Peptide Nucleic Acid (PNA)‐Guided Peptide Engineering of an Aptamer Sensor for Protease‐Triggered Molecular Imaging

Protease-triggered control of functional DNA has remained unachieved, leaving a significant gap in activatable DNA biotechnology. Herein, we report the design of a protease-activatable aptamer system that can perform molecular sensing and imaging in a tumor-specific manner. The system is constructed by locking the structure-switching activity of an aptamer using a rationally designed PNA-peptide-PNA triblock copolymer. Highly selective protease-mediated cleavage of the peptide substrate results in reduced binding affinity of PNA to the aptamer module, with the subsequent recovery of its biosensing function. We demonstrated that the DNA/peptide/PNA hybrid system allows for tumor cell-selective ATP imaging in vitro and also produces a fluorescent signal in vivo with improved tumor specificity. This work illustrates the potential of bridging the gap between functional DNA and peptides for precise biomedical applications.