We have investigated the interaction of ohgonucleotides and their alkylating derivatives with mammalian cells. In experiments with L929 mouse fibroblast and Krebs 2 ascites carcinoma cells, it was found that cellular uptake of oligodeoxynucleotide derivatives is achieved by an endocytosis mechanism. Uptake is considerably more efficient at low oligomer concentration (<1 jAM), because at this concentration a significant percentage of the total oligomer pool is absorbed on the cell surface and internalized by a more efficient absorptive endocytosis process. Two modified proteins were detected in mouse fibroblasts that were treated with the alkylating oligonucleotide derivatives. The binding of the oligomers to the proteins is inhibited by other oligodeoxynucleotides, single-and double-stranded DNA, and RNA. The polyanions heparin and chondroitin sulfates A and B do not inhibit binding. These observations suggest the involvement of specific receptor proteins in binding of oligomers to mammalian cells.Antisense oligodeoxynucleotides and their derivatives have been shown to be specific inhibitors ofgene expression. They have been considered as a potential new generation of drugs, capable perhaps of inhibiting various pathogens and of regulating specific gene expression by inhibiting the translation of mRNA molecules in a highly specific manner (1-4). However, a prevailing view is that cells are not very permeable to oligonucleotides. Considerable efforts have been made to design nonionic membrane-permeable analogs (5) and to develop special delivery techniques (6, 7). Nevertheless, it has also been shown that normal unsubstituted oligonucleotides can cause hybridization arrest of specific mRNAs and viruses in cell culture (8-11). These observations provide evidence that oligodeoxynuicleotides do indeed enter cells. Recently, the ability of oligonucleotides to enter mammalian cells has been proved experimentally (9,(11)(12)(13)(14).The present investigation studied the interaction of oligodeoxynucleotide derivatives with mammalian cells. We examined the efficiency of oligomer binding and the time course of oligomer internalization under various conditions. We also investigated the stability of the internalized oligomers. Experiments involving the reactive 4-[N-(2-chloroethyl)-N-methyl]aminobenzyl phosphamide derivative of oligodeoxynucleotides [general formula ClRCH2NHpTTFr..., where R = -CH2CH2N(CH3)C6Hr---(1)]t were also undertaken: reagents of this type have recently been used by us as inhibitors of the influenza and tick-borne encephalitis viruses (4,15). In the present study, these reagents were used to chemically modify the putative cellular receptors binding oligonucleotides. MATERIALS AND METHODSOligodeoxynucleotides and Their Derivatives. The oligodeoxyribonucleotides pT, (n = 8, 9, 10, 16)

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Phosphodiester oligodeoxynucleotides bearing a 5' cholesteryl (chol) modification bind to low density lipoprotein (LDL), apparently by partitioning the chol-modified oligonucleotides into the lipid layer. Both HL60 cells and primary mouse spleen T and B cells incubated with fluorescently labeled chol-modified oligonucleotide showed substantially increased cellular association by flow cytometry and increased internalization by confocal microscopy compared to an identical molecule not bearing the chol group. Cellular internalization of chol-modified oligonucleotide occurred at least partially through the LDL receptor; it was increased in mouse spleen cells by cell culture in lipoprotein-deficient medium and/or lovastatin, and it was decreased by culture in high serum medium. To determine whether chol-modified oligonucleotides are more potent antisense agents, we titered antisense unmodified phosphodiester and chol-modified oligonucleotides targeted against a mouse immunosuppressive protein. Murine spleen cells cultured with 20 microM phosphodiester antisense oligonucleotides had a 2-fold increase in RNA synthesis, indicating the expected lymphocyte activation. Antisense chol-modified oligonucleotides showed an 8-fold increase in relative potency: they caused a 2-fold increase in RNA synthesis at just 2.5 microM. The increased efficacy was blocked by heparin and was further increased by cell culture in 1% (vs. 10%) fetal bovine serum, suggesting that the effect may, at least in part, be mediated via the LDL receptor. Antisense chol-modified oligonucleotides are sequence specific and have increased potency as compared to unmodified oligonucleotides.

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