Use of Polyethylenimine-Adenovirus Complexes to Examine Triplex Formation in Intact Cells

Abstract

Triplex-forming oligonucleotides (TFOs) show potential for sequence-specific DNA binding and inhibition of gene expression. We have applied this antigene strategy using a TFO incorporating an Auger-emitting radio-nucleotide, ¹²⁵I, to study the production of double-strand breaks (dsb) in the rat aquaporin 5 (rAQP5) cDNA. ¹²⁵I-TFO bound to the pCMVrAQP5 plasmid in vitro in a dose-dependent manner and formed stable triplexes up to 65°C and in the presence of 140 mM KCl. Further, ¹²⁵I-TFO resulted in a predictable dsb when analyzed by Southern hybridization. To deliver TFOs to epithelial cells, we employed ¹²⁵I-TFO-polyethyleneimine-adenovirus (¹²⁵I-TFO-PEI-Ad) complexes. We hypothesized that these complexes would take advantage of adenoviral characteristics to transfer ¹²⁵I-TFO to the cell nucleus. Adenovirus-containing complexes brought about greater uptake and nuclear localization of TFOs compared with delivery with ¹²⁵I-TFO-PEI complexes alone. No significant degradation of ¹²⁵I-TFO was found after delivery into cells using PEI-Ad complexes and freezing and thawing. We next used PEI-Ad complexes to deliver ¹²⁵I-TFO and pCMVrAQP5 separately to epithelial cells to determine if triplexes can form de novo within cells, resulting in the specific dsb in the rAQP5 cDNA. After delivery, cell pellets were stored at -80°C for more than 60 days. Thereafter, plasmid DNA was isolated from cells and analyzed for dsb by Southern hybridization. However, none were detected. We conclude that under the experimental conditions employed, effective triplexes, with ¹²⁵I-TFO and pCMVrAQP5, do not form de novo inside cells.