Da. Brown et al., EFFECT OF PHOSPHOROTHIOATE MODIFICATION OF OLIGODEOXYNUCLEOTIDES ON SPECIFIC PROTEIN-BINDING, The Journal of biological chemistry, 269(43), 1994, pp. 26801-26805
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.