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Several oligodeoxynucleotides (ODNs) were designed in order to interact with the purine rich element of the IRE (Interferon Responsive Element) of the 6-16 gene by triplex formation. An ODN of 21 bases, the sequence being identical to that of the purine strand of the IRE (48% G), but in reverse orientation, was able to interact with the IRE (KD: 20 nM). The binding was Mg2+ dependent. The two purine strands of the triplex were oriented antiparallel as confirmed by DNAase I and copper-phenanthroline footprinting experiments. An ODN in which A were replaced by T, also interacted with the same target, but with a lower affinity. Exonuclease III action indicated that the two IRE repeats of the 6-16 promoter interacted with each other through Hoogsteen base pairing, the third strand being parallel to the paired Watson-Crick strand. This led to a potential H-DNA structure which could be destabilized by adding ODNs able to form a triplex structure. 6-16 IRE driven-reporter gene constructs lost their interferon stimulability when co-transfected with triplex forming ODNs. The range of effective ODN concentrations was compatible with the affinity determined when measuring their direct interactions with the DNA.

Inhibition of gene transcription by purine rich triplex forming oligodeoxyribonucleotides