In vitro selection and characterization of deoxyribonucleic acid aptamers for digoxin

Kiani, Z. and Shafiei, M. and Rahimi-Moghaddam, P. and Karkhane, A.A. and Ebrahimi, S.A. (2012) In vitro selection and characterization of deoxyribonucleic acid aptamers for digoxin. Analytica Chimica Acta, 748. pp. 67-72.

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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�10-9M, 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. © 2012 Elsevier B.V.

Item Type: Article
Additional Information: cited By 11
Uncontrolled Keywords: Aptamers; Binding affinities; Binding studies; Digoxin; Dissociation constant; DNA aptamers; In-vitro; Inhibitory effect; Magnetic beads; PCR amplification; PCR products; Serum levels; Streptavidin; Synthetic DNA; Systematic evolution of ligands by exponential enrichments; Therapeutic agents; Therapeutic index, Binding energy; Cloning; Dissociation; DNA; Electrophoresis; Iterative methods; Monoclonal antibodies; Organic acids; Proteins; Sugars; Tissue, Polymerase chain reaction, aptamer; bovine serum albumin; deoxyribonucleic acid aptamer; digoxin; fluorescein; monoclonal antibody; ouabain; streptavidin; unclassified drug, animal tissue; article; binding affinity; biotinylation; chemical interaction; chemical labeling; combinatorial chemistry; controlled study; dissociation constant; DNA library; drug determination; drug structure; electrophoresis; elution; enzyme linked immunosorbent assay; experimental guinea pig; female; heart atrium muscle; incubation time; male; material coating; nonhuman; polymerase chain reaction; priority journal; sequence analysis, Animals; Aptamers, Nucleotide; Biotinylation; Cardiotonic Agents; Cattle; Clinical Chemistry Tests; Digoxin; Guinea Pigs; Heart; Models, Molecular; Oxidation-Reduction; SELEX Aptamer Technique; Serum Albumin, Bovine, Cavia; Strophanthus gratus
Depositing User: EPrints System Admin
Date Deposited: 19 Jul 2017 10:13
Last Modified: 19 Jul 2017 10:13

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