The objective of the work described in this proposal is to study, using high field 1H and some 31P NMR spectroscopy, the range of conformations which short synthetic DNA oligomers adopt in solution in a variety of conditions, as model systems for DNA conformations which may exist in vivo. We also will explore the role of this conformational variability in the sequence specific recognition of DNA by selected drugs. Our emphasis is on DNA oligomers which may adopt alternative (non-B-DNA) structures under appropriate conditions. We propose to investigate the following systems: 1. Triple-stranded DNA structures formed from two homopyrimidine and one homopurine strand or two homopurine and one homopyrimidine strand. We will investigate the equilibrium between the triplex and duplex DNA structures, factors which stabilize the triplex, the structures of the triplexes, and the structure of the DNA in a duplex-triplex junction. 2. Structures of echinomycin-DNA complexes. We will continue our investigations of echinomycin complexes with different DNA sequences, with specific emphasis on formation, stability, and propagation of Hoogsteen base pairs next to echinomycin binding sites and elucidation of structural changes other than Hoogsteen base pairing which occur upon drug binding. 3. Z-B junctions and the nature of the B-Z transition. DNA sequences in which part of the molecule could potentially form Z-DNA will be investigated in conditions which favor Z-DNA in order to study the conformation of DNA at a junction between Z and B DNA and to give insight into the mechanism of the B-Z transition. We will also investigate the pH dependence of the B-Z transition. The oligomers will be studied in a variety of different salt and solvent conditions. One- and two-dimensional NMR techniques will be used to assign the resonances and obtain information on the stability of the DNA structures observed under different conditions. Distance constraints derived from NOEs and coupling constants will be used to build models of these structures. For selected cases, complete three-dimensional structures will be determined. These studies should help lead to an understanding of the role of alternative DNA conformation in protein and drug recognition and in genetic regulation. The studies on DNA triplexes should help delineate the structures of and factors determining triplex formation in vivo and provide information on the relative stabilities and sequence specificities of triplexes used as sequence specific probes for genomic sequencing and as antisense DNA to repress transcription.
Showing the most recent 10 out of 54 publications
