Proton NMR study of the base-pairing reactions of d(GGAATTCC): salt and polyamine effects on the imino proton exchange

Abstract

Salts and polyamines have a variety of effects on the physical properties of DNA, including stabilization against thermal melting. We wished to gain greater insight into the mechanism of this stabilization by ascertaining its effect on the dynamics of base opening and closing reactions, as measured by NMR. Since the binding of spermidine(3+) is influenced by salt, and since spermidine may act as a base catalyst in proton exchange reactions, we have undertaken a study of salt and base catalyst effects on the imino proton exchange kinetics of a model oligomeric DNA. The selective longitudinal NMR relaxation rates of the hydrogen-bonded imino protons of the self-complementary octadeoxyribonucleotide d(GGAATTCC) monitor the rate of the base-catalyzed chemical exchange of these protons with solvent water. The exchange rates thus obtained provide a sensitive measure of the base-pair opening reactions of the DNA duplex. Under conditions of low pH and no added base catalyst, the NMR relaxation rates allow the determination of kd, the rate constant for the dissociation of the octameric duplex into single strands. Titration with the base catalyst tris(hydroxymethyl)aminomethane allows the determination of kop, the rate constant for the localized opening of individual base pairs, prior to dissociation. A significant Na+ concentration dependence is found for kd. From an analysis of this dependence, it is determined that 0.6 .+-. 0.1 sodium ion is released during the dissociation event. The activation energy for helix dissociation (200 .+-. 5 kJ/mol) is not dependent on the sodium ion concentration, indicating that the dissociation is entropically driven by the release of bound sodium ions. In agreement with previous results, no measurable salt dependence is found for kop, which is equal to about 100 s-1 at 25.degree.C. Under low-salt conditions, the trivalent cation spermidine decreases the rate of helix dissociation, again without affecting the activation energy for this process. Diprotonated spermidine(2+) acts as an extremely effective catalyst of imino proton exchange.