Nucleic acid aptamers are promising candidates for therapeutic and diagnostic agents owing to their high affinity and selectivity to target molecules. Thrombin-binding DNA aptamers are important in the medical and clinical fields, as thrombin is a multifunctional serine protease that plays a key role in thrombosis, homeostasis, and inflammation. Herein, we successfully identified new classes of thrombin-binding aptamers with high affinity by the systematic evolution of ligands by exponential enrichment (SELEX) with microbeads-assisted capillary electrophoresis (MACE). Due to the low partitioning efficiency of target/aptamer complexes from unbound oligonucleotides, traditional SELEX generally requires laborious selection cycles up to twenty. MACE is a novel CE-partitioning method for SELEX and able to separate microbead-coupled target/aptamer complexes from a library of unbound nucleic acids by CE, where the aptamer/target complexes can be detected reliably and partitioned with high purity even in the first selection cycle. In the present study, MACE was applied to the selection of DNA aptamers against human α-thrombin, and showed a more than 250-fold enrichment of the aptamers relative to that of conventional CE-SELEX. MACE-SELEX afforded five thrombin-binding aptamers (TBAs) with a nanomolar affinity (Kd = 4.5-8.2 nM) that surpasses the high affinity of previously reported HD22 (29 mer, Kd = 13 nM). Among these TBAs, only two exhibited homologous motifs with HD22, indicating that MACE-SELEX identified a new class of TBAs within only three selection cycles. Analyses of the aptamer-thrombin complexes using a coated capillary suggested that a number of high-affinity aptamers can be collected only by MACE-SELEX, due to the dominant mobility of the microbeads. TBAs discovered in the present study could serve as new candidates for anti-coagulant drugs. Furthermore, MACE separation is simple and applicable to a wide range of targets, which should facilitate the identification of aptamers with high affinity in early selection rounds.

Identification of new classes of thrombin-binding aptamers by microbeads-assisted capillary electrophoresis (MACE) SELEX

255th National Meeting (2018)

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