Enriching thermodynamically best aptamers by addressing the kinetic aspect of the DNA strand-displacement reaction

Selection of high affinity aptamers is the basis for all aptamer-based applications. Most aptamer selections prioritize thermodynamic factors such as the target concentration yet neglect binding kinetics. Using ampicillin as a target, a library-immobilization based aptamer selection was carried out. A low-affinity aptamer (Kd = 12.7 µM) dominated under the typical condition of gravity flow with a short interaction time of 1–2 min, whereas a high affinity aptamer (Kd = 1.8 µM) dominated the library with a 10 min incubation. These two aptamers differ only in three positions and the strand-displacement kinetics was determined to be responsible for the selection outcomes. Kinetic effects were also observed in the selection of aptamers for adenosine, underscoring the generality of this observation. Aptamers for a challenging target, voriconazole, were also enriched by extending the incubation time. To reliably obtain thermodynamically best aptamers, a combination of low target concentration and long incubation time is recommended. The effect of aptamer binding kinetics in biosensor development is also discussed. This work not only yields a high-affinity and selective aptamer for ampicillin but also highlights the interplay between thermodynamics and kinetics in aptamer selection.