Artificial MetalloDNAzymes with High-Density, Near-Atomic Precision Organization of Metal Cofactors for Enhanced Bioorthogonal Catalysis

Bioorthogonal catalysis mediated by immobilized transition metal catalysts (TMCs) offers an enzyme-complementary approach for prodrug activation, but is often constrained by a trade-off between high catalyst loading and precise spatial organization. Here, we report an artificial metalloDNAzyme (Ru-ac@TDF) by incorporating acridine-conjugated ruthenium complexes (Ru-ac) into a tetrahedral DNA framework (TDF) functionalized with the AS1411 aptamer for enhanced bioorthogonal prodrug activation. By intercalating between base pairs of DNA duplexes, Ru-ac is spatially organized within the TDF scaffold, achieving high-density, near-atomic precision organization while simultaneously gaining enhanced solubility and protection from nonspecific deactivation. Under biologically relevant conditions, high-density Ru-ac loading within the TDF boosts the catalytic efficiency by over 1000-fold relative to the bare catalyst. Following nucleolin-mediated selective uptake by cancer cells, Ru-ac@TDF efficiently catalyzes intracellular activation of an alloc-caged doxorubicin prodrug, producing pronounced antiproliferative effect in vitro and potent tumor suppression in vivo, with enhanced intratumoral drug exposure and minimal systemic toxicity. Overall, this work establishes programmable DNA nanostructures as architecturally defined platform for organizing TMCs with high density and spatial precision, providing a conceptually new route toward enhanced bioorthogonal catalysis.