Acidic in vitro selection of metal-specific deoxyribozymes

Deoxyribozymes (DNAzymes) are in vitro selected catalytic DNA molecules that recruit metal ions to function. However, nearly all previous DNAzymes generated through conventional selection methods exhibit poor metal selectivity. Here, we report an acidic in vitro selection strategy for isolating truly metal-specific DNAzymes. By using Ca²⁺ as the target in positive selection and a mixture of competing metal ions in counter-selection, and conducting the selections under acidic conditions to suppress metal hydrolysis, we have successfully selected an acidic RNA-cleaving DNAzyme, termed aRCD-Ca2, which is only activated by Ca²⁺ and shows no response to all other tested metal ions, including monovalent ions and chemically similar competing divalent ions (Mg²⁺, Cu²⁺, Zn²⁺, Co²⁺, Ni²⁺, Mn²⁺ and Pb²⁺). This represents the first acidic DNAzyme with exclusive metal selectivity. Moreover, aRCD-Ca2 exhibits fast catalytic activity, with a k _obs of 0.026 min^-1 toward Ca²⁺. A trans-acting aRCD-Ca2TCQ was also engineered from aRCD-Ca2 that enabled highly specific and sensitive monitoring of Ca²⁺ dynamics in HT22 cell lysosomes through a fluorescent probe. We envision that the described acidic in vitro selection strategy can be readily adapted to obtain more new DNAzymes with high specificity for other metal ions and advance the development of nucleic acid catalysts for a wide range of applications.