Rapidly Neutralizable and High Anticoagulant Thrombin-Binding DNA Aptamer Discovered by Microbeads-Assisted Capillary Electrophoresis (MACE) SELEX

Abstract We present a rapidly neutralizable and high anticoagulant thrombin binding aptamer with a short toehold sequence, originally discovered by the systematic evolution of ligands by exponential enrichment (SELEX) with microbeads-assisted capillary electrophoresis (MACE). MACE is a novel CE-partitioning method for SELEX and able to separate aptamers from a library of unbound nucleic acids, where the aptamer/target complexes can be detected reliably and partitioned with high purity even in the first selection cycle. Three selection rounds of MACE-SELEX discovered several TBAs with a nanomolar affinity (Kd = 4.5-8.2 nM) that surpasses the previously reported TBAs such as HD1, HD22, and NU172 (Kd = 118, 13, and 12 nM respectively). One of the obtained aptamers, M08, showed a 10 to 20-fold more prolonged clotting time than other anticoagulant TBAs such as HD1, NU172, RE31 and RA36. Analyses of the aptamer/thrombin complexes using both of bare and coated capillaries suggested that a large number of efficient aptamers are missed in conventional CE-SELEX, due to the increased interaction between the complex and the capillary. In addition, the toehold-mediated rapid antidote was designed for safe administration. The efficient aptamer/antidote system developed in the present study could serve as a new candidate for anticoagulant therapy.