Use of polyethylenimine-adenovirus complexes to examine tripler formation in intact cells

Tripler-forming oligonucleotides (TFOs) show potential for sequence-specifi c DNA binding and inhibition of gene expression. We have applied this antig ene strategy using a TFO incorporating an Auger-emitting radionucleotide, I -125, to study the production of double-strand breaks (dsb) in the rat aqua porin 5 (rAQP5) cDNA, I-125-TFO bound to the pCMVrAQP5 plasmid in vitro in a dose-dependent manner and formed stable triplexes up to 65 degrees C and in the presence of 140 mM KCl. Further, I-125-TFO resulted in a predictable dsb when analyzed by Southern hybridization. To deliver TFOs to epithelial cells, we employed I-125-TFO-polyethyleneimine-adenovirus (I-125-TFO-PEI-A d) complexes. We hypothesized that these complexes would take advantage of adenoviral characteristics to transfer I-125-TFO to the cell nucleus. Adeno virus-containing complexes brought about greater uptake and nuclear localiz ation of TFOs compared with delivery with I-125-TFO-PEI complexes alone. No significant degradation of I-125-TFO was found after delivery into cells u sing PEI-Ad complexes and freezing and thawing. We next used PEI-Ad complex es to deliver I-125-TFO and pCMVrAQP5 separately to epithelial cells to det ermine if triplexes can form de novo within cells, resulting in the specifi c dsb in the rAQP5 cDNA, After delivery, cell pellets were stored at -80 de grees C for more than 60 days. Thereafter, plasmid DNA was isolated from ce lls and analyzed for dsb by Southern hybridization, However, none were dete cted, We conclude that under the experimental conditions employed, effectiv e triplexes, with I-125-TFO and pCMVrAQP5, do not form de novo inside cells .