Bivalent Ligands with Long Nanometer-Scale Flexible Linkers
Bivalent Ligands with Long Nanometer-Scale Flexible Linkers
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DOI：
10.1021/bi801630b
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发表时间：
2009-01-20
期刊：
影响因子：
2.9
通讯作者：
Heyduk, Tomasz
中科院分区：
文献类型：
--
作者：
Tian, Ling;Heyduk, Tomasz
高亲和力配体对生物分子的识别具有高特异性，对药物发现和生物分子检测至关重要。我们在这里描述了一种简单的方法来制备具有增强结合亲和力的适配体基配体。在这种方法中，两个具有次优结合性能的适体配体与一个长柔性连接体共价连接，以创建具有显著提高结合亲和力的二价配体。我们首先使用了一个简单的基于寡核苷酸的模型，该模型模拟了二价配体与其靶分子之间的相互作用，以研究亲和性增强的原理。这些实验表明，只要单个配体具有至少亚微摩尔的结合亲和力，它们就可以与纳米尺度的柔性连接体连接，从而产生具有更高结合亲和力和特异性的二价配体。此外，将实验数据与虫链模型预测的二价配体性质进行了比较，表明该模型能很好地逼近纳米级柔性二价配体的结合性质。为了验证具有纳米级柔性连接体的二价配体的实用性，我们构建了基于适配体的人α -凝血酶二价配体。与模型系统的预测一致，凝血酶二价配体的结合亲和力和抗凝血活性比单个配体显著提高。
High-affinity ligands recognizing biomolecules with high specificity are crucial for drug discovery and biomolecule detection. We describe here a simple approach to preparing aptamer-based ligands with enhanced binding affinity. In this approach, two aptamer ligands with suboptimal binding properties are covalently linked with a long flexible linker to create a bivalent ligand with significantly improved binding affinity. We first used a simple oligonucleotide-based model, which mimicked the interaction between bivalent ligands and their target molecules, to investigate the principles governing the affinity enhancement. These experiments showed that as long as the individual ligands had at least submicromolar binding affinities, they could be linked with a nanometer-scale flexible linker to produce bivalent ligands with improved binding affinity and specificity. Furthermore, comparison of the experimental data with the bivalent ligand properties predicted by a wormlike chain model showed that this model provided a good approximation of the binding properties of nanometer-scale flexible bivalent ligands. To verify the practicality of bivalent ligands with nanometer-scale flexible linkers, we constructed aptamer-based bivalent ligands for human alpha-thrombin. In agreement with the predictions derived from the model system, the binding affinities and the anticlotting activities of thrombin bivalent ligands were significantly improved compared to those of the individual ligands.