Characterization of Binding Sites,Extent of Binding,and Drug Interactions of Oligonucleotides with Albumin

Abstract

Phosphorothioate oligonucleotides (S-ODNs) have the ability to modulate gene expression selectively and thus have potential therapeutic capabilities. This potential led us to investigate the protein binding characteristics 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 × 10^-5 and 2.4-3.1 × 10^-4 M, respectively. The K_m for an unrelated 15 mer S-ODN binding with HSA ranges between 3.7 and 4.8 × 10^-5 M. Studies with a fluorescently 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 followed by the addition of selected drugs known to be highly protein bound (nifedipine, warfarin, midazolam, probenecid, indomethacin, and mitoxantrone). Up to 30% of S-ODN was displaced by warfarin in competition binding assays. Conversely, HSA-bound warfarin was incubated with a variety of oligonucleotides, including RNA and genomic dsDNA. Maximum displacement of warfarin-bound HSA was observed following incubation with 5′-cholesterol-conjugated 20 mer S-ODN. In summary, S-ODNs are likely to interact and displace other therapeutic agents that bind to albumin, particularly those binding at site I.