Synthetic Protein-to-DNA Input Exchange for Protease\nActivity Detection Using CRISPR-Cas12a

We present a novel\nactivity-based detection strategy for matrix\nmetalloproteinase 2 (MMP2), a critical cancer protease biomarker,\nleveraging a mechanism responsive to the proteolytic activity of MMP2\nand its integration with CRISPR-Cas12a-assisted signal amplification.\nWe designed a chemical translator comprising two functional unitsa\npeptide and a peptide nucleic acid (PNA), fused together. The peptide\npresents the substrate of MMP2, while the PNA serves as a nucleic\nacid output for subsequent processing. This chemical translator was\nimmobilized on micrometer magnetic beads as a physical support for\nan activity-based assay. We incorporated into our design a single-stranded\nDNA partially hybridized with the PNA sequence and bearing a region\ncomplementary to the RNA guide of CRISPR-Cas12a. The target-induced\nnuclease activity of Cas12a results in the degradation of FRET-labeled\nDNA reporters and amplified fluorescence signal, enabling the detection\nof MMP2 in the low picomolar range, showing a limit of detection of\n72 pg/mL. This study provides new design principles for a broader\napplicability of CRISPR-Cas-based biosensing.