretical tools for studies of DNA and RNA, including a polarizable force field and novel sampling methodologies, and yield insights into the intrinsic and environmental contributions to both canonical and non-canonical structures of DNA and RNA. These tools and knowledge will greatly facilitate studies of ODNs by other laboratories, including the potential for targeting RNA for the development of novel antibiotics.