title: Thermodynamic properties of a conformationally constrained intramolecular DNA triple helix.
abstract: We describe the thermodynamic properties of an intramolecular triple helix with two all-thymine linker loops in which the Hoogsteen strand is 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 triplex without the disulfide crosslink. Optical and calorimetric measurements reveal that the uncrosslinked parent triplex melts in a biphasic manner above pH 6, with the initial triplex to duplex transition (Hoogsteen strand release) occurring at lower temperatures than subsequent melting of the hairpin helix. By contrast, crosslinking increases the thermal stability of the Hoogsteen transition such that the triplex and underlying hairpin duplex melt as a single transition under all conditions studied. Model independent thermodynamic data obtained by differential scanning calorimetry reveals the crosslink-induced increase in triplex thermal stability corresponds 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 triplex stabilization of 3 kcal/mol due to a reduction in the entropy change associated with triplex melting. In an effort to define the origin(s) of this entropic impact, we measured the pH and ionic strength dependence of the melting transitions. From a comparison of the melting transitions at different pH values and ionic strengths, we estimate that 0.4 more protons are associated with the crosslinked triplex state than with the uncrosslinked triplex, and 1.3 fewer counterions are released on melting the crosslinked triplex. We discuss how such crosslink-induced 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.
authors.affiliation: Department of Chemistry, Rutgers, State University of New Jersey, Piscataway 08855-0939, USA.
authors.forename: J
authors.initials: J
authors.lastname: Völker
authors.forename: S E
authors.initials: SE
authors.lastname: Osborne
authors.forename: G D
authors.initials: GD
authors.lastname: Glick
authors.forename: K J
authors.initials: KJ
authors.lastname: Breslauer
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011522:Protons; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D013816:Thermodynamics
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D009838:Oligodeoxyribonucleotides; D011522:Protons; D004247:DNA