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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