During the early 1980s there were two developments which lead to our studies of sequence specific DNA ligands. The first was the development of sequential assignment methods based on 2D NMR spectra which allowed complete assignment of resonances for proteins (Wüthrich, 1986). The assignments in turn allowed determination of many structural restraints through interpretation of NOESY crosspeaks and coupling constants from COSY type spectra. The second advance was the improvement of the chemistry for direct synthesis of DNA oligomers. With multimilligram samples of DNA oligomers available sequential assignment methods for DNA, paralleling those for proteins, were also worked out. Again with assignments came the possibility of determining DNA structures in solution. Howeverfor double stranded, Watson-Crick paired DNAs the structure can be reasonably approximated by the standard B-form model derived from fiber diffraction. The accurate determination of local conformational features has been somewhat difficult using NMR since tertiary contacts (as are so valuable in determining protein structures) do not occur. However with careful quantitative analysis some of the local details of structure can be determined. These NMR methods also offered the possibility of trying to understand the structural basis for binding of ligands to DNA oligomers. In order to make welldefined complexes we wanted to start with a compound that showed some sequence specificity in binding, and selected distamycin (shown below), a polypyrrole antibiotic which was known to have preference for binding to A-T rich DNA sequences. A close relative, netropsin, had been studied by Dinshaw Patel who showed that the binding is in the minor groove by identifying an NOE between a proton of the ligand and an adenosine H2 in the center of the minor groove (Patel, 1982). We began by making a complex with the self-complementary DNA oligomer: 5'-CGCGAATTCGCG-3', which had been studied extensively by X-- ray crystallography, and also by NMR. Distamycin did form a well-defined complex with this DNA, which was is slow exchange with free DNA during titrations (Klevit et al., 1986).
Limiting Behavior of the Target-Dependent Stochastic Sequential Assignment Problem
