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 oligodeoxyribonucleotides (ODN), 3'-fluorescence-tagged phosphodiester (P) and phosphorothioate (S) analogs of a series of model and vimentin and actin antisense ODN were applied to digitonin-permeabilized fibroblast and epithelial PtK2 cells. Fluorescence microscopy revealed binding of the ODN to intermediate filaments (Ifs) with a preference for cytokeratin Ifs, cytoplasmic membranes (endoplasmic reticulum), and, above all, the nuclear interior. The affinity of the ODN for these cellular substructures was dependent on their base composition, and the S-ODN were by far superior to the corresponding P-ODN in binding activity. Fluorescence polarization measurements of the interaction of ODN with purified IF proteins in vitro confirmed the differential, high-affinity binding 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 uptake of the ODN was considerably and differentially inhibited by wheat germ agglutinin. High-affinity S-ODN, but not P-ODN, additionally reacted with a structure presumably identical with the nuclear lamina. Simultaneously, 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 effects were not observed, although the nucleic acids rapidly moved into the nucleus and condensed into a large number of well-defined, spherical speckles or longitudinal rodlets. The methylphosphonate analogs of some of the ODN used exhibited only extremely low affinities for intracellular constituents. These results show that excess amounts of S-ODN saturate a host of both low-affinity and high-affinity binding sites on cellular substructures, whereas limited quantities as used for microinjection 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 components and substructures involved in replicational, transcriptional, and translational processes. On the other hand, the association of the ODN with membranes and cytoskeletal and karyoskeletal elements may serve to optimize their sequence-specific interaction with their intended target sites and also increase their cellular retention potential. These cellular structures would thus fulfill a depot function.