FLUORESCENCE MICROSCOPIC COMPARISON OF THE BINDING OF PHOSPHODIESTER AND PHOSPHOROTHIOATE (ANTISENSE) OLIGODEOXYRIBONUCLEOTIDES TO SUBCELLULAR STRUCTURES, INCLUDING INTERMEDIATE FILAMENTS, THE ENDOPLASMIC-RETICULUM, AND THE NUCLEAR INTERIOR

To detect potential intracellular binding sites for antisense oligodeo xyribonucleotides (ODN), 3'-fluorescence-tagged phosphodiester (P) and phosphorothioate (S) analogs of a series of model and vimentin and ac tin antisense ODN were applied to digitonin-permeabilized fibroblast a nd epithelial PtK2 cells. Fluorescence microscopy revealed binding of the ODN to intermediate filaments (Ifs) with a preference for cytokera tin Ifs, cytoplasmic membranes (endoplasmic reticulum), and, above all , the nuclear interior. The affinity of the ODN for these cellular sub structures was dependent on their base composition, and the S-ODN were by far superior to the corresponding P-ODN in binding activity. Fluor escence polarization measurements of the interaction of ODN with purif ied IF proteins in vitro confirmed the differential, high-affinity bin ding of S-ODN to Ifs. In permeabilized cells, the ODN readily migrated into the nucleus where, at ambient temperature, preferentially the S- ODN gave rise to a multitude of large, irregular aggregates. Nuclear u ptake of the ODN was considerably and differentially inhibited by whea t germ agglutinin. High-affinity S-ODN, but not P-ODN, additionally re acted with a structure presumably identical with the nuclear lamina. S imultaneously, they cause decompaction of chromatin, whereby the S-ODN aggregates appeared as compact inclusions in homogeneously dispersed chromatin. After microinjection of S-ODN into intact cells, these effe cts were not observed, although the nucleic acids rapidly moved into t he nucleus and condensed into a large number of well-defined, spherica l speckles or longitudinal rodlets. The methylphosphonate analogs of s ome of the ODN used exhibited only extremely low affinities for intrac ellular constituents. These results show that excess amounts of S-ODN saturate a host of both low-affinity and high-affinity binding sites o n cellular substructures, whereas limited quantities as used for micro injection recognize only the high-affinity binding sites. The results support the notion that the nonsequence-specific, often toxic effects of antisense S-ODN result from their strong binding to cellular compon ents and substructures involved in replicational, transcriptional, and translational processes. On the other hand, the association of the OD N with membranes and cytoskeletal and karyoskeletal elements may serve to optimize their sequence-specific interaction with their intended t arget sites and also increase their cellular retention potential. Thes e cellular structures would thus fulfill a depot function.