Kinetic and thermodynamic characterization of DNA duplex-hairpin interconversion for two DNA decamers: d(CAACGGGTTG)...

The duplex-hairpin interconversion of two DNA decamers, d(CAACGGGTTG) and d(CAACCCGTTG), has been characterized thermodynamically and kinetically by using uv-melting and nmr relaxation methods. Separately, each decamer shows slow exchange between hairpin and duplex conformations. The hairpin conformations have melting points of 47 and 50 degrees C, respectively, and exhibit similar thermodynamic stabilities. The enthalpies of duplex formation, measured by nmr, were found to be very similar (delta HDH = 26 +/- 3 kcal/mole) for both decamers at low salt concentrations (< 50 mM NaCl). However, as the salt concentration was increased the behavior of delta HDH and kinetics is significantly different for each decamer. The d(CAACGGGTTG) decamer forms a duplex containing two central G.G mismatches at high salt and DNA concentration. Based upon the measurement of high interconversion activation energies and a decrease in hairpin formation rate with increasing salt, the interconversion between hairpin and duplex was concluded to proceed by complete strand dissociation. In contrast, the d(CAACCCGTTG) decamer was determined to form a duplex with two centrally located C.C mismatches at pH values less than 6.2, consistent with the formation of a hemiprotonated C+.C mismatch. At pH values greater than 6.4, the hairpin-duplex equilibrium is almost completely shifted toward the hairpin conformation at DNA concentrations of 0.5-7.0 mM and salt concentrations of 10-100 mM. The interconversion of duplex and hairpin conformations was ascertained by means of both kinetic and thermodynamic measurements to proceed by a slightly different mechanism than its complementary decamer. Although the interconversion proceeds by complete strand separation as suggested by high duplex-hairpin interconversion activation enthalpies, the increasing hairpin formation rate with increasing ionic strength as well as the delta HDH dependence on salt indicate that an intermediate internally bulged duplex (no C+.C formation) is stabilized by increasing ionic strength. These data support an interconversion mechanism where an intermediate internally bulged duplex may be the rate limiting step before strand separation.