DNA-Peptide Nanoassembly for Mitochondria-Specific and Programmable Subcellular Apoptotic Regulation.

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 H2O2-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.