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영어

Aptamers are artificial nucleic acid ligands, specifically generated to recognize target molecules, such as amino acids, drugs, proteins or other molecules. Some aptamers can even discriminate optica isomers of a target molecule such as L-arginine, L-tyrosinamide, and D-vasopressin. These artificial ligands have mostly been screened from combinatorial libraries of synthetic nucleic acids by a method, known as systematic evolution of ligands by exponential enrichment (SELEX), comprising an in vitro iterative process of adsorption, recovery and amplification. Verapamil, a calcium entry blocker, has been used in the treatment of hypertension, angina pectoris and cardiac arrhythmia. Verapamil formulations for drug administration usually consist of a blend of R-(+) and S-(−) enantiomers pharmacokinetic and/or pharmacodynamic properties of which are radically different. In addition, an overdose injection of verapamil may cause serious side effects. Therefore, identification of aptamers specifically cognitive of S-(−)-verapamil would facilitate an efficient separation of verapamil enantionmers, furthermore rendering controlled administration of S-(−)-verapamil or close monitoring of drug concentration in order to avoid toxic effects. In the present study, owing to the recent advances in SELEX methodology, we took advantage of chromatography-based in-vitro selection using FPLC to select aptamers capable of specifically binding to S-(−) verapamil from an 86-bp random single-stranded DNA pool. Pre-negative and negative screening were implemented with bare column and R-(−)-verapamil immobilized column in the recycle mode to eliminate the non-specific binders before the main screening. The percentage of ssDNA eluted measured for the fractionated DNA pool following each round of main selection increased with selection rounds,
indicating the successful enrichment of aptamers with enhanced affinity to the target. Finally, an
aptamer sequence, exhibiting the highest affinity and specificity to S-(−) verapamil following the
assessment of Kd for the enriched aptamer sequences, was demonstrated to effect a high-efficiency chiral separation of verapamil enantiomers.