EFFECT OF COMPETING SELF-STRUCTURE ON TRIPLEX FORMATION WITH PURINE-RICH OLIGODEOXYNUCLEOTIDES CONTAINING GA REPEATS

Competition between tripler formation with double-stranded DNA and oligonucleotide self-association was investigated in 23mer GA and GT oligonucleotides containing d(GA)(5) or d(GT)(5) repeats. Whereas tripler formation with GT oligonucleotides was diminished when temperature increased from 4 to 37 degrees C, tripler formation with GA oligonucleotides was enhanced when temperature increased within the same range due to the presence of competing intermolecular GA oligonucleotide self-structure, This self-structure was determined to be a homoduplex stabilized by the internal GA repeats. UV spectroscopy of these homoduplexes demonstrated a single sharp transition with rapid kinetics (Tm = 38,5-43,5 degrees C over strand concentrations of 0.5-4 mu M, respectively, with transition enthalpy, Delta H = -89 +/- 7 kcal/mol) in 10 mM MgCl2, 100 mM NaCl, pH 7.0, Homoduplex formation was strongly stabilized by multivalent cations (spermine > Mg2+ = Ca2+) and destabilized by low concentrations of monovalent cations (K+ = Li+ = Na+) in the presence of divalent cations, However, unlike GA or GT oligonucleotide-containing triplexes, the homoduplex formed even in the absence of multivalent cations, stabilized by only moderate concentrations of monovalent cations (Li+ > Na+ > K+). Through the development of multiple equilibrium states and the resulting depletion of free oligonucleotide, it was found that the presence of competing self-structure could decrease triplex formation under a variety of experimental conditions.