Slow conformational exchange in DNA minihairpin loops: a conformational study of the circular dumbbell d.

In recent years various examples of highly stable two-residue hairpin loops (miniloops) in DNA have been encountered. As the detailed structure and stability of miniloops appear to be determined not only by the nature and sequence of the two bases in the loop, but also by the closing base pair, it is desirable to carry out in-depth studies of especially designed small model DNA compounds. Therefore, a circular DNA dumbbell-like molecule is tailored to consist of a stem of three Watson-Crick base pairs, flanked on each side by a minihairpin loop. The resulting circular DNA decamer 5'-d-3' (I) is studied in solution by means of nmr spectroscopy. At a temperature of 269 K the molecule occurs in a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4). L2L2 contains three Watson-Crick C-G base pairs and two two-residue loops (H2-family type) in opposite parts of the molecule. On raising the temperature from 269 to 314 K, the L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted closing G-C base pair in the 5'-GTTC-3' loop with only one remaining solvent-accessible hydrogen bond between NH alpha of the cytosine C(1) and O6 of the guanine G(8), whereas the opposite 5'-CTTG-3' loop remains stable. The disruption of the C(1)-G(8) base pair in the L2L4 form is correlated with the presence of a syn orientation for the C(1) base at the 5'-3' loop-stem junction in the 5'-GTTC-3' loop. The two conformers, L2L2 and L2L4, occur in slow equilibrium (2-20 s-1). Moderate line broadening of specific 1H, 13C, and 31P resonances of residues C(1), G(8), T(9), and T(10) at low temperatures, due to chemical exchange between L2L2 and L2L4, show that the interconversion from an anti to syn conformer in residue C(1) has a small local effect on the structure of the dumbbell. T1 relaxation measurements, chemical-shift considerations, and complete band-shape calculations of the exchange process of the G(8) imino proton reveal a possibility for the existence of multiconformational states in the anti-syn equilibrium.