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Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding
D A Brown1, S H Kang, S M Gryaznov
1Department of Neuropharmacology, Scripps Research Institute, La Jolla, California 92037.
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.
Insights
Phosphorothioate modification enhances oligodeoxynucleotide (ODN) stability but increases nonspecific protein binding. This binding may negate the therapeutic benefits of fully modified ODNs.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Phosphorothioate modification stabilizes oligodeoxynucleotides (ODNs) for therapeutic use.
- This modification can lead to increased nonspecific effects, which are not well understood.
Purpose of the Study:
- To investigate the relationship between the extent of phosphorothioate modification and cellular retention and protein binding of ODNs.
- To determine if increased protein binding affects ODN potency and specificity.
Main Methods:
- Flow cytometry was used to assess cellular association of fluorescein-labeled ODNs with varying phosphorothioate modifications.
- Gel shift assays were employed to evaluate cytoplasmic and nuclear protein binding.
- Gel shift competition assays were performed to assess transcription factor binding specificity.
Main Results:
- Fully phosphorothioate-modified ODNs showed significantly greater cellular association compared to 3'-terminally modified ODNs.
- Fully modified ODNs exhibited increased cytoplasmic and nuclear protein binding.
- Transcription factor binding by fully modified ODNs was found to be nonspecific.
Conclusions:
- Cellular retention and protein binding of ODNs are dependent on the degree of phosphorothioate modification.
- Increased nonspecific protein binding associated with full phosphorothioate modification may counteract the therapeutic advantages of ODNs.