An engineered ClyA nanopore detects folded target proteins by selective external association and pore entry.
Mikhael Soskine,Annemie Biesemans,Benjamien Moeyaert,Stephen Cheley,Hagan Bayley,Giovanni Maglia +5 more
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TL;DR: A biological nanopore ClyA is introduced that is wide enough to sample and distinguish large analyte proteins, which enter the pore lumen, and selectively captured and internalized cognate protein analytes but excluded noncognate analytes.
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Abstract: 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 analyte 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 covalently attached aptamers. These nanopores selectively captured and internalized cognate protein analytes but excluded noncognate analytes, in a process that resembles transport by nuclear pores.
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Citations
Electro-Osmotic Vortices Promote the Capture of Folded Proteins by PlyAB Nanopores.
TL;DR: It is found that the precise engineering of the inner surface charge of the PlyAB induced electro-osmotic vortices that allowed the electrophoretic capture of the proteins and provided a practical method to promote the capture and analysis of folded proteins by nanopores.
Rapid and Accurate Determination of Nanopore Ionic Current Using a Steric Exclusion Model.
TL;DR: A robust and inexpensive computational approach to the steric exclusion model (SEM) of nanopore conductance is demonstrated that is orders of magnitude more efficient than all-atom MD and yet is sensitive enough to account for the atomic structure of the nanopore and the analyte.
Electrostatic Interactions between OmpG Nanopore and Analyte Protein Surface Can Distinguish between Glycosylated Isoforms.
Monifa A. Fahie,Min Chen +1 more
TL;DR: In this article, the authors demonstrate that the direct interaction between the nanopore and analyte surface, induced by the electrostatic attraction between the two molecules, is essential for protein isoform detection.
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The Utility of Nanopore Technology for Protein and Peptide Sensing.
TL;DR: A review of developments in the area of nanopore‐based protein and peptide detection highlights resistive pulse nanopore sensing, which enables label‐free single‐molecule analysis of a wide range of analytes.
64
Detection of Two Isomeric Binding Configurations in a Protein-Aptamer Complex with a Biological Nanopore
TL;DR: In this paper, the interaction between the G-quadruplex fold of the thrombin binding aptamer (TBA) and human Thrombin (HT) was studied using ClyA nanopores and showed that the internalization of the HT:TBA complex inside the nanopore induced two types of current blockades with distinguished residual current and lifetime.
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