Intracellular distribution of digoxigenin-labeled phosphorothioate oligonucleotides

The great potential of antisense oligonucleotides as selective inhibitors of gene expression has led to their development and use for therapeutic purposes. Antisense technology has had to satisfy several conditions, such as stability to nucleases, enhancement of cellular permeability, and selectivity for the target sequence. Some of these requirements have been addressed by the chemical development of nucleic acid analogs. Although modifications such as phosphorothioate substitutions have been used mainly for improved stability and C-5 propyne or morpholino modifications have been used for higher affinity, the major concern is, at present, inefficient cellular uptake. Carrier systems such as cationic liposomes, nanoparticles, or supramolecular biovectors have been proposed to enhance cellular uptake and nuclear accumulation. Nevertheless, the characterization of intracellular distribution is important in assessing an effective antisense effect. As an alternative approach to the study of the distribution of antisense oligonucleotides, this chapter describes the synthesis and purification of digoxigenin-labeled phosphorothioate oligonucleotides, and their use in the characterization of intracellular distribution in human Epstein-Barr virus-transformed B cells using immunocytochemistry with both confocal and electron microscopy.