THERMODYNAMIC PROPERTIES OF A CONFORMATIONALLY CONSTRAINED INTRAMOLECULAR DNA TRIPLE-HELIX

We describe the thermodynamic properties of an intramolecular triple h elix with two all-thymine linker loops in which the Hoogsteen strand i s covalently crosslinked to the underlying Watson-Crick hairpin duplex by means of a disulfide bridge. We compare these properties to those of the corresponding intramolecular tripler without the disulfide cros slink. Optical and calorimetric measurements reveal that the uncrossli nked parent tripler melts in a biphasic manner above pH 6, with the in itial tripler to duplex transition (Hoogsteen strand release) occurrin g at lower temperatures than subsequent melting of the hairpin helix. By contrast, crosslinking increases the thermal stability of the Hoogs teen transition such that the tripler and underlying hairpin duplex me lt as a single transition under all conditions studied. Model independ ent thermodynamic data obtained by differential scanning calorimetry r eveals the crosslink-induced increase in tripler thermal stability cor responds to a free energy stabilization of about 3 kcal/mol, with this stabilization being entirely entropic in origin. In other words, the crosslink is enthalpically neutral, but nevertheless, induces a triple r stabilization of 3 kcal/mol due to a reduction in the entropy change associated with tripler melting. In an effort to define the origin(s) of this entropic impact, we measured the pH and ionic strength depend ence of the melting transitions. From a comparison of the melting tran sitions at different pH values and ionic strengths, we estimate that 0 .4 more protons are associated with the crosslinked tripler state than with the uncrosslinked tripler, and 1.3 fewer counterions are release d on melting the crosslinked tripler. We discuss how such crosslink-in duced changes in proton binding and counterion release, in conjunction with potential changes in hydration and conformational freedom, could combine to give rise to the observed changes in entropy.