A high-affinity dopamine aptamer: implications of library diversity and negative selection

Abstract

In 2018, a DNA aptamer for dopamine was isolated after extensive negative selection steps. Herein, a new selection experiment was carried out under the same conditions except that no negative selections were performed. This selection yielded a single family of binding sequences, with a K_d of 424 nM from isothermal titration calorimetry, 6-fold lower compared to the previous aptamer, although they differ only by one nucleotide. Using the fluorescence strand-displacement reaction, the newly selected aptamer had a 4-fold faster release rate, 2-fold lower affinity, and 15-fold lower apparent K_d compared to a quencher-labeled DNA acting as a capture strand surrogate. All these properties would favor the selection of this new higher affinity aptamer. This aptamer was missed in the previous work likely by the overly stringent negative selection. Positive selection needs to be carried out at a concentration a few times higher than the K_d of the best aptamer, whereas negative selection needs to be done at a negative target concentration that does not induce significant removal of the best aptamers. This work not only identifies a high-affinity dopamine aptamer mutant but also prompts rethinking of strategies to increase library diversity and optimize the concentration of negative targets in aptamer selection.