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 Kd of 90 μM for l-lactate and a detection limit of 0.21 mM, while isothermal titration calorimetry gave a Kd of 113 μM. Using 2-aminopurine fluorescence spectroscopy, both the previously reported Lac201 aptamer and the current Lac2059 were found to require Mg²⁺ for binding. Notably, Lac2059 was less sensitive to Mg²⁺ fluctuations at low Mg²⁺ concentrations, facilitating improved signal stability for l-lactate sensing under physiological conditions.