An efficient method to evaluate experimental factor influence on in vitro binding of aptamers

Abstract Nucleic acid-based aptamers are promising alternative to antibodies, however, their selection process (SELEX) is challenging. A number of simulations and few experiments have been reported offering insights into experimental factors (EFs) that govern the effectiveness of the selection process. Though useful, these previous studied were either lack of experimental confirmation, or considered limited EFs. A more efficient experimental method is highly desired. In this study, we developed a fast method that is capable to quantitatively probe the influence of multiple EFs. Based on the fact that the aptamer enrichment efficiency is highly affected by background binding, the binding ratio between the numbers of specific aptamer binders and nonspecific (unselected library) binders per bead was used to quantitatively evaluate EF effects. Taking thrombin and streptavidin as models, three previously studied EFs (surface coverage, buffer composition, and DNA concentration) and four never-studied ones (surface chemistry, heat treatment, elution methodology and pool purity) were investigated. The EFs greatly affected binding ratio (ranging from 0.03 ± 0.03 to 14.60 ± 2.30). The results were in good agreement with the literature, suggesting the good feasibility of our method. Our study provides guidance for the choice of EFs not only for aptamer selection, but also for binding evaluation of aptamers.