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Single-molecule transport across an individual biomimetic nuclear pore complex

Abstract

Nuclear pore complexes regulate the selective exchange of RNA and proteins across the nuclear envelope in eukaryotic cells1. Biomimetic strategies offer new opportunities to investigate this remarkable transport phenomenon2. Here, we show selective transport of proteins across individual biomimetic nuclear pore complexes at the single-molecule level. Each biomimetic complex is constructed by covalently tethering either Nup98 or Nup153 (phenylalanine-glycine (FG) nucleoporins) to a solid-state nanopore3. Individual translocation events are monitored using ionic current measurements with sub-millisecond temporal resolution. Transport receptors (Impβ) proceed with a dwell time of 2.5 ms for both Nup98- and Nup153-coated pores, whereas the passage of non-specific proteins is strongly inhibited with different degrees of selectivity. For pores up to 25 nm in diameter, Nups form a dense and low-conducting barrier, whereas they adopt a more open structure in larger pores. Our biomimetic nuclear pore complex provides a quantitative platform for studying nucleocytoplasmic transport phenomena at the single-molecule level in vitro.

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Figure 1: Biomimetic NPC.
Figure 2: Conductance measurements and models.
Figure 3: Single-molecule translocation events.
Figure 4: Event frequencies through bare and Nup-modified pores, showing NPC-like selectivity.
Figure 5: Nanopore array.

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Acknowledgements

The authors thank K.A. Williams for suggestions on the chemistry, A.R. Hall, M. van den Hout and X. Janssen for membrane fabrication and discussions, D. Grünwald and G.V. Soni for help with TIRF measurements, Y. Rabin and A.Y. Grosberg for help with theoretical work, and T. Dange, N.H. Dekker, D. Grünwald, P.L. Hagedoorn, G.F. Schneider and G.M. Skinner for discussions. This research was funded by the ‘Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)’, programme NanoSci E+ of the European Commission, and the READNA (REvolutionary Approaches and Devices for Nucleic Acid analysis) project of the European Commission. L.K. and R.Y.H.L. are supported by the National Center of Competence in Research ‘Nanoscale Science’ (NCCR-Nano), the Swiss National Science Foundation, the Biozentrum and the Swiss Nanoscience Institute.

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S.W.K., R.Y.H.L. and C.D. devised the experiments. L.K. cloned, purified and labelled proteins and carried out SPR analysis. S.W.K., T.R.B., T.M. and P.V.N. carried out the experiments and analysed data. S.W.K., R.Y.H.L. and C.D. wrote the manuscript.

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Correspondence to Roderick Y. H. Lim or Cees Dekker.

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Kowalczyk, S., Kapinos, L., Blosser, T. et al. Single-molecule transport across an individual biomimetic nuclear pore complex. Nature Nanotech 6, 433–438 (2011). https://doi.org/10.1038/nnano.2011.88

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