Oligonucleotide inhibitors of human thrombin that bind distinct epitopes
Oligonucleotide inhibitors of human thrombin that bind distinct epitopes
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DOI：
10.1006/jmbi.1997.1275
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发表时间：
1997-10-10
影响因子：
5.6
通讯作者：
Steiner, W
中科院分区：
文献类型：
--
作者：
Tasset, DM;Kubik, MF;Steiner, W
凝血酶是一种多功能丝氨酸蛋白酶，识别多种大分子底物，并在proc凝和抗凝剂功能中起关键作用。凝血酶的底物特异性涉及两个电阳性表面，纤维蛋白原 - 识别和肝素结合外粒。 SELEX过程是一种强大的组合方法，用于将高亲和力的寡核苷酸配体识别到任何所需的靶标。 SELEX过程已用于将单链DNA配体与人凝血酶分离。在这里，描述了一个29-核苷酸单链DNA配体，指定为60-18 [29]，K-D约为0.5 nm，DNA 60-18 [29]抑制了凝血酶催化的凝血酶纤维蛋白凝结蛋白凝块在体外。先前描述的DNA配体结合了纤维蛋白原 - 识别外部，而竞争和光链接实验表明DNA配体60-18 [29]结合了肝素结合的外观。 DNA 60-18 [29]是一种四链体/双链体，具有15-核苷酸“核心”序列，与先前描述的DNA配体与凝血酶具有惊人的相似性，但亲和力高20至50倍。 15-核苷酸核心序列具有八个高度保守的鸟嘌呤残基，并形成了G四链体结构。 G-四链体结构中的唱片LE核苷酸可以将DNA引导到不同的表位。配体60-18的双链区域中的其他序列信息[29]有助于更大的稳定性和与凝血酶结合的亲和力。已经提出了DNA 60-18 [29]与人凝血酶相互作用的低分辨率模型。 （c）1997 Academic Press Limited。
Thrombin, a multifunctional serine protease, recognizes multiple macromolecular substrates and plays a key role in both procoagulant and anticoagulant functions. The substrate specificity of thrombin involves two electropositive surfaces, the fibrinogen-recognition and heparin-binding exosites. The SELEX process is a powerful combinatorial methodology for identifying high-affinity oligonucleotide Ligands to any desired target. The SELEX process has been used to isolate single-stranded DNA Ligands to human thrombin. Here, a 29-nucleotide single-stranded DNA ligand to human thrombin, designated 60-18[29], with a K-d of approximately 0.5 nM is described, DNA 60-18[29] inhibits thrombin-catalyzed fibrin clot formation in vitro. Previously described DNA ligands bind the fibrinogen-recognition exosite, while competition and photocrosslinking experiments indicate that the DNA ligand 60-18[29] binds the heparin-binding exosite. DNA 60-18[29] is a quadruplex/duplex with a 15-nucleotide ''core'' sequence that has striking similarity to previously described DNA ligands to thrombin, but binds with 20 to 50-fold higher affinity. The 15-nucleotide core sequence has eight highly conserved guanine residues and forms a G-quadruplex structure. A sing le nucleotide within the G-quadruplex structure can direct the DNA to a distinct epitope. Additional sequence information in the duplex regions of ligand 60-18[29] contribute to greater stability and affinity of binding to thrombin. A low-resolution model for the interaction of DNA 60-18[29] to human thrombin has been proposed. (C) 1997 Academic Press Limited.