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

Abstract

In 2018, a DNA aptamer for dopamine was selected 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, which has a Kd 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 4-fold faster release rate, 2-fold lower affinity, and 15-fold lower apparent Kd to a quencher-labeled DNA acting as a capture strand surrogate. All these properties would favor the selection of this new aptamer. We rationalize the missing of this aptamer in the previous work by the too stringent negative selections. The positive selection needs to be carried out at a concentration a few folds higher the Kd of the best aptamer, whereas the negative selection need to be done at a negative target concentration that does not induce significant removal of the best aptamers. This work not only discovers a high affinity dopamine aptamer mutant but prompts thinking of increased library diversity via mutation and the concentration of negative targets for aptamer selection.