Selection and characterization of a DNA aptamer inhibiting coagulation factor XIa.
Selection and characterization of a DNA aptamer inhibiting coagulation factor XIa.
复制标题
DOI：
10.1038/s41598-017-02055-x
复制
发表时间：
2017-05-18
影响因子：
4.6
通讯作者：
Sheffield WP
中科院分区：
文献类型：
--
作者：
Donkor DA;Bhakta V;Eltringham-Smith LJ;Stafford AR;Weitz JI;Sheffield WP
因子XIa （FXIa）是一种丝氨酸蛋白酶，在凝血级联中催化因子IX （FIX）的激活。FXIa及其前体FXI是开发更安全的抗凝药物的新兴治疗靶点。在这里，我们寻找一种新的基于dna的药物来抑制FXIa。为此，我们筛选了一个80碱基的单链DNA适体文库（包含一个40碱基的随机核心），通过10轮阳性和阴性选择来筛选fxia结合候选物。经筛选，89个不同序列中有6个抑制fxia介导的显色底物S2366的裂解。最活跃的抗fxia适配体中心序列为5 ' - aacctatcggactattgttagtgattttttatagtgt -3 '，命名为因子11抑制适配体（FELIAP）。FELIAP，但不是打乱适体控制（SCRAPT），竞争性地抑制fxia催化的S2366裂解、FIX激活和与抗凝血酶的复合物形成。FELIAP对FXI的激活没有影响。FELIAP对血浆凝血和凝血酶生成的抑制程度明显大于SCRAPT。固定化FELIAP结合FXIa具有较强的亲和力，并且通过表面等离子体共振测定了低纳摩尔范围内的平衡结合常数（KD）。FELIAP是第一个被描述的fxia抑制适体，并构成了一个先导化合物，用于开发体内使用的相关适体。
Factor XIa (FXIa) is a serine protease that catalyzes the activation of Factor IX (FIX) in the blood coagulation cascade. FXIa and its precursor FXI are emergent therapeutic targets for the development of safer anticoagulant agents. Here, we sought a novel DNA-based agent to inhibit FXIa. Towards this goal, an 80 base, single-stranded DNA aptamer library (containing a 40 base randomized core) was screened for FXIa-binding candidates, using ten rounds of positive and negative selection. After selection, 6 of 89 different sequences inhibited FXIa-mediated chromogenic substrate S2366 cleavage. The most active anti-FXIa aptamer had a hypervariable central sequence 5′-AACCTATCGGACTATTGTTAGTGATTTTTATAGTGT-3′ and was designated Factor ELeven Inhibitory APtamer (FELIAP). FELIAP, but not a scrambled aptamer control (SCRAPT), competitively inhibited FXIa-catalyzed S2366 cleavage, FIX activation, and complex formation with antithrombin. No effect of FELIAP on FXI activation was observed. FELIAP inhibited plasma clotting and thrombin generation assays to a significantly greater extent than SCRAPT. Immobilized FELIAP bound FXIa with strong affinity and an equilibrium binding constant (KD) in the low nanomolar range determined using surface plasmon resonance. FELIAP is the first FXIa-inhibitory aptamer to be described and constitutes a lead compound to develop related aptamers for in vivo use.