Selection is more intelligent than design: improving the affinity of a bivalent ligand through directed evolution.
Selection is more intelligent than design: improving the affinity of a bivalent ligand through directed evolution.
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DOI：
10.1093/nar/gks899
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发表时间：
2012-12
影响因子：
14.9
通讯作者：
Soh HT
中科院分区：
文献类型：
--
作者：
Ahmad KM;Xiao Y;Soh HT
可以利用多价分子相互作用来显著增强亲和试剂的性能。用多价构建体实现的亲和力和特异性的增强关键取决于连接配体的支架的有效性，因为这决定了它们相对于靶分子的位置和取向。目前，没有可推广的设计规则存在用于构建具有已知结构的靶的最佳多价配体，并且对于大量结构未知的蛋白质，设计挑战仍然是不可逾越的障碍。作为这种基于设计的策略的替代方案，我们在这里报告了一种基于定向进化的方法，用于生成最佳的二价适体。为了证明这种方法，我们将两个凝血酶适体与随机DNA序列融合，并使用微流体体外选择策略来分离具有极高亲和力的支架。在五轮选择中，我们产生了结合凝血酶的二价适体，其表观解离常数（Kd）<10 pM，这表示结合亲和力比单体适体提高了200倍，比最佳设计的二价构建体提高了2015倍。这里描述的过程可用于生产高亲和力多价适体，并可能适用于其他类型的生物分子。
Multivalent molecular interactions can be exploited to dramatically enhance the performance of an affinity reagent. The enhancement in affinity and specificity achieved with a multivalent construct depends critically on the effectiveness of the scaffold that joins the ligands, as this determines their positions and orientations with respect to the target molecule. Currently, no generalizable design rules exist for construction of an optimal multivalent ligand for targets with known structures, and the design challenge remains an insurmountable obstacle for the large number of proteins whose structures are not known. As an alternative to such design-based strategies, we report here a directed evolution-based method for generating optimal bivalent aptamers. To demonstrate this approach, we fused two thrombin aptamers with a randomized DNA sequence and used a microfluidic in vitro selection strategy to isolate scaffolds with exceptionally high affinities. Within five rounds of selection, we generated a bivalent aptamer that binds thrombin with an apparent dissociation constant (Kd) <10 pM, representing a ∼200-fold improvement in binding affinity over the monomeric aptamers and a ∼15-fold improvement over the best designed bivalent construct. The process described here can be used to produce high-affinity multivalent aptamers and could potentially be adapted to other classes of biomolecules.
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DOI：
10.3390/s8021090
发表时间：
2008-02-19
期刊：
Sensors (Basel, Switzerland)
影响因子：
--
作者：
Hasegawa H;Taira KI;Sode K;Ikebukuro K
通讯作者：
Ikebukuro K
影响因子：
3.7
作者：
Ahmad KM;Oh SS;Kim S;McClellen FM;Xiao Y;Soh HT
通讯作者：
Soh HT
影响因子：
5.6
作者：
Tasset, DM;Kubik, MF;Steiner, W
通讯作者：
Steiner, W
影响因子：
13.8
作者：
Liu, Fen;Walters, Kylie J.
通讯作者：
Walters, Kylie J.
影响因子：
2.9
作者：
Tian, Ling;Heyduk, Tomasz
通讯作者：
Heyduk, Tomasz