Engineering a Biomimetic Biological Nanopore to Selectively Capture Folded Target Proteins

Nanopores have been used in label-free single-molecule studies, including investigations of chemical reactions, nucleic acid analysis and applications in sensing. Biological nanopores generally perform better than artificial nanopores as sensors, but they have disadvantages including a fixed diameter. Here we introduce a biological nanopore ClyA that is wide enough to sample and distinguish large analytes proteins, which enter the pore lumen. Remarkably, human and bovine thrombins, despite 86% sequence identity, elicit characteristic ionic current blockades, which at −50 mV differ in their main current levels by 26 ± 1 pA. The use of DNA aptamers or hirudin as ligands further distinguished the protein analytes. Finally, we constructed ClyA nanopores decorated with aptamers covalently attached to the nanopore exterior. Like nuclear-pore complexes (NPC), these nanopores selectively captured and translocated cognate protein analytes into their interiors, but excluded non-cognate analytes.View Large Image | View Hi-Res Image | Download PowerPoint Slide