Benzoquinoquinoxaline derivatives stabilize and cleave H-DNA and repress transcription downstream of a triplex-forming sequence.

Oligopyrimidine*oligopurine sequences with potential to form intramolecular triple helix structures (H-DNA) have been found mainly in high eukaryote genomes. However, the natural occurrence and function of H-DNA remains elusive largely because we lack appropriate reagents to demonstrate the formation of these structures in cells. We examined whether a triple-helix specific stabilizing compound, benzoquinoquinoxaline (BQQ), and its 1,10-phenanthroline derivative can be efficiently utilized to study the formation and stabilization of an intramolecular triple-helical DNA structure in growing Escherichia coli cells and in vitro. Cell uptake of BQQ was confirmed by fluorescence microscopy. A plasmid carrying an H-DNA forming sequence upstream of a reporter gene was used to assess the effects of H-DNA formation and stabilization in growing cells. The presence of the H-DNA forming sequence dramatically repressed beta-lactamase expression, and sub-growth-inhibitory doses of BQQ caused a further 40% reduction. Most importantly, repression was dependent on the triple-helix forming sequence and correlated with the addition of BQQ. As the abundance of the H-DNA forming plasmid was not affected by the addition of BQQ, the dose-dependent reduction at the protein level observed here is likely caused by repression of transcription. Finally, the triple-helix specific interaction of BQQ with the target DNA sequence was demonstrated using a triple-helix directed cleavage assay by BQQ-1,10-phenanthroline conjugate in vitro.