The overall goal of the work described in this proposal is aimed at understanding the range of conformations that DNA can adopt, both in vivo and in vitro, and the principles underlying the folding of DNA into these non-B-DNA conformations. To this end, we propose to investigate two classes of folded DNA molecules: (1) DNA triplexes and (2) DNA aptamers. Multidimensional proton and heteronuclear NMR methods will be used to study the conformation and dynamics of the DNA oligonucleotides. DNA triplexes: In order to elucidate the factors governing sequence specific recognition of DNA by third strand binding to form triplexes, we propose to investigate the structures and cation dependence of several different triplexes and related structures. These include DNA triplexes with modified bases, ribose and 2'Omethyl ribose third strands, purine third strands, and crossover triplexes. We also propose to determine optimal loop sequences for intramolecular triplexes. Studies of a novel triplex-guanine 'clamp' are also planned. DNA aptamers: Aptamers are RNA or DNA molecules which have been selected, from a large pool of oligonucleotides containing a region of random nucleotide sequence, for binding to a specific target molecule. In collaboration with the Szostak laboratory, we will determine the structures of a variety of DNA aptamers which have been selected for binding to the cofactors ATP. GTP, biotin, and riboflavin. These will provide insight into the tertiary structure of nucleic acid binding pockets for protein cofactors. Ultimately, we also plan to determine the structures of DNA catalysts which contain cofactor binding sites. Since many of the folded oligonucleotides which we propose to study are too large to obtain well-defined structures using 1H spectroscopy alone, we plan to develop and optimize methods for synthesizing uniformly 15N and/or 13C labeled DNA oligonucleotides. Once the labeled DNA oligonucleotides are obtained, optimal methods for assignment and structure determination using double and triple-resonance NMR experiments will be developed, similar to those already widely used in protein NMR spectroscopy and more recently applied to labeled RNA oligonucleotides. Where possible, complete three-dimensional structures will be refined from starting structures generated by metric matrix distance geometry calculations. An understanding of the three-dimensional non-B-DNA structures that DNA can adopt and the factors that stabilize these conformations is important for a number of reasons. The solution of new DNA structures should lead to a better understanding of the principles of nucleic acid folding, stability, and sequence specific recognition by ligands including other nucleic acids. These studies should also provide a structural basis for potential applications of nucleic acids as pharmaceuticals, biosensors, and diagnostics.
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