An engineered ClyA nanopore detects folded target proteins by selective external association and pore entry.

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.

[1]  T. G. Martin,et al.  DNA origami gatekeepers for solid-state nanopores. , 2012, Angewandte Chemie.

[2]  C. Dekker,et al.  Translocation of RecA-coated double-stranded DNA through solid-state nanopores. , 2009, Nano letters.

[3]  P. Borer,et al.  Engineering a rigid protein tunnel for biomolecular detection. , 2012, Journal of the American Chemical Society.

[4]  Sheereen Majd,et al.  Controlling the translocation of proteins through nanopores with bioinspired fluid walls , 2011, Nature nanotechnology.

[5]  S. Howorka,et al.  Sequence-specific detection of individual DNA strands using engineered nanopores , 2001, Nature Biotechnology.

[6]  Johan Desmet,et al.  The dead-end elimination theorem and its use in protein side-chain positioning , 1992, Nature.

[7]  Hongzhi Xie,et al.  A Genetically Encoded Pore for the Stochastic Detection of a Protein Kinase , 2006, Chembiochem : a European journal of chemical biology.

[8]  C. Dekker,et al.  Single-molecule transport across an individual biomimetic nuclear pore complex. , 2011, Nature nanotechnology.

[9]  Marcus Mueller,et al.  The structure of a cytolytic α-helical toxin pore reveals its assembly mechanism , 2009, Nature.

[10]  Stefan Howorka,et al.  Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore , 2000, Nature Biotechnology.

[11]  R. Glockshuber,et al.  Cytotoxin ClyA from Escherichia coli assembles to a 13‐meric pore independent of its redox‐state , 2006, The EMBO journal.

[12]  M. Niederweis,et al.  Single-molecule DNA detection with an engineered MspA protein nanopore , 2008, Proceedings of the National Academy of Sciences.

[13]  C. Dekker.  Solid-state nanopores. , 2007, Nature nanotechnology.

[14]  J. Danesh,et al.  C-reactive protein concentration and risk of coronary heart disease, stroke, and mortality: an individual participant meta-analysis , 2010, The Lancet.

[15]  Ohad Medalia,et al.  Functional architecture of the nuclear pore complex. , 2012, Annual review of biophysics.

[16]  N. D. de Rooij,et al.  Label-free detection of single protein molecules and protein-protein interactions using synthetic nanopores. , 2008, Analytical chemistry.

[17]  H. Bayley,et al.  Stochastic sensors inspired by biology , 2001, Nature.

[18]  W. Goebel,et al.  Analysis of the SlyA‐controlled expression, subcellular localization and pore‐forming activity of a 34 kDa haemolysin (ClyA) from Escherichia coli K‐12 , 1999, Molecular microbiology.

[19]  D. McNabb,et al.  Electrical characterization of protein molecules by a solid-state nanopore. , 2007, Applied physics letters.

[20]  Li-Qun Gu,et al.  Single-molecule observation of the catalytic subunit of cAMP-dependent protein kinase binding to an inhibitor peptide. , 2005, Chemistry & biology.

[21]  Nicholas A W Bell,et al.  DNA origami nanopores. , 2012, Nano letters.

[22]  Friedrich Förster,et al.  Snapshots of nuclear pore complexes in action captured by cryo-electron tomography , 2007, Nature.

[23]  Ruoshan Wei,et al.  Stochastic sensing of proteins with receptor-modified solid-state nanopores. , 2012, Nature nanotechnology.

[24]  H. Bayley,et al.  Single-molecule kinetics of two-step divalent cation chelation. , 2010, Angewandte Chemie.

[25]  Stefan Howorka,et al.  Stochastic detection of monovalent and bivalent protein-ligand interactions. , 2004, Angewandte Chemie.

[26]  H. Bayley,et al.  Electroosmotic enhancement of the binding of a neutral molecule to a transmembrane pore , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[27]  J. Segura,et al.  Recombinant Erythropoietin and Analogues: A Challenge for Doping Control , 2004, Therapeutic drug monitoring.

[28]  S. Riaz,et al.  Proteomic identification of human serum biomarkers in diabetes mellitus type 2. , 2010, Journal of pharmaceutical and biomedical analysis.

[29]  H. Bayley,et al.  Protein Detection by Nanopores Equipped with Aptamers , 2012, Journal of the American Chemical Society.

[30]  S. Howorka,et al.  Stochastic detection of motor protein-RNA complexes by single-channel current recording. , 2007, Chemphyschem : a European journal of chemical physics and physical chemistry.

[31]  T. Schwartz,et al.  3D ultrastructure of the nuclear pore complex. , 2012, Current opinion in cell biology.

[32]  H. Bayley,et al.  Single-molecule detection of nitrogen mustards by covalent reaction within a protein nanopore. , 2008, Journal of the American Chemical Society.

[33]  H. Bayley,et al.  Analysis of single nucleic acid molecules with protein nanopores. , 2010, Methods in enzymology.

[34]  W. Goebel,et al.  ClyA cytolysin from Salmonella: distribution within the genus, regulation of expression by SlyA, and pore-forming characteristics. , 2009, International journal of medical microbiology : IJMM.

[35]  U. Rant,et al.  Electrically facilitated translocations of proteins through silicon nitride nanopores: conjoint and competitive action of diffusion, electrophoresis, and electroosmosis. , 2010, Nano letters.

[36]  M. Tainsky,et al.  Genomic and proteomic biomarkers for cancer: a multitude of opportunities. , 2009, Biochimica et biophysica acta.

[37]  H. Ogawa,et al.  Significance of a multiple biomarkers strategy including endothelial dysfunction to improve risk stratification for cardiovascular events in patients at high risk for coronary heart disease. , 2009, Journal of the American College of Cardiology.

[38]  B. Chait,et al.  Artificial nanopores that mimic the transport selectivity of the nuclear pore complex , 2009, Nature.

[39]  Xiaofeng Lu,et al.  Simultaneous stochastic sensing of divalent metal ions , 2000, Nature Biotechnology.