Solution structure of the parallel-stranded hairpin d(T8C4A8) as determined by two-dimensional NMR

The structure of the oligodeoxynucleotide (3')T8(5')-(5')C4A8(3') hairpin in aqueous solution was studied by two-dimensional (2D) proton and phosphorus nuclear magnetic resonance (NMR) spectroscopy. At 2.5 mM and 10 degrees C, the molecule exists predominantly as a monomolecular hairpin with a C4 loop. At higher concentrations and lower temperatures, NMR signals from multimers are obvious. They account for approximately 25% of the total population at 4 mM and 10 degrees C. Nearly all of the proton NMR signals for the hairpin could be assigned using 2D COSY, HOHAHA, and NOESY experiments. 2D 1H-31P correlation experiments were used to assign all the phosphorus resonances and to provide an additional check for the sequential assignments. A parallel-stranded T8.A8 stem can be formed in the hairpin due to the presence of the unusual 5'-5' linkage in the loop. 2D NOESY experiments indicate that the A H2 and its 5'-end neighbor base pair T methyl protons are within 5 A of each other. This is in accord with reverse Watson-Crick base pairing between T and A, which locates the A H2 and the T methyl protons in the same groove of the duplex. The chemical shifts of A H1', H2', and H2" sugar and the H2 base protons are quite different compared to normal B-DNA. Analysis of the 2D COSY and NOESY cross peak patterns indicates that the deoxyribose rings are mainly in the C2'-endo conformation and that the stem forms a right-handed helix, with the two strands held together by eight reverse Watson-Crick A.T base pairs to form a parallel-stranded duplex. The backbone torsion angles, as determined from the 31P chemical shifts, are slightly different for the A and the T residues. A molecular model was constructed, using a total of 336 proton NOE cross peak intensities as proton-proton distance constraints. In the refined structure, the conformations of the sugar-phosphate linkage, the deoxyribose rings, and the glycosyl bonds for the two parallel strands of the hairpin are close to a regular B-DNA structure. The base-stacking and the hydrogen-bonding interactions are well optimized; however, the two grooves are of approximately equal width. Thus, compared to B-DNA, the parallel-stranded duplex has a very different surface shape, and because of the reverse Watson-Crick base pairing, it has different groups exposed in each groove.