Single OmpG Nanopore Gating Regulated by Linker Length for Protease Detection

Nanopore sensing is a powerful technology for single-molecule analysis. The OmpG protein is particularly suitable for occlusion-based detection owing to its inherent gating properties. However, insertion of long sequences into the internal loops of OmpG can compromise structural stability. In this study, we investigated an approach of fusing functional linkers to the N-terminus of OmpG, a method that preserves structural integrity while allowing for the introduction of specific protease cleavage sites for TEV protease, thrombin, and HRV3C. Using a planar lipid bilayer system, we analyzed the ionic currents of the OmpG variants before and after protease treatment. Our results showed that N-terminal linker length significantly modulated gating dynamics. Variants with a linker exhibited high gating frequencies, characterized by over 3.6 events/s of a close event rate and mean interevent intervals below 300 ms. Protease-mediated cleavage effectively reversed these behaviors, allowing for clear discrimination of the linker presence with accuracies of up to 90.9%. Molecular dynamics simulations corroborated these findings, showing that longer linkers had a higher probability of occluding pore entrances. Furthermore, mismatch experiments confirmed high specificity and analysis of short-duration signals revealed that the mean interevent interval is a reliable index for high-throughput screening. This study suggests that N-terminal linker-fused OmpG platforms can be used for specific detection of proteases.