INTRACELLULAR ROUTING AND INHIBITORY ACTIVITY OF OLIGONUCLEOPEPTIDES CONTAINING A KDEL MOTIF

On internalization, oligonucleotides (ODN) remain mostly sequestered i n endocytic compartments. To increase their delivery into the cytosol and/or nucleus, which contain their targets, we attempted to guide the m into compartments containing the KDEL receptor. Antisense ODN, phosp hodiester protected at both ends, that are complementary to the AUG in itiation site of gag(HIV-1) mRNA (odn) were linked to a peptide ending with the Lys-Asp-Glu-Leu (KDEL) motif in a carboxyl-terminal position (odn-p-KDEL) or with the Lys-Asp-Glu-Ala (odn-p-KDEA) as a control. T he effect of odn substitution with a peptide was examined with regard to its accumulation, subcellular location, and activity in HepG2 cells . Although odn-p-KDEL was internalized 4-fold less than the correspond ing peptide-free odn, it was 5-fold more efficient in inhibiting gag(H IV-1) gene expression in HepG2 cells. The internalization of odn-p-KDE A was as low as that of odn-p-KDEL, but its biological activity was lo wer, close to that of the peptide-free odn. On endocytosis at 37 degre es, both conjugates as well as the peptide-free odn were found in a ne utral environment. However, the substitution of an odn with a KDEL mot if altered its intracellular trafficking; most of the odn-p-KDEL was f ound in the endoplasmic reticulum and in the intermediate compartment as identified by colabeling with either anti-ERGlC-53 or anti-KDEL rec eptor antibodies. Conversely, odn-p-KDEA and peptide-free odn were loc alized in vesicular compartments not labeled with these antibodies. In addition, pulse-chase experiments showed that odn-p-KDEL and odn-p-KD EA had a lower efflux than peptide-free odn. Therefore, the large incr ease in efficiency was due to the KDEL motif.