A SIMPLE METHOD FOR THE STUDY OF THE 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 loca ted in the cytoplasm (messenger RNA) or the nucleus (pre-messenger RNA or DNA). This approach is conditioned by the presence of the antisens e molecule inside the cell at sufficient concentrations and in the app ropriate compartments. We propose in this paper a simple method for th e study of the cytosolic content of internalized oligonucleotides. Thi s method is based on the selective permeabilization of the plasmic mem brane by the detergent digitonin. By complexing to membrane cholestero l, the detergent creates pores through which soluble and diffusible sp ecies can escape outside the cell. The selectivity of membrane permeab ilization war controlled by wing compartment markers: lactate dehydrog enase (LDH) for cytosol, dextrane-rhodamine (DEX) and hexosaminidase ( HAM) for endocytic vesicles and lysosomes, respectively. Optimal digit onin concentrations and incubation times have been defined to reach th e following pattern of membrane permeabilization: LDH > 80%; DEX and H AM < 15%. The method was applied to monitor the quantity of extractibl e oligonucleotides from cells after endocytosis. The results showed th at phosphodiester and phosphorothioate oligomers are readily available in the cytosol (60-50% of the internalized species), whereas those be aring a hydrophobic moiety (fluorescein, cholesterol) are less diffusi ble probably owing to membrane binding. Internalization and cytosol pa rtition were found to depend an the chemical nature of the oligonucleo tide, 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 cell, and for a more appropriate choice of control sequences in antisense studies.