Conformationally Locked Peptide–DNA Nanostructures for CRISPR‐Amplified Activity‐Based Sensing

We introduce a new class of chemical probes for activity-based sensing of proteases, termed cleavable, locked initiator probes (CLIPs). CLIPs contain a protease-cleavable peptide linked between two programmable DNA strands-an "initiator" DNA and a shorter "blocking" DNA. These DNA sequences are designed to hybridize, creating a "locked" hairpin-like structure. Upon proteolytic cleavage, the initiator strand is released, triggering the activation of CRISPR-Cas12a enzymes and producing an amplified fluorescence response. CLIPs generate more than 20-fold turn-on signals at room temperature (25 °C), significantly outperforming commercial probes by yielding ∼40-fold lower limits of detection (LOD) at 100-fold lower concentrations. Their versatility enables the detection of various disease-relevant proteases-including the SARS-CoV-2 main protease, caspase-3, matrix metalloproteinase-7, and cathepsin B-simply by altering the peptide sequence. Importantly, CLIPs detect cathepsin B in four different colorectal cancer cell lines, highlighting their clinical potential. Taken together, the sensitivity (LOD: ∼88 pM), selectivity, and rapid assay time (down to 35 min), combined with the ability to operate in complex biological media with minimal sample preparation, position CLIPs as powerful chemical tools for activity-based sensing of functional enzymes.