title: In vitro selection and characterization of deoxyribonucleic acid aptamers for digoxin.
abstract: The low therapeutic index of digoxin necessitates careful monitoring of its serum levels. Most of digoxin immunoassays suffer from interferences with digoxin-like immunoreactive substances. Since aptamers have been shown to be highly specific for their targets, the aim of this study was to develop DNA aptamers for this widely used cardiac glycoside. Digoxin was coated onto the surface of streptavidin magnetic beads. DNA aptamers against digoxin were designed using Systematic Evolution of Ligands by Exponential enrichment method (SELEX) by 11 iterative rounds of incubation of digoxin-coated streptavidin magnetic beads with synthetic DNA library, DNA elution, electrophoresis and PCR amplification. The PCR product was cloned and sequenced. Binding affinity was determined using digoxin-BSA conjugate, coated onto ELISA plate. Inhibitory effect of anti-digoxin aptamer was conducted using isolated guinea-pig atrium. Three aptamers (D1, D2 and D3) were identified. Binding studies of fluorescein-labeled truncated (without primer binding region) D1 and D2 and full length D1 anti-digoxin aptamers were performed and their corresponding dissociation constants values were 8.2×10(-9), 44.0×10(-9) and 17.8×10(-9) M, respectively. This is comparable to what other workers have obtained for interaction of monoclonal antibodies raised against digoxin. There was little difference in binding affinity between full length and truncated anti-digoxin D1 aptamer. D1 anti-digoxin aptamer also inhibited the effects of digoxin on the isolated guinea-pig atrium. D1 anti-digoxin aptamer distinguished between digoxin and ouabain in both tissue study and binding experiments. Our finding indicated that D1 anti-digoxin aptamer can selectively bind to digoxin. Further studies might show its suitability for use in digoxin assays and as a therapeutic agent in life-threatening digoxin toxicity.
authors.affiliation: Tehran University of Medical Sciences, School of Medicine, Department of Pharmacology, Tehran, Iran.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Kiani
authors.forename: Massoumeh
authors.initials: M
authors.lastname: Shafiei
authors.forename: Parvaneh
authors.initials: P
authors.lastname: Rahimi-Moghaddam
authors.forename: Ali Asghar
authors.initials: AA
authors.lastname: Karkhane
authors.forename: Soltan Ahmed
authors.initials: SA
authors.lastname: Ebrahimi
mesh_terms: D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D019921:Biotinylation; D002316:Cardiotonic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D019963:Clinical Chemistry Tests; D004077:Digoxin / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D006168:Guinea Pigs; D006321:Heart / Q000187:drug effects; D008958:Models, Molecular; D010084:Oxidation-Reduction; D052156:SELEX Aptamer Technique; D012710:Serum Albumin, Bovine / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D002316:Cardiotonic Agents; D012710:Serum Albumin, Bovine; D004077:Digoxin