On‐Demand Regulation of Catalytic DNA Circuits Using Phosphorylated Charge Reversal Peptides

Catalytic DNA circuits have emerged as a powerful tool for high-performance biosensing application; however, the establishment of a safe and efficient in vivo delivery system remains a critical bottleneck. Peptides serve as attractive carriers due to their rich chemical diversity, excellent biocompatibility, high loading capacity, and specific binding ability, making them ideal candidates for the on-demand regulation of DNA circuits-yet remains largely unexplored. In this study, we developed a multifunctional enzyme-responsive peptide (ERP) for the efficient loading and specific intracellular delivery and release of catalytic circuitry probes through a phosphorylation-based charge reversal procedure. This ERP-programmed catalytic DNA circuit enables the precise, spatially controllable in vivo imaging of microRNA (miRNA). The multifunctional cationic peptide formed a stable nanocomplex with anionic DNA cargo via strong electrostatic interactions, thus protecting the DNA probes from degradation in biological environments. Moreover, with the ability to actively targeting tumor cells and facilitate endogenous phosphorylation-guided release of DNA probes, this multifunctional peptide could significantly reduce the nonspecific delivery of probes to healthy tissues, thereby minimizing unwanted off-site signal leakage. By the integration of cell-selective delivery and site-specific stimulation, this endogenously regulated and multiply guaranteed DNA circuit system paves a simple yet effective way for disease diagnosis.