EFFECT OF PHOSPHOROTHIOATE MODIFICATION OF OLIGODEOXYNUCLEOTIDES ON SPECIFIC PROTEIN-BINDING

Phosphorothioate modification of internucleoside linkages is widely us ed to prevent degradation of oligodeoxynucleotide (ODN) therapeutic ag ents in serum and cells. This modification generally increases ODN pot ency, but in many instances it is associated with an increase of poorl y understood nonspecific effects. In this study, we have found that bo th cellular retention and nonspecific protein binding are dependent up on the extent of the oligonucleotide's modification. Flow cytometry of cells treated with fluorescein-labeled single-stranded (ss) or double -stranded (ds) ODNs demonstrated that fully phosphorothioate-modified ODNs exhibit much greater cellular association than 3'-terminally modi fied ODNs (with three 3'-terminal phosphorothioate linkages). Addition ally gel shift assays with either ss- or ds-probes showed that fully p hosphorothioate-modified ODNs also exhibit much greater cytoplasmic an d nuclear protein binding than either 3'-terminally modified or unmodi fied ODNs. However, gel shift competition assays showed that transcrip tion factor binding by fully phosphorothioate-modified ds-ODNs was com pletely nonspecific relative to 3'-terminally modified and unmodified ds-ODNs. These results suggest that the benefits derived from full pho sphorothioate modification of ODNs may be negated by increases of nons pecific protein binding and associated sequence-independent effects.