Beyond Volume Exclusion-Nanopore-Based Protein Sequencing

Abstract

The amino acid sequence and structure of proteins largely determine the intermolecular interaction, protein functions and dynamic process. Nanopore technology has been developing as a powerful single-molecule approach, which has been widely used in biomolecule sensing and achieved DNA sequencing, now aiming at protein sequencing. Compared to the four kinds bases of DNA on nanopore sequencing, the twenty natural amino acids own non-uniform charges and small volumes. Hence, the discrimination of all amino acids is one of the main challenges in protein sequencing. Based on volume exclusion sensing mechanism, most efforts have been taken to explore the nanopore which owns a narrow sensing point with short length to match a single amino acid. However, apart from the fast translocation speed, such a sensing point actually has its limitation theoretically, which could not detect volume difference below 0.003 nm3. Hence, new sensing mechanism needs develop for accurate identification of single amino acid based on confined effect interaction. The long (∼ 10 nm) and narrow (∼ 1.0 nm) aerolysin nanopore could identify single nucleotide and single amino acid differences with long duration, indicating an entirely new sensing mechanism. Although the contribution of the tripeptide volume to the current was only about 0.8%, through reasonably construct mutant aerolysin nanopore sensing interface by modifying and regulating the channels interaction such as hydrogen bonds, hydrophilic and hydrophobic interactions, electrostatic interactions, van der Waals force, etc., the event duration is prolonged to 2.41ms in N226Q mutant nanopore and the small volume tripeptide could be recognized with about 33% ionic blockage. This new mechanism based nanopore holds great promise for the advancement of nanopore based protein sequencing.