COMPARISON OF ANTIPARALLEL A-CENTER-DOT-AT AND T-CENTER-DOT-AT TRIPLETS WITHIN AN ALTERNATE STRAND DNA TRIPLE-HELIX

We have examined the formation of alternate strand triple-helices at the target sequence A(11)(TC)(6)-(GA)(6)T-11 using the oligonucleotides T-11(AG)(6) and T-11(TG)(6), by DNase I footprinting. These third strands were designed so as to form parallel T-AT triplets together with antiparallel G-GC and A-AT or T-AT triplets. We find that, although both oligonucleotides yield clear footprints at similar concentrations (0.3 mu M) in the presence of manganese, only T-11(TG)(6) forms a stable complex in magnesium-containing buffers, albeit at a higher concentration (10-30 mu M). Examination of the interaction of (AG)(6) and (TG)(6) with half the target site confirmed that the complex containing A-AT triplets was only stable in the presence of manganese. In contrast no binding of (TG)(6) was detected in the presence of either metal ion, suggesting that the reverse-Hoogsteen T-AT triplet is less stable that G-GC. We suggest that, within the context of G-GC triplets, the rank order of antiparallel triplet stability is A-AT (Mn2+) > T-AT (Mn2+) > T-AT (Mg2+) > A-AT (Mg2+). Third strands containing a single base substitution in the centre of either the parallel or antiparallel portion showed a (10-fold) weaker interaction in manganese-containing buffers, and no interaction in the presence of magnesium.