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)2GGX(GGA)2GG.(CCT)2CCZ(CCT)2CC (where X.Z is each base pair in turn), with the GA- and GT-rich oligonucleotides, (GGA)2GGN(GGA)2GG and (GGT)2GGN(GGT)2GG (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. At 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 triplet 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 GT-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.