Triple helix formation by (G,A)-containing oligonucleotides: Asymmetric sequence effect

Sequence effects on the stability of purine-motif (also called (G,A)-motif) triple helix have been investigated through mio symmetry-related systems: one of them had a 5'(GGA)(4)3' core sequence of triplex-forming oligonucleo tides (TFOs), whereas the other one had a reversed 5'(AGG)(4)3' core sequen ce. These (G,A)-containing TFOs were prone to self-associate into intermole cular complexes at room temperature. The competition of TFOs' self-associat ion with triple helix formation was assessed, and minimized. By varying the lengths and the terminal base sequences of TFOs, the following were found that (1) The stability of two triple helices with identical length and base composition but reverse strand orientation may be significantly different (up to a factor of 6). (2) When the 5'(GGA)(4)3' core sequence was extended at the 3'-end by a G, the 13-nt TFO exhibited 3- and 5-fold higher affinit y toward the target double-stranded DNA (dsDNA) than the longer 14-nt and 1 5-nt TFOs in which one and two A(s) were added at the 3'-end of the 13-nt T FO, respectively. In contrast, when the similar extensions occurred at the 5'-end of the 5'(AGG)(4)3' core sequence, the length increase provided a hi gher binding affinity of TFOs toward the target duplex. (3) The nature of t he base triplets involved at the ends of triple helices may have great infl uence on triplex stability. The observed asymmetric sequence effect of the (G,A)motif triple helix formation is discussed in terms of the binding stre ngth of the first base triplet(s) at the 3' end which seems to be deeply in volved in the nucleation step of triple helix formation and therefore to be a determining factor for triplex stability.