ACTIVE NUCLEAR IMPORT OF SINGLE-STRANDED OLIGONUCLEOTIDES AND THEIR COMPLEXES WITH NON-KARYOPHILIC MACROMOLECULES

The objective of this investigation was to characterize intranuclear a ccumulation of oligonucleotides and their adducts with non-karyophilic compounds in cultured animal cells and thus to present a model system for nucleic acid-mediated nuclear import. Ln digitonin-permeabilized cells, nuclear uptake of 3'-FITC-labeled, single-stranded 25-mer oligo deoxyribonucleotides was independent of added cytosolic protein, large ly energy-dependent, inhibitable by wheat germ agglutinin but not by N -ethylmaleimide, and a function of their base composition. When couple d to FITC-labeled streptavidin or streptavidin-bovine serum albumin co njugates, the oligonucleotides delivered the proteins to the nuclear i nterior with rates roughly proportional to their karyophilicity as fre e molecules. Transport activity was also demonstrated for single-stran ded oligoribonucleotides. The transport was energy-dependent, inhibite d by GMP-PNP and wheat germ agglutinin, but unaffected by N-ethylmalei mide. Nuclear import of oligo(dG)(25)/protein adducts needed 3 to 4 ol igonucleotide signals per complex and the signal had to be at least 15 nucleotides long. Microinjection experiments showed that the results obtained with digitonin-permeabilized cells are not artifacts of a qua si-intact cellular system. These data were confirmed by electron micro scopy employing complexes of oligodeoxyribonucleotides with streptavid in-peroxidase-bovine serum albumin-1 nm gold. In permeabilized cells, the complexes docked to the cytoplasmic face of the nuclear pore compl exes, were translocated through the central pore channel and accumulat ed in large quantities in the nuclear baskets before they were release d into the nucleoplasm. These results demonstrate that nuclear uptake of oligonucleotides and their complexes is an active process mediated by nuclear pore complexes, which, at least regarding its cytoplasmic c omponent, is different from the pathway requiring classical nuclear lo calization signals.