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.
Olivas
W M
WM
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805.
Maher
L J
LJ
D001483:Base Sequence; D002414:Cations, Monovalent; D004591:Electrophoresis, Polyacrylamide Gel; D006147:Guanine / Q000737:chemistry*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*
D002414:Cations, Monovalent; D009838:Oligodeoxyribonucleotides; D006147:Guanine