A Cascaded DNA Nanocircuit for Multi‐Signal‐Responsive Precision siRNA Delivery in Cancer Therapy

Precision control over nucleic acid delivery remains a critical challenge in cancer therapy, particularly for siRNA-based gene silencing, where off-target effects limit clinical translation. Herein, we report a programmably engineered DNA nanocircuit with cascaded dual-AND logic gates, which enables the development of a spatiotemporally controlled siRNA delivery strategy for precision cancer therapy. The DNA nanocircuit is engineered to respond to three tumor-specific signals in a sequential manner: extracellular acidic pH, membrane-overexpressed nucleolin (NCL), and intracellular glutathione (GSH). The first AND gate is activated by the co-occurrence of acidic pH and NCL, triggering a conformational rearrangement that generates a molecular output. This integrated output, combined with intracellular GSH, serves as the dual input to co-activate the second AND gate, initiating siRNA release via a cascade reaction inherent to the DNA circuit. As a proof-of-concept, when harnessing this DNA circuit in a temozolomide (TMZ)-resistant glioblastoma (GBM) mouse model, we demonstrate that this design ensures highly selective release of siPARP1 in GBM cells, achieving efficient PARP1 silencing, reversed TMZ resistance, and minimized off-target toxicity. Collectively, the cascaded dual-AND logic, enabled by precise DNA sequence programming, represents a generalizable strategy for multi-signal-responsive delivery systems, highlighting the potential of DNA circuits in precision cancer therapy.