Berezovski Maxim, Drabovich Andrei, Krylova Svetlana M, Musheev Michael, Okhonin Victor, Petrov Alexander, Krylov Sergey N
Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada.
J Am Chem Soc. 2005 Mar 9;127(9):3165-71. doi: 10.1021/ja042394q.
Aptamers are DNA (or RNA) ligands selected from large libraries of random DNA sequences and capable of binding different classes of targets with high affinity and selectivity. Both the chances for the aptamer to be selected and the quality of the selected aptamer are largely dependent on the method of selection. Here we introduce selection of aptamers by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). The new method has a number of advantages over conventional approaches. First, NECEEM-based selection has exceptionally high efficiency, which allows aptamer development with fewer rounds of selection. Second, NECEEM can be equally used for selecting aptamers and finding their binding parameters. Finally, due to its comprehensive kinetic capabilities, the new method can potentially facilitate selection of aptamers with predefined K(d), k(off), and k(on) of the aptamer-target interaction. In this proof-of-principle work, we describe the theoretical bases of the method and demonstrate its application to a one-step selection of DNA aptamers with nanomolar affinity for protein farnesyltransferase.
适体是从随机DNA序列的大型文库中筛选出的DNA（或RNA）配体，能够以高亲和力和选择性结合不同类型的靶标。适体的筛选机会和所选适体的质量在很大程度上取决于筛选方法。在此，我们介绍通过平衡混合物的非平衡毛细管电泳（NECEEM）筛选适体。该新方法相对于传统方法具有许多优势。首先，基于NECEEM的筛选具有极高的效率，这使得适体开发所需的筛选轮次更少。其次，NECEEM可同样用于筛选适体并确定其结合参数。最后，由于其全面的动力学能力，该新方法有可能促进对适体-靶标相互作用具有预定义解离常数（K(d)）、解离速率常数（k(off)）和结合速率常数（k(on)）的适体的筛选。在这项原理验证工作中，我们描述了该方法的理论基础，并展示了其在一步筛选对蛋白质法尼基转移酶具有纳摩尔亲和力的DNA适体中的应用。
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