DNA‐Peptide Nanoassembly for Mitochondria‐Specific and Programmable Subcellular Apoptotic Regulation

ABSTRACT Effective tumor cell apoptosis critically depends on the efficient delivery of pro‐apoptotic agents to mitochondria. While mitochondria‐targeted peptide‐based nanoassemblies offer a promising platform for controllable delivery, achieving mitochondria‐specific responses and programmable subcellular apoptotic regulation remains a critical yet underexplored challenge. Herein, we report controllable mitochondria‐targeted DNA‐peptide nanoassemblies co‐assembled from an enterokinase (ENTK)‐responsive cationic peptide—incorporating a DDDDK substrate motif targeted to the perimitochondrial ENTK—and a multifunctional DNAzyme chimera (DC) through DNAzyme‐catalyzed dityrosine crosslinking, thereby enabling mitochondria‐specific and programmable subcellular apoptotic regulation. In addition, the DC integrates a hemin‐intercalated G‐quadruplex (G4/hemin) that catalyzes H 2 O 2 ‐dependent reactive oxygen species (ROS) generation and an antisense oligonucleotide (ASO) targeting antisense long noncoding mitochondrial RNAs (ASncmtRNAs). Upon selective peptide cleavage by perimitochondrial ENTK, the DNA‐peptide nanoassemblies undergo subcellular‐specific disassembly and payload release, resulting in mitochondria‐localized ROS generation and synergistic ASO‐mediated ASncmtRNA knockdown, accompanied by suppression of the anti‐apoptotic protein survivin. These coordinated effects collectively induce mitochondria‐mediated apoptosis and achieve effective tumor suppression. This work establishes a DNAzyme‐catalyzed covalent assembly strategy that integrates the molecular programmability of nucleic acids with the biological specificity of peptides, providing a modular and highly tunable platform for organelle‐targeted nanosystems and enabling sophisticated spatiotemporal combination therapy.