Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
5372155 | Biophysical Chemistry | 2007 | 11 Pages |
The thermal stability and ligand binding properties of the l-argininamide-binding DNA aptamer (5â²-GATCGAAACGTAGCGCCTTCGATC-3â²) were studied by spectroscopic and calorimetric methods. Differential calorimetric studies showed that the uncomplexed aptamer melted in a two-state reaction with a melting temperature Tm = 50.2 ± 0.2 °C and a folding enthalpy ÎH°fold = â 49.0 ± 2.1 kcal molâ 1. These values agree with values of Tm = 49.6 °C and ÎH°fold = â 51.2 kcal molâ 1 predicted for a simple hairpin structure. Melting of the uncomplexed aptamer was dependent upon salt concentration, but independent of strand concentration. The Tm of aptamer melting was found to increase as l-argininamide concentrations increased. Analysis of circular dichroism titration data using a single-site binding model resulted in the determination of a binding free energy ÎG°bind = â 5.1 kcal molâ 1. Isothermal titration calorimetry studies revealed an exothermic binding reaction with ÎH°bind = â 8.7 kcal molâ 1. Combination of enthalpy and free energy produce an unfavorable entropy of â TÎS° = + 3.6 kcal molâ 1. A molar heat capacity change of â 116 cal molâ 1 Kâ 1 was determined from calorimetric measurements at four temperatures over the range of 15-40 °C. Molecular dynamics simulations were used to explore the structures of the unligated and ligated aptamer structures. From the calculated changes in solvent accessible surface areas of these structures a molar heat capacity change of â 125 cal molâ 1 Kâ 1 was calculated, a value in excellent agreement with the experimental value. The thermodynamic signature, along with the coupled CD spectral changes, suggest that the binding of l-argininamide to its DNA aptamer is an induced-fit process in which the binding of the ligand is thermodynamically coupled to a conformational ordering of the nucleic acid.