Two-dimensional NMR and restrained molecular dynamics studies of the hairpin d(T8C4A8): detection of an extraloop cytosine.

The 1H and 31P NMR resonances of the partly self-complementary 20-mer DNA d(T8C4A8) were assigned by two-dimensional HOHAHA, NOESY, and heteronuclear COSY NMR spectroscopy. The chemical shifts, NOEs, and H-H coupling patterns are indicative of the formation of a hairpin structure with the four C residues forming a loop and the T8.A8 portion of a double-stranded stem. The observation of unusual across-strand NOEs between the A H2 and the T H1' of the corresponding 3'-end neighboring base pairs of the stem residues suggests that the structure of the hairpin stem deviates from regular B-DNA. A total number of 296 interproton NOEs were used as approximate proton-proton distance constraints in restrained molecular dynamics calculations. Several different starting models, all generated manually from standard B-DNA coordinates, gave rise to virtually the same refined hairpin structure. In the final structure, the interior A-T base pairs of the hairpin stem show a high degree of propeller twist as well as base pair buckle, while the minor groove is slightly narrower compared with a normal B-DNA structure; these features are all common to bent DNA. The first three A-T pairs from the end of the hairpin have a propeller twist and base pair buckle which more closely resemble those of regular B-DNA. The four-residue loop was formed mainly by variations in the phosphate backbone torsion angle epsilon at the loop-stem junctions (residues 8 and 13) and at the first C residue (C 9). The base of the first C residue is positioned outside of the loop.(ABSTRACT TRUNCATED AT 250 WORDS)