Protease probes that enable excimer signaling upon scission.

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Protease activity monitoring is crucial in biological research and diagnostics.
• Fluorescent probes offer sensitive detection but face challenges in complex media like blood serum due to autofluorescence.
• Pyrene excimer signaling provides advantages like a large Stokes shift and long emission lifetime, ideal for such media.

Purpose of the Study:

• To develop a novel probe architecture for protease activity detection using pyrene excimer signaling.
• To overcome the limitation of proteolytic cleavage abolishing proximity required for excimer formation.
• To enable "turn-on" fluorescence detection of protease activity in biological samples.

Main Methods:

• Design of hairpin-structured peptide nucleic acid (PNA)/peptide hybrids.
• Incorporation of pyrene units and anthraquinone-based quenchers in a zipper-like arrangement within the PNA stem.
• Utilizing time-resolved measurements for signal detection.

Main Results:

• The developed excimer hairpin peptide beacons demonstrated up to a 50-fold enhancement in pyrene excimer emission upon proteolytic cleavage.
• Successful detection of matrix metalloprotease 7 (MMP-7) in human blood serum was achieved.
• The probe architecture effectively switched on pyrene excimer emission upon proteolytic scission.

Conclusions:

• The novel hairpin-structured PNA/peptide hybrid probes enable "turn-on" pyrene excimer fluorescence signaling upon protease activity.
• This approach overcomes previous limitations and allows for sensitive protease detection in complex biological matrices.
• The probes show promise for real-time monitoring of specific protease activities in clinical diagnostics.