Detection of competing DNA structures by thermal gradient gel electrophoresis: from self-association to triple helix formation by (G,A)-containing oligonucleotides

Sequence-specific recognition of DNA can be achieved by triple helix-forming oligonucleotides that bind to the major groove of double-helical DNA. These oligonucleotides have been used as sequence-specific DNA ligands for various purposes, including sequence-specific gene regulation in the so-called 'antigene strategy'. In particular, (G,A)- containing oligonucleotides can form stable triple helices under physiological conditions. However, triplex formation may be in competition with self- association of these oligonucleotides. For biological applications it would be interesting to identify the conditions under which one structure is favoured as compared to the other(s). Here we have directly studied competition between formation of a parallel (G,A) homoduplex and that of a triple helix by a 13 nt (G,A)-containing oligonucleotide. Temperature gradient gel electrophoresis allows simultaneous detection of competition between the two structures, because of their different temperature dependencies and gel electrophoretic mobilities, and characterisation of this competition. stability of a (C,T) motif triple helix is pH dependent. In the purine motif (G,A)-containing oligonucleotides bind in an antiparallel orientation to the oligopurine strand of the duplex by forming C·GG and T·AA base triplets through reverse Hoogsteen hydrogen bonding interactions. (G,T)-containing oligonucleotides can form triplexes involving either Hoogsteen or reverse Hoogsteen hydrogen bonds and have a parallel or antiparallel orientation with respect to the target oligopurine strand sequence, respectively. It has been shown that the preferred strand orientation depends on the sequence of (G,T)- containing oligonucleotides (5). The formation of both (G,A) and (G,T) motif triple helices is pH independent but requires the presence of divalent cations in the millimolar concentration