How to Engineer Biological Nanopores for Single-Molecule Capture and Discrimination

Protein-based biological nanopores have emerged as versatile tools for sequencing biopolymers, identifying molecules, and probing reactions at the single-molecule level. Many biological nanopores have been characterized, revealing the significance of nanopore size, shape, and chemical composition in molecular detection and discrimination. Advancements in understanding the nanofluidic properties of nanopores have complemented engineering approaches expanding the possible applications of nanopore technology. Through a combination of structural insights, rational design, systematic screening, and molecular dynamics simulations, researchers have developed nanopores capable of distinguishing between ions, nucleic acids, peptides, and folded proteins with remarkable specificity and sensitivity. Building upon three decades of progress, this Review highlights recent engineering innovations, discusses ongoing challenges, and explores the prospects in the field.