Abstract
Phosphorothioate modification of internucleoside linkages is widely used to prevent degradation of oligodeoxynucleotide (ODN) therapeutic agents in serum and cells. This modification generally increases ODN potency, but in many instances it is associated with an increase of poorly understood nonspecific effects. In this study, we have found that both cellular retention and nonspecific protein binding are dependent upon 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 modified ODNs (with three 3'-terminal phosphorothioate linkages). Additionally, gel shift assays with either ss- or ds-probes showed that fully phosphorothioate-modified ODNs also exhibit much greater cytoplasmic and nuclear protein binding than either 3'-terminally modified or unmodified ODNs. However, gel shift competition assays showed that transcription factor binding by fully phosphorothioate-modified ds-ODNs was completely nonspecific relative to 3'-terminally modified and unmodified ds-ODNs. These results suggest that the benefits derived from full phosphorothioate modification of ODNs may be negated by increases of nonspecific protein binding and associated sequence-independent effects.
Highlights
Whereas some studieshave shown that phosphorothioatebindingbyfully phosphorothioate-modifiedds-ODNs modified ODNs appear to acbt y sequence-specific mechanisms wascompletelynonspecificrelativeto3’-terminally
(15-181, other studies have showthnat phosphorothioate-modimodified and unmodified ds-ODNs. These results sug- fied ODNs cause a wide range of nonspecific effects
For exgest that the benefits derived from full phosphorothio- ample, phosphorothioate-modified ODNs lacking sequence ate modification of ODNs may be negated by increaspsecsifiocfity have been shown to result ineffects on cell growth, nonspecific protein binding and associated sequenceindependent effects
Summary
0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Val. 269, No 43, Issue of October 28,pp. 26801-26805,1994 Printed in U.S.A. Lysis I1 software after gating on lo[4] living cells using forward versus right angle scatter and further exclusion of propidium iodide positive more potent therapeutic agents than unmodified ODNs, presumably because they exhibit increased cellular uptake and resistance t o degradation by intracellular nucleases(10-12).In this study, we have end-labeled both ss- and ds-ODNs with fluorescein and analyzed cells by fluorescence-activated cell sorting to determine theeffect of the extenot f phosphorothioate cells. These data were expressed as the meanfluorescence intensity of each given cell population corrected for cellular autofluorescence. In cases of longer incubation time (up to[10] h) and higher concentration (up5 1to1~)of ODNs, the increase of mean fluorescence intensity waseven greater with
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