d -Lactate SELEX Yielding Aptamers Binding Stronger to l -Lactate: Implication of Intrinsic Chiral Preference

Chiral molecules play vital roles in life sciences and medicine. For example, l -lactate is the predominant form in human blood, whereas elevated d -lactate levels are indicative of certain diseases. Aptamers are capable of chiral-selective binding, and to date, aptamers selected against a chiral target consistently exhibit a stronger affinity for that target than for its enantiomer. We previously selected aptamers using l -lactate and obtained sequences that bind l -lactate more strongly than they do d -lactate. Here, d -lactate was used as the selection target; however, the resulting aptamers still showed a higher affinity for l -lactate, suggesting an intrinsic chiral preference of natural D-DNA for l -lactate. A new aptamer, Lac2059, was characterized using a fluorescence strand-displacement assay, yielding a K d of 90 μM for l -lactate and a detection limit of 0.21 mM, while isothermal titration calorimetry gave a K d of 113 μM. Using 2-aminopurine fluorescence spectroscopy, both the previously reported Lac201 aptamer and the current Lac2059 were found to require Mg 2+ for binding. Notably, Lac2059 was less sensitive to Mg 2+ fluctuations at low Mg 2+ concentrations, facilitating improved signal stability for l -lactate sensing under physiological conditions.