Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures: A Universal Tool for Development of Aptamers

Abstract

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.

Micromagnetic selection of aptamers in microfluidic channels

“…The CMACS device was operated under typical conditions, in which buffer was pumped at 5 mL/hr and samples containing 1 M ssDNA and magnetic beads (2 ϫ 10 6 beads per milliliter) were pumped at 1 mL/hr. The measured PE of CMACS was (1.4 Ϯ 0.6) ϫ 10 6 , which significantly exceeded most conventional separation methods (e.g., filters and columns) and is comparable with that of capillary electrophoresis (23).…”

“…Multiple rounds of selection (typically [8][9][10][11][12][13][14][15] are generally necessary to isolate aptamers with sufficient specificity and binding affinity (e.g., nanomolar dissociation constant (K d ) for proteins and micromolar for small molecules), which therefore requires significant resources and time (17). To accelerate this lengthy discovery process, a wide variety of molecular separation techniques-beyond traditional filter-binding assays (18) and affinity chromatography (19)-have been explored as means to enhance the efficiency of aptamer selection, including surface plasmon resonance (20), flow cytometry (21), and capillary electrophoresis (CE) (22)(23)(24)(25). In particular, CE-based separation methods have shown remarkable selection efficiencies for protein targets that exhibit significant shifts in the electrophoretic mobility ( e ) upon binding to aptamers; proteinbinding aptamers with K d in the low-nanomolar range have been isolated after a small number (Ϸ1-4) of selection rounds (22)(23)(24)(25).…”

“…To accelerate this lengthy discovery process, a wide variety of molecular separation techniques-beyond traditional filter-binding assays (18) and affinity chromatography (19)-have been explored as means to enhance the efficiency of aptamer selection, including surface plasmon resonance (20), flow cytometry (21), and capillary electrophoresis (CE) (22)(23)(24)(25). In particular, CE-based separation methods have shown remarkable selection efficiencies for protein targets that exhibit significant shifts in the electrophoretic mobility ( e ) upon binding to aptamers; proteinbinding aptamers with K d in the low-nanomolar range have been isolated after a small number (Ϸ1-4) of selection rounds (22)(23)(24)(25). However, the method is less effective for other classes of targets that do not induce sufficient e shift of aptamers (e.g., small molecules and cell surfaces), or those that cannot be accommodated in the separation apparatus (e.g., whole organisms).…”

Tau protein binds single‐stranded DNA sequence specifically – the proof obtained in vitro with non‐equilibrium capillary electrophoresis of equilibrium mixtures

“…Moreover, they facilitate a number of other applications ranging from quantitative analyses of proteins, DNA, and RNA [14] to selection of aptamers [17].…”

Quantitative selection and parallel characterization of aptamers

“…For example, novel separation methodologies such as capillary electrophoresis (13,14) and microfluidic devices (15)(16)(17) can isolate high-affinity aptamers after a minimal number of selection rounds. This increased efficiency is achieved through tighter control over selection stringency by applying rigorous washing to isolate aptamers with slow off-rates or by using minimal target quantities to establish a highly competitive binding environment.…”