Triplex-forming oligonucleotides are able to modulate gene expression by site-specific binding to genomic DNA. Their use as therapeutic agents is limited by inefficient cellular uptake, scarce nuclear internalization, and oligonucleotide self-aggregation. In this study, we demonstrate that a 13-mer AG motif oligonucleotide covalently linked to a high-molecular mass (9000 Da) polyethylene glycol (PEG ODN(13)) exhibits uptake and biological properties that are superior to those of the nonconjugated isosequence analogue (free ODN(13)). Band-shift and footprinting experiments showed that PEG ODN(13) forms a stable triple helix (apparent K(d) between 10(-6) and 10(-7) M in 50 mM Tris-acetate, 10 mM MgCl(2), pH 7.4, 37 degrees C) with a natural polypurine-polypyrimidine target located in the 5' flanking region of the human bcr/abl oncogene. Confocal laser microscopy performed on unfixed live cells stained with hexidium iodide as well as on glass-fixed cells stained with propidium iodide showed that fluorescein-labeled PEG ODN(13) is far more efficiently taken up and internalized in the nucleus by K562 and HeLa cells than the nonconjugated free ODN(13). It was found that PEG ODN(13) specifically downregulated the transcription of bcr/abl mRNA at 65 +/- 5% with respect to control and inhibited cell growth by 32 +/- 3% in a 3 day liquid culture assay. Moreover, PEG ODN(13) was more resistant against S1 and fetal bovine serum nucleases than free ODN(13), and less inclined to self-associate into multistrand structures in solution. Taken together, these results provide useful elements for designing artificial transcription repressors with enhanced potency in vivo.