BINDING, UPTAKE, AND INTRACELLULAR TRAFFICKING OF PHOSPHOROTHIOATE-MODIFIED OLIGODEOXYNUCLEOTIDES

An enhanced appreciation of uptake mechanisms and intracellular traffi cking of phosphorothioate modified oligodeoxynucleotides (P-ODN) might facilitate the use of these compounds for experimental and therapeuti c purposes, We addressed these issues by identifying cell surface prot eins with which P-ODN specifically interact, studying P-ODN internaliz ation mechanisms, and by tracking internalized P-ODN through the cell using immunochemical and ultrastructural techniques, Chemical cross-li nking studies with a biotin-labeled P-ODN (P-b-ODN), revealed the exis tence of five major cell surface P-ODN binding protein groups ranging in size from similar to 20-143 kD, Binding to these proteins was compe titively inhibited with unlabeled P-ODN, but not free biotin, suggesti ng specificity of the interactions, Additional experiments suggested t hat binding proteins likely exist as single chain structures, and that carbohydrate moieties may play a role in P-ODN binding, Uptake studie s with S-35-labeled P-ODN revealed that endocytosis, mediated by a rec eptor-like mechanism, predominated at P-ODN concentrations <1 mu M, wh ereas fluid-phase endocytosis prevailed at higher concentrations, Cell fractionation and ultrastructural analysis demonstrated the presence of ODN in clathrin coated pits, and in vesicular structures consistent with endosomes and lysosomes, Labeled ODN were also found in signific ant amounts in the nucleus, while none was associated with ribosomes, or ribosomes associated with rough endoplasmic reticulum (ER), Since n uclear uptake was not blocked by wheat germ agglutinin or concanavalin A, a nucleoporin independent, perhaps diffusion driven, import proces s is suggested, These data imply that antisense DNA may exert their ef fect in the nucleus, They also suggest rational ways to design ODN whi ch might increase their efficiency.