Competitive Triplex/Quadruplex Equilibria Involving Guanine-Rich Oligonucleotides

Oligonucleotide-directed triple helix formation in the purine motif involves the binding of guanine-rich oligonucleotides to duplex DNA. Although this approach has been proposed for in vivo gene inhibition, triple helix formation by guanine-rich oligonucleotides is severely inhibited by physiological concentrations of certain monovalent cations (M⁺), especially K⁺. To clarify the mechanism of this inhibition, electrophoretic gel mobility shift titrations were performed to analyze the formation and stability of a purine motif triple helix in the presence of M⁺ and to monitor oligonucleotide aggregation under these conditions. M⁺ inhibition of triplex formation exhibited a concentration and ionic radius dependence that correlates with the ability of M⁺ to stabilize guanine quartet structures. In the presence of inhibitory [M⁺], guanine-rich oligonucleotides formed aggregates having characteristics consistent with the involvement of guanine quartets. The inhibitory effects of K⁺ on triplex formation could not be reversed by addition of the physiological polyamines spermidine³⁺ or spermine⁴⁺. M⁺ reduced the equilibrium concentration of the triplex primarily by decreasing the rate of triplex formation, but M⁺ also caused a detectable increase in the rate of triplex dissociation. Together, these results suggest that triplex inhibition under physiological ionic conditions is caused by competing equilibria wherein guanine-rich oligonucleotides form aggregates involving guanine quartets. Approaches to destabilizing aggregates of guanine-rich oligonucleotides under physiological conditions will be required before in vivo applications can be realistically considered.