Improving the Sensitivity of a Mn(II)-Specific DNAzyme for Cellular Imaging Sensor through Sequence Mutations

Detection of Mn²⁺ in living cells is important in understanding the roles of Mn²⁺ in cellular processes and investigating its potential implications in various diseases and disorders. Toward this goal, we have previously selected a Mn²⁺-specific 11-5 DNAzyme through an in vitro selection method and converted it into a fluorescence sensor for intracellular Mn²⁺ sensing. Despite the progress, the nucleotides responsible for the activity are unclear, and the performance of the DNAzyme needs to be improved in order for more effective applications in biological systems. To address these issues, we herein report site-specific mutations within the catalytic domain of the selected 11-5 DNAzyme. As a result, we successfully identified a variant DNAzyme, designated as Mn5V, which exhibited a twofold increase in activity compared to the original 11-5 DNAzyme. Importantly, Mn5V DNAzyme maintained its high selectivity for Mn²⁺ over other competing metal ions. Upon the addition of Mn²⁺, Mn5V DNAzyme exhibited a higher fluorescence signal within the tumor cells compared to that of the 11-5 DNAzyme. This study therefore provides a better understanding of how the DNAzyme functions and a more sensitive probe for investigating Mn²⁺ in biological systems.