DNA‐Encoded Noncanonical Substrate Library for Protease Profiling

Abstract Profiling the substrate sequence preferences of proteases is important for understanding both biological functions as well as for designing protease inhibitors. Several methods are available for profiling the sequence specificity of proteases. However, there is currently no rapid and high‐throughput method to profile specificity of proteases for noncanonical substrates. In this study, we described a strategy to use a DNA‐encoded noncanonical substrate library to identify the protease substrates composed of both canonical and noncanonical amino acids. This approach uses a DNA‐encoded peptide library and introduces a biotin molecule at the N‐terminus to immobilize the library on a solid support. Upon protease hydrolysis, the released DNA tag of the substrate peptides can be sequenced to identify the substrate structures. Using this approach, we profiled trypsin and fibroblast activation protein α and discovered noncanonical substrates that were more efficiently cleaved than the commonly used substrates. The identified substrates of FAP were further used to design corresponding covalent inhibitors containing non‐canonical sequences with high potency for the target protease. Overall, our approach can aid in the development of new protease substrates and inhibitors.