ELECTRON MICROGRAPHIC STUDIES OF TRANSPORT OF OLIGODEOXYNUCLEOTIDES ACROSS EUKARYOTIC CELL-MEMBRANES

Unmodified oligodeoxynucleotides (ODNs) were synthesized and tested fo r their ability to cross external eukaryotic cell membranes and to ent er the cytosol and nucleus in tissue cultures. The ODNs were labeled w ith high-specific-activity [H-3]thymidine (greater-than-or-equal-to 10 0 Ci/mmol), or [alpha-P-32]ATP or [gamma-P-32]ATP (300-1000 Ci/mmol; 1 Ci = 37 GBq), and the label was either in the central portion of the molecule or at the 3' or 5' end. The cells employed were for the most part 3T6 murine fibroblasts, grown in monolayers, either semiconfluent or confluent, but some experiments were carried out with chicken embr yo fibroblasts or human HeLa cells. Parallel wells in the same experim ent were prepared for electron microscopy or for cell fractionation an d radioactivity assays. Electron microscopic autoradiography indicated that ODNs cross the external cell membrane, traverse the cytosol, and begin to enter the cell nucleus within a few seconds to 5 min at 37-d egrees-C in Dulbecco's medium without added serum. After 30-60 min of incubation with ODNs, abundant silver grains were observed at or just inside the nuclear membrane or well distributed across the nucleus, pa rticularly in association with euchromatin. There was a paucity of sil ver grains associated with nucleoli. Cell entry of oligomer was relate d to cell cycling events and was energy dependent. Degradation of olig omer to monomers, with reincorporation into DNA, does not appear to ex plain these results. No sequestration of labeled oligomer in cytoplasm ic vesicles en route from the exterior of the cell to the nucleus was observed. The observations are more suggestive of internalization of o ligonucleotide by a mechanism as yet unclear or, alternatively, by a c aveolar, potocytotic mechanism rather than by endocytosis.