Triplex formation by morpholino oligodeoxyribonucleotides in the HER-2/neupromoter requires the pyrimidine motif

Triplex-forming oligonucleotides (TFOs) are good candidates to be used as s ite-specific DNA-binding agents. Two obstacles encountered with TFOs are su sceptibility to nuclease activity and a requirement for magnesium for tripl ex formation. Morpholino oligonucleotides were shown in one study to form t riplexes in the absence of magnesium. In the current study, we have compare d phosphodiester and morpholino oligonucleotides targeting a homopurine-hom opyrimidine region in the human HER2/neu promoter. Using gel mobility shift analysis, our data demonstrate that triplex formation by phosphodiester ol igonucleotides at the HER-2/neu promoter target is possible with pyrimidine -parallel, purine-antiparallel and mixed sequence (GT)-antiparallel motifs. Only the pyrimidine-parallel motif morpholino TFO was capable of efficient triple helix formation, which required low pH. Triplex formation with the morpholino TFO was efficient in low or no magnesium. The pyrimidine motif T FOs with either a phosphodiester or morpholino backbone were able to form t riple helices in the presence of potassium ions, but required low pH. We ha ve rationalized the experimental observations with detailed molecular model ing studies. These data demonstrate the potential for the development of TF Os based on the morpholino backbone modification and demonstrate that the p yrimidine motif is the preferred motif for triple helix formation by morpho lino oligonucleotides.