通过动力学毛细管电泳方法筛选智能适配体。

Selection of smart aptamers by methods of kinetic capillary electrophoresis.

作者信息

Drabovich Andrei P, Berezovski Maxim, Okhonin Victor, Krylov Sergey N

机构信息

Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada.

出版信息

Anal Chem. 2006 May 1;78(9):3171-8. doi: 10.1021/ac060144h.

Abstract

We coin the term "smart aptamers" -- aptamers with predefined binding parameters (k(on), k(off), Kd) of aptamer-target interaction. Aptamers, in general, are oligonucleotides, which are capable of binding target molecules with high affinity and selectivity. They are considered as potential therapeutic targets and also thought to rival antibodies in immunoassay-like analyses. Aptamers are selected from combinatorial libraries of oligonucleotides by affinity methods. Until now, technological limitations have precluded the development of smart aptamers. Here, we report on two kinetic capillary electrophoresis techniques applicable to the selection of smart aptamers. Equilibrium capillary electrophoresis of equilibrium mixtures was used to develop aptamers with predefined equilibrium dissociation constants (Kd), while nonequilibrium capillary electrophoresis of equilibrium mixtures facilitated selection of aptamers with different dissociation rate constants (k(off)). Selections were made for MutS protein, for which aptamers have never been previously developed. Both theoretical and practical aspects of smart aptamer development are presented, and the advantages of this new type of affinity probes are described.

摘要

我们创造了“智能适配体”这一术语,即具有适配体与靶标相互作用的预定义结合参数(结合速率常数k(on)、解离速率常数k(off)、解离常数Kd)的适配体。一般来说,适配体是寡核苷酸,能够以高亲和力和选择性结合靶分子。它们被视为潜在的治疗靶点,并且在类似免疫分析的检测中也被认为可与抗体相媲美。适配体通过亲和方法从寡核苷酸组合文库中筛选获得。到目前为止,技术限制阻碍了智能适配体的开发。在此,我们报道了两种适用于筛选智能适配体的动力学毛细管电泳技术。利用平衡混合物的平衡毛细管电泳来开发具有预定义平衡解离常数(Kd)的适配体,而平衡混合物的非平衡毛细管电泳则有助于筛选具有不同解离速率常数(k(off))的适配体。我们针对MutS蛋白进行了筛选,此前从未开发过针对该蛋白的适配体。本文介绍了智能适配体开发的理论和实际方面,并描述了这种新型亲和探针的优势。

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