Conformational differences between bulged pyrimidines (C-C) and purines (A-A, I-I) at the branch point of three-stranded DNA junctions.

We have synthesized DNA oligomers that can combine to form three-way junctions containing six base pairs in each stem and two unpaired bases at the branch point. Gel electrophoresis experiments indicate that the oligomers form stable complexes with equimolar stoichiometry. Using two- and three-dimensional proton nuclear magnetic resonance spectroscopy, we have completed nonexchangeable proton chemical shift assignments for three junctions which differ only in the identity of the unpaired bases (C-C, A-A, or I-I) at the branch point. Our results indicate that unpaired pyrimidines at the branch point of junctions behave differently than do unpaired purines. In a junction with two unpaired cytidines, the 5' base loops out from the molecule to lie along the minor groove of the preceding duplex stem of the junction. The 3' unpaired cytidine also demonstrates an unusual pattern of NOE connectivities with detected cross peaks to the subsequent base in the 3' direction. Junctions with unpaired purines at the branch point exhibit different behavior. Our data suggests that in these molecules the unpaired bases participate in stacking interactions among themselves and with the neighboring bases in the molecule. Despite these differences, the NOE patterns from each junction suggest the presence of a preferred, pair-wise stacking between two of the helices within the molecule. The structural differences between bulge-pyrimidine and bulge-purine junctions are discussed in light of the functional significance unpaired bases might have in the structure and dynamics of multistranded DNA junctions and, by extension, to junctions within cellular RNAs.