Selection of Smart Aptamers by Methods of Kinetic Capillary Electrophoresis

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.