Synthetic protein-conductive membrane nanopores built with DNA

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Abstract

Nanopores are key in portable sequencing and research given their ability to transport elongated DNA or small bioactive molecules through narrow transmembrane channels. Transport of folded proteins could lead to similar scientific and technological benefits. Yet this has not been realised due to the shortage of wide and structurally defined natural pores. Here we report that a synthetic nanopore designed via DNA nanotechnology can accommodate folded proteins. Transport of fluorescent proteins through single pores is kinetically analysed using massively parallel optical readout with transparent silicon-on-insulator cavity chips vs. electrical recordings to reveal an at least 20-fold higher speed for the electrically driven movement. Pores nevertheless allow a high diffusive flux of more than 66 molecules per second that can also be directed beyond equillibria. The pores may be exploited to sense diagnostically relevant proteins with portable analysis technology, to create molecular gates for drug delivery, or to build synthetic cells.

Abstract

Nanopores have a wide range of applications in the field of sensing. Here the authors report on synthetic nanopores made of DNA and designed for the transit of folded proteins across membranes to allow for biosensing.

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Most cited references 70

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Self-assembly of DNA into nanoscale three-dimensional shapes

Shawn M. Douglas, Hendrik Dietz, Tim Liedl … (2009)

Molecular self-assembly offers a ‘bottom-up’ route to fabrication with subnanometre precision of complex structures from simple components1. DNA has proven a versatile building block2–5 for programmable construction of such objects, including two-dimensional crystals6, nanotubes7–11, and three-dimensional wireframe nanopolyhedra12–17. Templated self-assembly of DNA18 into custom two-dimensional shapes on the megadalton scale has been demonstrated previously with a multiple-kilobase ‘scaffold strand’ that is folded into a flat array of antiparallel helices by interactions with hundreds of oligonucleotide ‘staple strands’19, 20. Here we extend this method to building custom three-dimensional shapes formed as pleated layers of helices constrained to a honeycomb lattice. We demonstrate the design and assembly of nanostructures approximating six shapes — monolith, square nut, railed bridge, genie bottle, stacked cross, slotted cross — with precisely controlled dimensions ranging from 10 to 100 nm. We also show hierarchical assembly of structures such as homomultimeric linear tracks and of heterotrimeric wireframe icosahedra. Proper assembly requires week-long folding times and calibrated monovalent and divalent cation concentrations. We anticipate that our strategy for self-assembling custom three-dimensional shapes will provide a general route to the manufacture of sophisticated devices bearing features on the nanometer scale.

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Continuous base identification for single-molecule nanopore DNA sequencing.

James Clarke, Hai-Chen Wu, Lakmal Jayasinghe … (2009)

A single-molecule method for sequencing DNA that does not require fluorescent labelling could reduce costs and increase sequencing speeds. An exonuclease enzyme might be used to cleave individual nucleotide molecules from the DNA, and when coupled to an appropriate detection system, these nucleotides could be identified in the correct order. Here, we show that a protein nanopore with a covalently attached adapter molecule can continuously identify unlabelled nucleoside 5'-monophosphate molecules with accuracies averaging 99.8%. Methylated cytosine can also be distinguished from the four standard DNA bases: guanine, adenine, thymine and cytosine. The operating conditions are compatible with the exonuclease, and the kinetic data show that the nucleotides have a high probability of translocation through the nanopore and, therefore, of not being registered twice. This highly accurate tool is suitable for integration into a system for sequencing nucleic acids and for analysing epigenetic modifications.

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Solid-state nanopores.

Cees Dekker (2007)

The passage of individual molecules through nanosized pores in membranes is central to many processes in biology. Previously, experiments have been restricted to naturally occurring nanopores, but advances in technology now allow artificial solid-state nanopores to be fabricated in insulating membranes. By monitoring ion currents and forces as molecules pass through a solid-state nanopore, it is possible to investigate a wide range of phenomena involving DNA, RNA and proteins. The solid-state nanopore proves to be a surprisingly versatile new single-molecule tool for biophysics and biotechnology.

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Author and article information

Contributors

Robert Tampé:

ORCID: http://orcid.org/0000-0002-0403-2160

tampe@em.uni-frankfurt.de

Stefan Howorka:

ORCID: http://orcid.org/0000-0002-6527-2846

s.howorka@ucl.ac.uk

Journal

Journal ID (nlm-ta): Nat Commun

Journal ID (iso-abbrev): Nat Commun

Title: Nature Communications

Publisher: Nature Publishing Group UK (London )

ISSN (Electronic): 2041-1723

Publication date (Electronic): 4 November 2019

Publication date PMC-release: 4 November 2019

Publication date Collection: 2019

Volume: 10

Electronic Location Identifier: 5018

Affiliations

[1 ]ISNI 0000 0004 1936 9721, GRID grid.7839.5, Institute of Biochemistry, Biocenter, , Goethe University Frankfurt, ; Max-von-Laue Str.9, 60438 Frankfurt/M., Germany

[2 ]ISNI 0000000121901201, GRID grid.83440.3b, Department of Chemistry, Institute of Structural Molecular Biology, , University College London, ; London, WC1H 0AJ UK

[3 ]ISNI 0000000123222966, GRID grid.6936.a, Molecular Electronics, , Technical University of Munich, ; Theresienstraße 90, 80333 Munich, Germany

[4 ]ISNI 0000 0004 0496 8414, GRID grid.469866.3, Fraunhofer Research Institution for Microsystems and Solid State Technologies (EMFT), ; Hansastraße 27d, 80686 Munich, Germany

[5 ]ISNI 0000 0004 1936 973X, GRID grid.5252.0, Center of Nanoscience (CeNS), , Ludwig-Maximilians-University, ; Schellingstraße 4, 80799 Munich, Germany

Author information

Tim Diederichs http://orcid.org/0000-0002-4603-6274

Genevieve Pugh http://orcid.org/0000-0002-9022-167X

Adam Dorey http://orcid.org/0000-0003-1771-9436

Yongzheng Xing http://orcid.org/0000-0002-6186-8028

Jonathan R. Burns http://orcid.org/0000-0003-0117-9658

Quoc Hung Nguyen http://orcid.org/0000-0002-2166-4741

Marc Tornow http://orcid.org/0000-0002-1860-2769

Robert Tampé http://orcid.org/0000-0002-0403-2160

Stefan Howorka http://orcid.org/0000-0002-6527-2846

Article

Publisher ID: 12639

DOI: 10.1038/s41467-019-12639-y

PMC ID: 6828756

PubMed ID: 31685824

SO-VID: e632e6fc-c3a0-4f6e-ac40-7c656f62ef8a

License:

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 18 June 2016

Date accepted : 23 September 2019

Funding

Funded by: German-Israeli Project Cooperation, DIP, TO 266/8-1

Funded by: FundRef https://doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft (German Research Foundation);

Award ID: SFB 807

Award ID: EXC 115

Award Recipient : Robert Tampé

Funded by: German-Israeli Project Cooperation (DIP, TO 266/8-1)

Funded by: FundRef https://doi.org/10.13039/501100000266, RCUK | Engineering and Physical Sciences Research Council (EPSRC);

Award ID: EP/N009282/1

Award Recipient : Stefan Howorka

Funded by: FundRef https://doi.org/10.13039/501100000268, RCUK | Biotechnology and Biological Sciences Research Council (BBSRC);

Award ID: BB/M025373/1, BB/N017331/1

Award Recipient : Stefan Howorka

Funded by: FundRef https://doi.org/10.13039/501100000275, Leverhulme Trust;

Award ID: RPG-2017-015

Award Recipient : Stefan Howorka

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ScienceOpen disciplines: Uncategorized

Keywords: biophysical chemistry,dna nanostructures

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ScienceOpen disciplines: Uncategorized

Keywords: biophysical chemistry, dna nanostructures

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Synthetic protein-conductive membrane nanopores built with DNA

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Most cited references 70

Self-assembly of DNA into nanoscale three-dimensional shapes

Continuous base identification for single-molecule nanopore DNA sequencing.

Solid-state nanopores.

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Cited by 29

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