[A simple method for the study of cytosolic content of oligonucleotides in cells]

Antisense oligonucleotides are currently used for the specific control of the expression of a selected gene. Their putative targets are located in the cytoplasm (messenger RNA) or the nucleus (pre-messenger RNA or DNA). This approach is conditioned by the presence of the antisense molecule inside the cell at sufficient concentrations and in the appropriate compartments. We propose in this paper a simple method for the study of the cytosolic content of internalized oligonucleotides. This method is based on the selective permeabilization of the plasmic membrane by the detergent digitonin. By complexing to membrane cholesterol, the detergent creates pores through which soluble and diffusible species can escape outside the cells. The selectivity of membrane permeabilization was controlled by using compartment markers: lactate dehydrogenase (LDH) for cytosol, dextrane-rhodamine (DEX) and hexosaminidase (HAM) for endocytic vesicles and lysosomes, respectively. Optimal digitonin concentrations and incubation times have been defined to reach the following pattern of membrane permeabilization: LDH > 80%; DEX and HAM < 15%. The method was applied to monitor the quantity of extractible oligonucleotides from cells after endocytosis. The results showed that phosphodiester and phosphorothioate oligomers are readily available in the cytosol (60-50% of the internalized species), whereas those bearing a hydrophobic moiety (fluorescein, cholesterol) are less diffusible probably owing to membrane binding. Internalization and cytosol partition were found to depend on the chemical nature of the oligonucleotide, and also on the sequence and the cell type. This method could be useful for the selection of antisense molecules that exhibit the best internalization and distribution in cells, and for a more appropriate choice of control sequences in antisense studies.