Conformationally Locked Peptide-DNA Nanostructures for CRISPR-Amplified Activity-Based Sensing

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Protease activity is crucial in biological processes and disease.
• Existing methods for protease sensing often lack sensitivity or require complex sample preparation.

Purpose of the Study:

• To introduce a novel class of chemical probes, cleavable, locked initiator probes (CLIPs), for activity-based sensing of proteases.
• To demonstrate the high sensitivity, selectivity, and versatility of CLIPs for detecting various disease-relevant proteases.

Main Methods:

• CLIPs utilize protease-cleavable peptides linked to DNA strands, forming a "locked" structure.
• Proteolytic cleavage releases an initiator DNA strand, activating CRISPR-Cas12a for amplified fluorescence detection.
• The peptide sequence can be modified to target specific proteases, including SARS-CoV-2 main protease, caspase-3, MMP-7, and cathepsin B.

Main Results:

• CLIPs exhibit a >20-fold signal turn-on at room temperature.
• CLIPs achieve a limit of detection (LOD) approximately 40-fold lower than commercial probes, at 100-fold lower concentrations (LOD: ~88 pM).
• CLIPs successfully detected cathepsin B in colorectal cancer cell lines, demonstrating clinical potential.

Conclusions:

• CLIPs represent a powerful new tool for activity-based sensing of functional enzymes.
• The probes offer high sensitivity, selectivity, rapid assay times (35 min), and compatibility with complex biological samples.
• CLIPs have significant potential for diagnosing and monitoring diseases associated with protease dysregulation.