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.

We propose kinetic capillary electrophoresis (KCE) as a conceptual platform for the development of kinetic homogeneous affinity methods. KCE is defined as the CE separation of species that interact during electrophoresis. Depending on how the interaction is arranged, different KCE methods can be designed. All KCE methods are described by the same mathematics: the same system of partial differential equations with only initial and boundary conditions being different. Every qualitatively unique set of initial and boundary conditions defines a unique KCE method. Here, we (i) present the theoretical bases of KCE, (ii) define four new KCE methods, and (iii) propose a multimethod KCE toolbox as an integrated kinetic technique. Using the KCE toolbox, we were able to, for the first time, observe high-affinity (specific) and low-affinity (nonspecific) interactions within the same protein-ligand pair. The concept of KCE allows for the creation of an expanding toolset of powerful kinetic homogeneous affinity methods, which will find their applications in studies of biomolecular interactions, quantitative analyses, and selecting affinity probes and drug candidates from complex mixtures.

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.

We recently introduced a new electrophoretic method, nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). NECEEM provides a unique way of finding kinetic and equilibrium parameters of the formation of intermolecular complexes from a single electropherogram and allows for the use of weak affinity probes in protein quantitation. In this work, we study theoretical bases of NECEEM by developing a mathematical model for the new method. By solving a system of partial differential equations with diffusion in linear approximation, we found the analytical solution for concentrations of components involved in complex formation as functions of time from the beginning of separation and position in the capillary. The nonnumerical nature of the solution makes it a powerful tool in studying the theoretical foundations of the NECEEM method and modeling experimental results. We demonstrate the use of the model for finding binding parameters of complex formation by nonlinear regression of NECEEM electropherograms obtained experimentally.

We introduce transverse diffusion of laminar flow profiles (TDLFP), the first generic method for mixing two or more reactants inside capillaries. Conceptually, solutions of reactants are injected inside the capillary by pressure as a series of consecutive plugs. Due to the laminar nature of flow inside the capillary, the nondiffused plugs have parabolic profiles with predominantly longitudinal interfaces between them. After injection, the plugs are mixed by transverse diffusion; longitudinal diffusion does not contribute to mixing. To prove the principle, we used TDLFP to mix two reactants-an enzyme and its substrate. After mixing the reactants by TDLFP, we incubated reaction mixtures for different periods of time and measured the reaction kinetics. We found that the reaction proceeded in time- and concentration-dependent fashion, thus confirming that the reactants were mixed by TDLFP. Remarkably, the experimental reaction kinetics were not only in qualitative agreement but also in good quantitative agreement with theoretically predicted ones. TDLFP has a number of enabling features. By facilitating the preparation of reaction mixtures inside the capillary, TDLFP lowers reagent consumption to nanoliters (microliters are required for conventionally mixing reagents in a vial). The reaction products can be then analyzed "on-line" by capillary separation coupled with optical, electrochemical, or mass spectrometric detection. The combination of TDLFP with capillary separation will be an indispensable tool in screening large combinatorial libraries for affinity probes and drug candidates: a few microliters of a target protein will be sufficient to screen thousands of compounds. The new method paves the road to a wide use of capillary nanoreactors in different areas of physical and life sciences.