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Abstract 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.
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Correlational networking guides the discovery of unclustered lanthipeptide protease-encoding genes
Abstract Bacterial natural product biosynthetic genes, canonically clustered, have been increasingly found to rely on hidden enzymes encoded elsewhere in the genome for completion of biosynthesis. The study and application of lanthipeptides are frequently hindered by unclustered protease genes required for final maturation. Here, we establish a global correlation network bridging the gap between lanthipeptide precursors and hidden proteases. Applying our analysis to 161,954 bacterial genomes, we establish 5209 correlations between precursors and hidden proteases, with 91 prioritized. We use network predictions and co-expression analysis to reveal a previously missing protease for the maturation of class I lanthipeptide paenilan. We further discover widely distributed bacterial M16B metallopeptidases of previously unclear biological function as a new family of lanthipeptide proteases. We show the involvement of a pair of bifunctional M16B proteases in the production of previously unreported class III lanthipeptides with high substrate specificity. Together, these results demonstrate the strength of our correlational networking approach to the discovery of hidden lanthipeptide proteases and potentially other missing enzymes for natural products biosynthesis.
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- Award ID(s):
- 1655740
- PAR ID:
- 10364430
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 13
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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