Intracellular DNA network assembly triggered by microRNA and telomerase for imaging-guided STING hyperactivation

Despite its immense promise for cancer immunotherapy, the clinical translation of cGAS-STING activation has been severely limited by nonspecific activation and associated off-target toxicities. Here, we designed a strategy for intracellular DNA network assembly triggered by microRNA (miRNA) and telomerase, enabling imaging-guided STING hyperactivation. The nanoplatform (DiG-DNA) consists of miRNA-responsive hairpin DNAs (H1-H3) and a telomerase-activatable linear double-stranded DNA (LL-DNA). It remains silent in normal cells but undergoes cascade activation in tumor cells overexpressing miRNA-155 and telomerase. MiRNA-155 initiates catalytic hairpin assembly (CHA), while telomerase drives strand elongation; together, these processes release DNA components that co-assemble into a dense three-dimensional DNA network (N-DNA) inside cancer cells. The entire assembly process is monitored in real time via multiplexed fluorescence recovery (Cy3/Cy5), providing an imaging readout to validate successful activation and guide subsequent treatment. The in situ-formed N-DNA acts as a potent cGAS agonist, promotes liquid-liquid phase separation (LLPS), and leads to robust STING activation and antitumor immunity. Triggered by endogenous biomarkers, this intracellular assembly strategy ensures high spatial specificity, as evidenced by strong tumor inhibition in vivo. It thus offers a new paradigm for imaging-guided and precisely controlled cancer immunotherapy.