Specificity of antiparallel DNA triple helix formation

We have used DNase I footprinting to examine the formation of antiparallel DNA triple helices on DNA fragments containing the homopurine target sites (GGA)(2)GGX(GGA)(2)GG .(CCT)(2)CCZ(CCT)(2)CC (where X . Z is each base pair in turn). with the GA- and GT-rich oligonucleotides, (GGA)(2)GGN(GGA)(2)GG and (GGT)(2)GGN(GGT)(2)GG (N = each base in turn). These were designed to form G . GC and A . AT or T . AT triplets with a central N . XZ mismatch, which should bind in an antiparallel orientation, We find that almost all combinations generate DNase I footprints at low micromolar concentrations, bt each target site, the relative binding of the GA- and GT-containing oligonucleotides was not the same, suggesting that these two triplexes adopt different conformations. For a central GC base pair, the most stable complex is observed with a third strand generating a G . GC tripler as expected. A . GC is also stable, especially in the GT oligonucleotides. For a central AT base pair, all four bases form stable complexes though T . AT is favored for the GA-rich thirds strands and A . AT for the CT-rich strands. For a central CG base pair, the stable complexes are seen with third strands generating T . CG triplets, though A . CG and C . CG are stable with GT- and GA-containing oligonucleotides. respectively. C . TA is the best triplet at a central TA base pair. The third strands with central guanines avoided the formation of G . YR triplets on the fragments containing central pyrimidines, producing DNase I footprints which had slipped relative to the target site. These oligonucleotides bound at a different location, generating complexes containing 11 contiguous stable triplets at the 3'-end of the third strand. The results suggest rules for designing the best third strand oligonucleotides for targeting sequences in which homopurine tracts are interrupted by pyrimidines.