Abstract:

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.