Effect of temperature and ionic strength on the dissociation kinetics and lifetime of PNA-DNA triplexes.

Abstract

Dissociation kinetics of triplexes formed by molecules of peptide nucleic acid (PNA) and DNA have been studied. The complexes consisted of oligomeric PNA containing 10 thymine bases and the dA(10) target incorporated in single-stranded (ssDNA) or double-stranded DNA (dsDNA). Their dissociation was followed by means of the gel mobility shift assay at various temperatures and sodium ion concentrations. In all experiments, the dissociation kinetics of triplexes were exponential; the effective lifetime of a triplex, tau, depended on temperature in accordance with the Arrhenius law. The tau values for T(10) PNA complexes with ss- and dsDNA were equal within the accuracy of experiments. The activation energy, U, value for T(10) PNA-DNA complexes did not change when the NaCl concentration was increased from 50 to 200 or 600 mM. Conversely, the tau values decreased with the increase in NaCl concentration. The equal lifetimes of the T(10) PNA-DNA triplexes containing ss- and dsDNA suggest that the loop formed in dsDNA does not noticeably affect the triplex structure. The decrease in the triplex lifetime tau with an increase in ionic strength was accounted for by the fact that the PNA backbone is neutral. The lack of relationship between the activation energy of dissociation and salt concentration suggests that the dissociation enthalpy does not depend on the ionic strength. Thus, the effect of ionic strength on the lifetime is entropic by its nature. Contrary to this, for complexes of ssDNA with bis-PNA 1743, which also consists of 10 thymine bases but contains 2 additional positive charges inside the sequence in 1 of the PNA arms, an increase of the dissociation enthalpy at low salt concentration was observed. We suggest that this effect is a result of a direct electrostatic interaction of the positive charges of the PNA with the DNA backbone. Finally, our results allow an estimate of the lifetime of a 10-mer triplex invasion complex in dsDNA at 37 degrees C in excess of several hundred days.