Thermodynamics of an intramolecular DNA triple helix: a calorimetric and spectroscopic study of the pH and salt dependence of thermally induced structural transitions.
We have characterized thermodynamically the melting transitions of a DNA 31-mer oligonucleotide (5'-GAAGAGGTTTTTCCTCTTCTTTTTCTTCTCC-3') which is designed to fold into an intramolecular triple helix. The first 19 residues fold back on themselves to form an antiparallel Watson-Crick hairpin duplex with a T5 loop. The 3'-terminal seven residues, which are connected to the Watson-Crick hairpin duplex by a second T5 loop, form Hoogsteen interactions in the major groove of the Watson-Crick hairpin. From ultraviolet (UV) melting studies we find that the 31-mer exhibits either one or two transitions, depending on solution conditions. We use pH- and temperature-dependent circular dichroism (CD) to assign the initial and final states associated with each transition. We find that the disruption of the Hoogsteen hairpin is accompanied by a release of protons and an uptake of sodium ions while the disruption of the Watson-Crick hairpin is accompanied by a release of sodium ions with no change in protonation state. From these data, we construct a phase diagram for this intramolecular DNA triple helix as a function of pH, sodium ion concentration, and temperature. We characterize the energetics of each transition using a van't Hoff analysis and differential scanning calorimetry (DSC). Significantly, the DSC data provide a model-independent thermodynamic characterization of the thermally induced transitions of this triplex. By combining the spectroscopic and calorimetric data, we develop a semi-empirical model which describes the state of the 31-mer as a function of pH, sodium ion concentration, and temperature. With this model we successfully predict characteristics of the 31-mer, which are beyond the data which are used in establishing the model (for example, the salt dependence of the apparent pKa of the Hoogsteen strand). This semi-empirical model may serve as a prototype for developing a method to predict the phase diagrams of intramolecular triple helix systems.
Plum
G E
GE
Department of Chemistry, Rutgers State University of New Jersey, New Brunswick 08903, USA.
Breslauer
K J
KJ
D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002412:Cations / Q000737:chemistry; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D012965:Sodium Chloride / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics
D002412:Cations; C051802:triplex DNA; D012965:Sodium Chloride; D004247:DNA