Flexible synthetic methodology has been developed for site-specifically modifying both DNA and RNA with structurally non-perturbing disulfide cross-links. This chemistry circumvents many of the problems commonly associated with other cross-linking protocols, and in preliminary experiments has been used to prepare cross-linked hairpins, duplexes, triplexes, non-ground-state DNA conformations, and RNA secondary structures. Because this modification removes the initiation site(s) for thermal denaturation, the cross-lined nucleic acids possess extraordinary long range stability. Most importantly, preliminary evidence clearly indicates that these disulfide cross-links do not appear to alter native conformation. Furthermore, in the reduced form the free thiol groups that form the cross-links do not compromise the thermodynamic stability or alter the denaturation pathway of the modified DNA/RNA relateive to the corresponding wild-type sequences. While these experiments have provided insights into both DNA and RNA that were not otherwise possible, routine application of this chemistry to many different problems necessitates a comprehensive understanding of the conformational, dynamic, and thermodynamic consequences of incorporating these cross-links. To meet this need three coordinated subprojects have been designed. First, using NMR spectroscopy we propose to elucidate the solution conformations and measure the base-pair opening kinetics for (bis)- and (mono)-cross-linked analogs of d(CGCGAATTCGCG)2. The thermodynamic stability of these duplexes in their reduced (free thiol) and oxidized (disulfide cross-linked) forms will be determined by UV thermal denaturation experiments and differential scanning calorimetry (DSC). The data from these experiments will be compared with those obtained using the parent duplex to judge the effects of the disulfide modifications. Second, the structural, dynamic, and thermodynamic properties of cross-linked triplexes based on the pyrimidine.purine-pyrimidine sequence motif will be examined under physiologically relevant conditions using NMR and CD spectroscopies and DSC. These studies will demonstrate the utility of disulfide cross-links in stabilizing conformationally-labile DNA structures, and the proposed experiments are not possible with the unmodified sequences. Lastly, in analogy to recent protein folding experiments, disulfide cross-links will be used to elucidate the fold pathway of small RNA's using tRNA (Phe as a model system. Specifically, four tRNA (Phe) molecules that each contain a single disulfide cross-link bridging two structural domains will be chemically synthesized. The solution conformation of these tRNA's (oxidized and reduced) will be probed by chemical modification experiments to verify that the thiol/disulfides do not alter RNA geometry. The free energy of folding and the kinetics of unfolding will then be studied by UV thermal denaturation and stopped-flow spectrophotometry, respectively. The free energies of these transitions will be interpreted in the context of a folding pathway for tRNA(Phe).
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