HELICAL STACKING IN DNA 3-WAY JUNCTIONS CONTAINING 2 UNPAIRED PYRIMIDINES - PROTON NMR-STUDIES

The proton NMR spectra of DNA three-way junction complexes (TWJ) havin g unpaired pyrimidines, 5'-TT- and 5'-TC- on one strand at the junctio n site were assigned from 2D NOESY spectra acquired in H2O and D2O sol vents and homonuclear 3D NOESY-TOCSY and 3D NOESY-NOESY in D2O solvent . TWJ are the simplest branched structures found in biologically activ e nucleic acids. Unpaired nucleotides are common features of such stru ctures and have been shown to stabilize junction formation. The NMR da ta confirm that the component oligonucleotides assemble to form confor mationally homogeneous TWJ complexes having three double-helical, B-fo rm arms. Two of the helical arms stack upon each other. The unpaired p yrimidine bases lie in the minor groove of one of the helices and are partly exposed to solvent. The coaxial stacking arrangement deduced is different from that determined by Rosen and Patel (Rosen, M. A., and D. J. Patel. 1993. Biochemistry. 32:6576-6587) for a DNA three-way jun ction having two unpaired cytosines, but identical to that suggested b y Welch et al. (Welch, J. B., D. R. Duckett, D. M. J. Lilley. 1993. Nu cleic Acids Res. 21:4548-4555) on the basis of gel electrophoretic stu dies of DNA three-way junctions containing unpaired adenosines and thy midines.