PHOSPHOROTHIOATE OLIGODEOXYRIBONUCLEOTIDES DISSOCIATE FROM CATIONIC LIPIDS BEFORE ENTERING THE NUCLEUS

Antisense oligonucleotides complementary to specific mRNA sequences ar e widely used inhibitors of gene expression in vitro and in vivo. In v itro cationic lipids have been demonstrated to increase the pharmacolo gical activity of antisense oligonucleotides by increasing cellular up take and facilitating nuclear accumulation. We have investigated the i ntracellular fate of oligonucleotide/cationic lipid complexes using fl uorescently labeled lipids and oligonucleotides targeted to protein ki nase C-alpha. After addition to cells the lipids initially co-localize d with the oligonucleotide on the cell surface and in fine punctate st ructures within the cytoplasm. At later times the oligonucleotide bega n to accumulate in the nucleus as well as the cytoplasm. In contrast, the cationic lipid remained localized to the cell surface and the cyto plasm and was never found in the nucleus. Expression of protein kinase C-alpha mRNA did not begin to decline until after oligonucleotide was seen in the nucleus. This was also coincident with the transient appe arance of a smaller mRNA transcript believed to result from RNase H cl eavage of protein kinase C-alpha mRNA. These data suggest that althoug h cationic lipids facilitate uptake of oligonucleotides, the complex m ust disassociate before the oligonucleotide can gain access to the nuc leus and induce degradation of targeted mRNA.