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

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.