An Orthogonal Nucleic Acid/Peptide Amplification Circuit Enables Protease‐Triggered, Cancer Cell‐Selective PD‐L1 Imaging

Abstract Molecular imaging offers a powerful approach for in situ detection of programmed death‐ligand 1 (PD‐L1), however, achieving cancer cell‐selective imaging that discriminates PD‐L1 expression on malignant versus normal cells remains a challenge. Here, we present an orthogonal nucleic acid/peptide amplification circuit that integrates protease‐activated hybridization chain reaction (HCR) with aptamer‐mediated target recognition for cancer‐selective PD‐L1 imaging. In the design, PD‐L1 aptamer is coupled with an HCR initiator for targeting PD‐L1, while PNA is employed as a bridge scaffold to engineer the initiator with protease‐responsive peptide substrate and thus block the HCR. Within the tumor microenvironment, protease‐mediated peptide cleavage liberates the initiator, thereby triggering localized HCR amplification at PD‐L1 sites. In contrast, in normal tissues lacking the relevant proteases, the initiator remains inactive, yielding markedly improved spatial selectivity for cancer cell‐specific PD‐L1 imaging. Using mouse models, we further demonstrate that this strategy allows for non‐invasive assessment of tumor responses to immune checkpoint blockade therapy. This methodology will build a bridge between DNA nanobiotechnology and peptide‐based biochemistry for diverse biomedical applications.