NMR studies of DNA three-way junctions containing two unpaired thymidine bases: the influence of the sequence at the junction on the stability of the stacking conformers.

DNA three-way junctions (TWJs) containing unpaired residues at the branch point can adopt a conformation in which one helix is stacked upon another, forming a coaxial, quasicontinuous double helix. As in four-way junctions (FWJs), two conformers with different stacking arrangements between the arms are possible. However, in both types of structures a markedly strong preference for one conformer has been observed. To investigate the basis for this preference, in particular the influence of the stacking proclivity of the base-pairs at the centre of the junction, two linear oligomers (36 nucleotides), TWJ1 and TWJ2, differing only in one base-pair (G.C versus C.G, respectively) at the branch point, were designed and chemically synthesized. Each one is expected to fold into a stable three-way junction, containing two unpaired thymidine bases at the junction region and two arms capped with a hairpin loop. The data obtained from 1H and 31P-NMR spectroscopy confirm that both oligomers are present as stable three-way junctions. In both TWJs two of the helical arms stack preferentially upon each other. However, the stacking arrangement is similar in both molecules. From this it is deduced that purine-purine stacking across the junction cannot be considered as a major factor that determines the preferred stacking arrangement.