Exploring the Lower Limit of Target Concentration in Capture‐SELEX Using Guanine as a Model Target

During an aptamer selection, using a lower target concentration may result in aptamers with a higher binding affinity. Consequently, this begs the question of whether there is a lower limit for target concentration. In this work, we conducted three aptamer selections using 5 μM, 500 nM and 50 nM guanine as the targets, respectively. Successful enrichment of the same guanine aptamers was achieved at both 5 μM and 500 nM guanine, but not with 50 nM. Using 5 μM guanine, the aptamer was enriched in eight rounds of selection, compared to that for 500 nM, which was accomplished in 17 rounds. We discuss the relation of optimal target concentration to the observed K_d value of the resulting aptamers, of which the highest affinity aptamer had a measured K_d of 200 nM. Additionally, we investigated the binding of the aptamers through mutation studies, revealing a critical cytosine. Mutating this cytosine to a thymine switched the selectivity from guanine to adenine, which is reminiscent of the guanine riboswitch. This study revealed a limit in using low target concentration, and the insights described in this article will be useful for guiding the choice of target concentration during capture-SELEX.