KINETIC AND THERMODYNAMIC CHARACTERIZATION OF DNA DUPLEX-HAIRPIN INTERCONVERSION FOR 2 DNA DECAMERS - D(CAACGGGTTG) AND D(CAACCCGTTG)

The duplex-hairpin interconversion of two DNA decamers, d(CAACGGGTTG) and d(CAACCCGTTG), has been characterized thermodynamically and kineti cally 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 enth alpies of duplex formation, measured by nmr, were found to be very sim ilar (Delta H-DH = 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 H-DH and kinetics is significantly dif ferent for each decamer. The d(CAACGGGTTG) decamer forms a duplex cont aining two central G.G mismatches ar 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 in terconversion between hairpin and duplex was concluded to proceed by c omplete strand dissociation. In contrast, the d(CAACGGGTTG) decamer wa s determined to form a duplex with two centrally located C.C mismatche s at pH values less than 6.2, consistent with the formation of a hemip rotonated C+.C mismatch. At pH values greater than 6.4, the hairpin-du plex equilibrium is almost completely shifted toward the hairpin confo rmation at DNA concentrations of 0.5-7.0 mM and salt concentrations of 10-100 mM. The interconversion of duplex and hairpin conformations wa s ascertained by means of both kinetic and thermodynamic measurements to proceed by a slightly different mechanism than its complementary de camer. Although the interconversion proceeds by complete strand separa tion as suggested by high duplex-hairpin infer-conversion activation e nthalpies, the increasing hairpin formation rate with increasing ionic strength as well as the Delta H-DH dependence on salt indicate that a n intermediate internally bulged duplex (no C+.C formation) is stabili zed by increasing ionic strength. These data support an interconversio n mechanism where an intermediate internally bulged duplex may be the rare limiting step before strand separation. (C) 1995 John Wiley & Son s, Inc.