Rapidly Neutralizable and Highly Anticoagulant Thrombin-Binding DNA Aptamer Discovered by MACE SELEX

Koji Wakui,Toru Yoshitomi,Akane Yamaguchi,Maho Tsuchida,Shingo Saito,Masami Shibukawa,Hitoshi Furusho,Keitaro Yoshimoto

doi:10.1016/j.omtn.2019.03.002

Abstract

We present a rapidly neutralizable and highly anticoagulant thrombin-binding aptamer with a short toehold sequence, originally discovered by systematic evolution of ligands by exponential enrichment (SELEX) with microbead-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 and 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 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 longer prolonged clotting time than other anticoagulant TBAs, such as HD1, NU172, RE31, and RA36. Analyses of the aptamer and thrombin complexes using both bare and coated capillaries suggested that a large number of efficient aptamers are missed in conventional CE-SELEX because of increased interaction between the complex and the capillary. In addition, the toehold-mediated rapid antidote was designed for safe administration. The efficient aptamer and antidote system developed in the present study could serve as a new candidate for anticoagulant therapy.

Highlights

Anticoagulant drugs have been the mainstay for the treatment and prevention of thrombotic disorders
capillary electrophoresis (CE)-systematic evolution of ligands by exponential enrichment (SELEX) We propose microbead-assisted capillary electrophoresis (MACE)-SELEX as a novel SELEX system that contains a sophisticated separation step with high sensitivity based on CE separation using target-coupled microbeads
Conventional CE-SELEX was performed for comparison with MACE-SELEX to evaluate efficiency

Summary

Introduction

Anticoagulant drugs have been the mainstay for the treatment and prevention of thrombotic disorders. Clinically used anticoagulant drugs have a risk of side effects, such as significant bleeding, that increase patient morbidity and mortality.[1,2] the lack of specific reversal agents limits their use. HD1 and NU172 have advanced to phases I and II of clinical trials, respectively, for coronary artery bypass graft surgery, no update is available regarding the current situation.[31] On the other hand, the REG1 anticoagulant system containing the factor Ixa-binding aptamer and the complementary sequence for the antidote proceeded to a phase III clinical trial as an anticoagulant agent for percutaneous coronary intervention.[22,32] the efficiency of typical Watson-Crick base-pairing antidotes is not sufficient, and they require much higher doses or continuous administration to neutralize the drug activity,[22,32] resulting in an increase in cost and burden on patients. If there were aptamer-based anticoagulants with higher affinity and more efficient reversal agents, they could be promising anticoagulant systems

Methods

Results

Conclusion