Cellular Internalization of Oligodeoxynucleotides
Although the addition of oligodeoxynucleotides to cell cultures has been demonstrated to effectively inhibit intracellular gene expression, such findings have been in contrast to the prevailing view that cells are impermeable to negatively charged large molecules. With this in mind, several groups have attempted to modify oligos in order to circumvent the problem of negative charge. Thus, methylphosphonates are ionically neutral, and poly-L-lysine has been linked to oligos to supply a strong positive charge. Others have made various oligonucleotide-protein conjugates to aid internalization, while the benefits of oligo encapsulation into liposomes have also been explored. During the last several years, it has become clear that even highly negatively charged oligonucleotides (i.e., phosphodiesters and phosphorothioates) are actively internalized by cells in culture. Furthermore, it has become equally apparent that backbone or pendant oligonucleotide modifications can alter olionucleotide uptake characteristics, although not always in the way intended. In this chapter, we will review the current state of knowledge concerning internalization pathways for modified and unmodified oligonucleotides and how these pathways may affect the intracellular trafficking of oligonucleotides.
KeywordsHerpes Simplex Virus Type Cellular Internalization Acidic Compartment Phosphorothioate Oligos Endocytic Uptake
Unable to display preview. Download preview PDF.
- Boutorin AS, Gus’kova, LV, Ivanova EM, Kobetz ND, Zarytova VF, Ryte AS, Yurchenko LV, Vlassov VV (1989): Synthesis of alkylating oligonucleotide derivatives containing cholesterol of phenazinium residues at their 3-terminus and their interaction with DNA within mammalian cells. FEBS Lett 254: 129–132PubMedCrossRefGoogle Scholar
- Dautry-Varsat A, Ciechanover A, Lodish HF (1983): pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci USA 80: 2258–2262Google Scholar
- Geselowitz DA, Neckers L (1992): Analysis of oligonucleotide binding, internalization and intracellular trafficking utilizing a novel radiolabeled crosslinker. Antisense Res Dev (In Press)Google Scholar
- Harel-Bellan A, Ferris DK, Vinocour M, Holt JT, FarrarWL (1988): Specific inhibition of c-myc protein biosynthesis using an antisense synthetic deoxy-oligonucleotide in human lymphocytes. J Immunology 140: 2431–2435Google Scholar
- Harewood K, Pape K, Gabel C, Suleske R, Cunningham A (1991): Cellular uptake and localization of fluorescein-labelled, 15 mer phosphorothioate and phosphodiester oligonucleotides. J Cell Biochem 15D: 35Google Scholar
- Kulka M, Smith CC, Aurelian L, Fishelevich R, Meade K, Miller P, Tso POP (1989): Site specificity of the inhibitory effects of oligo(nucleoside methylphosphonate)s complementary to the acceptor splice junction of herpes simplex virus type 1 immediate early mRNA 4. Proc Natl Acad Sci USA 86: 6868–6872PubMedCrossRefGoogle Scholar
- Lemaitre M, Bayard B, Lebleu B (1987): Specific antiviral activity of a poly(L-lysine)-conjugated oligodeoxyribonucleotide sequence complementary to vesicular stomatitis virus N protein mRNA initiation site. Proc Natl Acad Sci USA 84: 648–652Google Scholar
- Neckers LM (1989): Antisense oligonucleotides as a tool for studying cell regulation: mechanism of uptake and application to the study of oncogene function. In: Oligodeoxynucleotides: Antisense inhibitors of gene expression, Cohen J, ed. London: Macmillan PressGoogle Scholar
- Pastan I, Willingham M, Anderson W, Gallo M (1977): Localization of serum-derived a2 macroglobulin in cultured cells and decrease after Moloney sarcoma virus transformation. Cell 609–617Google Scholar
- Zhang XH, Loke SL, Neckers L (1990): unpublished observationsGoogle Scholar