Addition of antisense oligonucleotides to cell cultures has been used to specifically inhibit gene expression. We have investigated the mechanism by which oligonucleotides enter living cells. These compounds are taken up by cells in a saturable, size-dependent manner compatible with receptormediated endocytosis. Polynucleotides of any length are competitive inhibitors of oligomer transport, providing they possess a 5'-phosphate moiety. Using oligo(dT)-cellulose for affinity purification, we have identified an 80-kDa surface protein that may mediate transport. Knowledge of the oligonucleotide transport mechanism should facilitate the design of more effective synthetic antisense oligomers as potential clinical agents.When oligodeoxynucleotides [oligo(dN)s] complementary to the 5' region of c-myc mRNA are added to cells in culture, c-myc protein synthesis is specifically inhibited (1)(2)(3)(4)(5). Furthermore, addition ofantisense oligo(dN)s to cultures inhibits intracellular viral replication (6)(7)(8) Fig. 1 Top depicts a typical fluorescence histogram comparing cells incubated with no oligo(dN) to those incubated for 24 hr with either acridine-labeled oligo(dN) alone or in the presence of excess unlabeled oligo(dN). Intracellular localization of fluorescence was confirmed by fluorescence microscopy of similarly treated cells (Fig. 1 Middle and Bottom). When we examined the rates of accumulation of variously sized acridine-labeled oligo(dN)s we found that the accumulated intracellular fluorescence after incubation of HL60 cells with 12.5 AtM acridine-labeled oligomers [ranging in size from oligo(dT)3 to oligo(dT)20] increased gradually, plateauing within -50 hr after addition of acridine-labeled oligo(dN) to the culture medium ( Fig. 2A). This is in contrast to the 90 min required

Oligodeoxynucleotides with a phosphorus atom in which one of the non-bridging oxygen atoms is substituted by selenium were prepared and investigated with respect to their antisense properties. A general synthesis of phosphoroselenoate analogs of oligonucleotides is described using potassium selenocyanate as the selenium donor. The compounds, characterized by 31P NMR, were shown to decompose to phosphate with a half-life of ca. 30 days. Melting temperatures of duplexes between poly(rA) or poly(rI) with oligo(dT) and oligo(dC), respectively, indicate diminished hybridization capability of phosphoroselenoate oligomers relative to both the unmodified phosphodiester oligomers and the phosphorothioate congeners. A phosphoroselenoate 17-mer is a sequence specific inhibitor of rabbit beta-globin synthesis in wheat germ extract and in injected Xenopus oocytes. In contrast phosphoroselenoate analogs are potent non-sequence specific inhibitors in rabbit reticulocyte lysate. In vitro HIV assays were carried out on a phosphoroselenoate sequence and compared with a phosphorothioate analogue that has previously been shown to exhibit anti-HIV activity (Matsukura et al., Proc. Natl. Acad. Sci. (1987) 84, 7706-7710). The phosphoroselenoate was somewhat less active, and was much more toxic to the cells.

One mismatch is discriminated in a target mRNA sequence by an inducible alkylation system based on sulfide precursors to the nucleoside 2‐amino‐6‐vinylpurine (see scheme). The reactive oligonucleotides were delivered into the cell as poly(ethylene glycol) (PEG) conjugates in polyion‐complex (PIC) micelles and showed antisense activity of high selectivity and greater potency than that of the natural antisense oligonucleotide.

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We report here the synthesis of novel 5'-linked oligodeoxynucleotides, both normal phosphodiester and phosphorothioate analogs, in which a covalently attached group at the Y-terminus is an anthraquinone. These compounds represent a new class of antisense compounds in which the base sequence of the oligodeoxynucleotide serves to deliver a nuclease-resistant reactive drug-like molecule to a cellular target nucleic acid (mRNA or DNA).

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