An Exceptionally Selective DNA Cooperatively Binding Two Ca2+ Ions

Ca²⁺ is a highly important metal ion in biology and in the environment, and thus there is extensive work in developing sensors for Ca²⁺ detection. Although many Ca²⁺‐binding proteins are known, few nucleic acids can selectively bind Ca²⁺. DNA‐based biosensors are attractive for their high stability and excellent programmability. We report a RNA‐cleaving DNAzyme, EtNa, cooperatively binding two Ca²⁺ ions but to only one Mg²⁺. Four DNAzymes with known Ca²⁺‐dependent activity were compared, and the EtNa had the best selectivity for Ca²⁺. The EtNa is 90 times more active in Ca²⁺ than in Mg²⁺. Phosphorothioate (PS) modification showed that both non‐bridging oxygen atoms at the scissile phosphate contribute equally to Ca²⁺ binding. The pH–rate profile suggests two concurrent deprotonation reactions. EtNa was further engineered for Ca²⁺ sensing, and found to have a detection limit of 17 μm Ca²⁺ and excellent selectivity. The detection of Ca²⁺ in tap water was performed, and the result was comparable with that by ICP‐MS. This study offers new fundamental insights into Ca²⁺ binding by nucleic acids and improved metal selectivity by having multiple cooperative metal binding sites.