CHARACTERIZATION OF BINDING-SITES, EXTENT OF BINDING, AND DRUG-INTERACTIONS OF OLIGONUCLEOTIDES WITH ALBUMIN

Phosphorothioate oligonucleotides (S-ODNs) have the ability to modulat e gene expression selectively and thus have potential therapeutic capa bilities. This potential led us to investigate the protein binding cha racteristics of selected S-ODNs. We evaluated S-ODN interactions with bovine serum albumin (BSA) and human serum albumin (HSA) in vitro. The equilibrium dissociation constants K-m for the binding of a 20 mer S- ODN with BSA and HSA range between 1.1-5.2 X 10(-5) and 2.4-3.1 X 10(- 4) M, respectively. The K-m for an unrelated 15 mer S-ODN binding with HSA ranges between 3.7 and 4.8 X 10(-5) M. Studies with a fluorescent ly labeled 27 mer S-ODN suggest cooperative binding (Hill slope = 1.67 ) and/or the presence of secondary binding sites on the S-ODN. HSA or BSA linked to Sepharose was incubated with a 15, 20, or 24 mer S-ODN f ollowed by the addition of selected drugs known to be highly protein b ound (nifedipine, warfarin, midazolam, probenecid, indomethacin, and m itoxantrone). Up to 30% of S-ODN was displaced by warfarin in competit ion binding assays. Conversely, HSA-bound warfarin was incubated with a variety of oligonucleotides, including RNA and genomic dsDNA. Maximu m displacement of warfarin-bound HSA was observed following incubation with 5'-cholesterol-conjugated 20 mer S-ODN. In summary, S-ODNs are l ikely to interact and displace other therapeutic agents that bind to a lbumin, particularly those binding at site I.