Oligonucleotide Uptake and Delivery in Tissue Culture Cells

Triplex forming oligonucleotides (TF-ODNs) hold promise for the specific modulation of gene expression (1), and recent data indicate that intermolecular triplex formation can take place in living cells and in chromatin (2, 3, 4, 5, 6). However, TF-ODNs must overcome significant cellular barriers in order to arrive at the target gene in the nucleus. Oligonucleotides designed for triple helix anti-gene applications must cross the cell membrane, escape the endosome, and resist degradation by nucleases to be available for triplex formation in the nucleus. Oligonucleotides (ODNs) do not passively diffuse across cell membranes, and available data indicate uptake into cells by adsorptive endocytosis, fluid phase pinocytosis, or endocytosis after binding to specific receptors on the surface of cells, such as the integrin protein, Mac-1 (7). Uptake of ODNs from cell culture media varies widely by cell type (8, 9) and may be insufficient for the desired biological effects of the ODN because of low uptake efficiency and sequestration in endosomal vesicles (7).