A Selective Activity-Based Approach for Analysis of Enzymes with an OmpG Nanopore

Many enzymatic activity assays are based on either (i) identifying and quantifying the enzyme with methods such as western blot or enzyme linked substrate assay (ELISA) or (ii) quantifying the enzymatic reaction by monitoring the changing levels of either product or substrate with techniques such as mass spectrometry, absorption or fluorescence spectroscopy, thin layer chromatography, luminescence, and more. We have generated outer membrane protein G (OmpG)-based nanopore approaches to distinguish enzyme identity and analyze the enzyme's catalytic activity. Here, we engineered an OmpG nanopore with a peptide cut site inserted into one of its loops to detect proteolytic behavior. In addition, we generated an OmpG nanopore with a single-stranded DNA attached to a loop for analyzing nucleolytic cleavage. These OmpG nanopore approaches may be highly useful in analyzing specific enzymes in complex biological samples, or in directly determining kinetics of enzyme-substrate complex association and dissociation.