DNA-Encoded Noncanonical Substrate Library for Protease Profiling

Area of Science:

• Biochemistry and Molecular Biology
• Protease Function and Inhibition

Background:

• Understanding protease substrate specificity is crucial for biological insights and therapeutic development.
• Existing methods lack high-throughput capabilities for profiling noncanonical protease substrates.

Purpose of the Study:

• To develop a rapid, high-throughput strategy for identifying protease substrates with noncanonical amino acids.
• To discover novel substrates for trypsin and fibroblast activation protein α (FAP).

Main Methods:

• Utilized a DNA-encoded peptide library with N-terminal biotin for immobilization.
• Employed protease hydrolysis to release DNA tags, which were then sequenced to determine substrate structures.
• Applied the method to profile trypsin and FAP.

Main Results:

• Identified novel noncanonical substrates for trypsin and FAP, some cleaved more efficiently than standard substrates.
• Designed potent covalent inhibitors for FAP based on the identified noncanonical substrates.

Conclusions:

• The DNA-encoded library approach provides a powerful tool for profiling protease specificity with noncanonical substrates.
• This strategy facilitates the development of new protease substrates and highly potent protease inhibitors.