Enhancing DNAzyme Silencing via Phosphorothiolation to Reduce the Mg²⁺-Dependence

817 DNAzyme has emerged as a potent catalytic nucleic acid tool for gene expression silencing, offering distinct advantages including programmable target recognition, enzymatic turnover capability, and high biostability. Despite its therapeutic potential, clinical applications of this metalloenzyme have been constrained by suboptimal catalytic performance under physiological conditions, which is primarily attributed to insufficient intracellular Mg²⁺ concentrations (typically <1 mM). To address this critical limitation, we have developed an atomic probing approach through systematic nucleotide modifications at key catalytic residues, which has successfully reduced the Mg²⁺ dependency by 50%. The optimized DNAzyme has largely enhanced the RNA cleavage (by 1.7 fold) at physiological Mg²⁺ (0.5 mM), offering significantly higher gene silencing in 293T cells, compared to the wild-type. By enabling efficient gene silencing in native biological environments, this novel advancement in metalloenzyme engineering and DNAzyme catalysis has established a chemical approach to enhance the DNAzyme activity under physiological Mg²⁺ conditions, which is a critical prerequisite for future therapeutic applications and biotech developments.