Abstract：
Thrombosis leads to elevated mortality rates and substantial medical expenses worldwide. Human factor IXa (HFIXa) protease is pivotal in tissue factor (TF)-mediated thrombin generation, and represents a promising target for anticoagulant therapy. We herein isolated novel DNA aptamers that specifically bind to HFIXa through systematic evolution of ligands by exponential enrichment (SELEX) method. We identified two distinct aptamers, seq 5 and seq 11, which demonstrated high binding affinity to HFIXa (Kd = 74.07 ± 2.53 nM, and 4.93 ± 0.15 nM, respectively). Computer software was used for conformational simulation and kinetic analysis of DNA aptamers and HFIXa binding. These aptamers dose-dependently prolonged activated partial thromboplastin time (aPTT) in plasma. We further rationally optimized the aptamers by truncation and site-directed mutation, and generated the truncated forms (Seq 5-1t, Seq 11-1t) and truncated-mutated forms (Seq 5-2tm, Seq 11-2tm). They also showed good anticoagulant effects. The rationally and structurally designed antidotes (seq 5-2b and seq 11-2b) were competitively bound to the DNA aptamers and effectively reversed the anticoagulant effect. This strategy provides DNA aptamer drug-antidote pair with effective anticoagulation and rapid reversal, developing advanced therapies by safe, regulatable aptamer drug-antidote pair.
摘要：
血栓形成导致全球死亡率升高和大量医疗费用。人因子IXa（HFIXa）蛋白酶在组织因子（TF）介导的凝血酶生成中至关重要，并代表了抗凝治疗的一个有希望的目标。我们在本文中通过指数富集(SELEX)方法通过配体的系统进化分离特异性结合HFIXa的新型DNA适体。我们鉴定了两种不同的适体,seq5和seq11，显示对HFIXa的高结合亲和力(Kd=74.07±2.53nM，和4.93±0.15nM，分别)。计算机软件用于DNA适体和HFIXa结合的构象模拟和动力学分析。这些适体剂量依赖性地延长血浆中活化的部分凝血活酶时间(aPTT)。我们通过截短和定点突变进一步合理优化了适体，并生成了截断的形式(Seq5-1t，Seq11-1t)和截短的突变形式(Seq5-2tm，Seq11-2tm）。它们还显示出良好的抗凝血作用。合理和结构设计的解毒剂（seq5-2b和seq11-2b）竞争性地结合到DNA适体上，并有效地逆转了抗凝血作用。这种策略提供了DNA适体药物-解毒剂对有效抗凝和快速逆转，通过安全开发先进的疗法，可调节的适体药物-解毒剂对。
