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Spatially multiplexed single-molecule translocations through a nanopore at controlled speeds

Nature Nanotechnology volume 18, pages 1078–1084 (2023)Cite this article

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Abstract

In current nanopore-based label-free single-molecule sensing technologies, stochastic processes influence the selection of translocating molecule, translocation rate and translocation velocity. As a result, single-molecule translocations are challenging to control both spatially and temporally. Here we present a method using a glass nanopore mounted on a three-dimensional nanopositioner to spatially select molecules, deterministically tethered on a glass surface, for controlled translocations. By controlling the distance between the nanopore and glass surface, we can actively select the region of interest on the molecule and scan it a controlled number of times and at a controlled velocity. Decreasing the velocity and averaging thousands of consecutive readings of the same molecule increases the signal-to-noise ratio by two orders of magnitude compared with free translocations. We demonstrate the method’s versatility by assessing DNA–protein complexes, DNA rulers and DNA gaps, achieving down to single-nucleotide gap detection.

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Fig. 1: Controlled translocations of single molecules with nanopore-based SICS.
Fig. 2: Controlled translocation of custom-designed DNA rulers increases SNR, precision and accuracy.
Fig. 3: Spatial addressability of single-molecule translocations demonstrated on DNA gaps.
Fig. 4: Detection of single-nucleotide gaps with SICS.

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Data availability

The data that support the plots in Figs. 1b,c, 2b–g, 3c–e and 4b,d are available via Zenodo at https://doi.org/10.5281/zenodo.7834215. Source data are provided with this paper.

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Acknowledgements

S.M.L. and G.E.F. acknowledge support from the Swiss Commission for Technology and Innovation under grant CTI-18330.1, and the European Research Council under grant no. ERC-2017-CoG, InCell. V.N., H.M. and A.R. acknowledge support from the National Center of Competence in Research (NCCR) Bio-Inspired Materials and Max-Planck-EPFL Center of Molecular Nanoscience and Technology. K.C. and U.F.K. were funded by PoreDetect (ERC-2019-POC, 899538) and EarlyPore (ERC-2022-POC1, 101069324).

Author information

Authors and Affiliations

  1. Laboratory for Bio- and Nano-Instrumentation, Institute of Bioengineering, School of Engineering, EPFL, Lausanne, Switzerland

    S. M. Leitao, B. Drake & G. E. Fantner

  2. Laboratory of Nanoscale Biology, Institute of Bioengineering, School of Engineering, EPFL, Lausanne, Switzerland

    V. Navikas, H. Miljkovic, S. Marion, S. F. Mayer & A. Radenovic

  3. Laboratory of Molecular Biology, Institute of Life Technologies, School of Engineering, HES-SO Valais-Wallis, Sion, Switzerland

    G. Pistoletti Blanchet & A. Kuhn

  4. Central Environmental Laboratory, Institute of Environmental Engineering, ENAC, EPFL, Sion, Switzerland

    G. Pistoletti Blanchet

  5. Cavendish Laboratory, University of Cambridge, Cambridge, UK

    K. Chen & U. F. Keyser

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  1. S. M. Leitao
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Contributions

S.M.L. developed the SICS system and performed the SICS measurements. S.M.L. and V.N. performed the data analysis with support from S.M. V.N. and H.M. prepared the single-molecule samples and fabricated the nanocapillaries. G.P.B. and A.K. created the DNA gap molecules. H.M. and S.F.M. created the oligonucleotides–DNA gap templates. K.C. and U.F.K. created the DNA rulers and provided free translocation data. S.M.L. and B.D. built the original SICM setup used in this work. A.R., G.E.F., S.M.L. and V.N. designed the experiments with input from all the authors. A.R. conceived the method. G.E.F. and A.R. supervised the project. S.M.L., G.E.F. and A.R. wrote the manuscript with input from all the authors.

Corresponding authors

Correspondence to G. E. Fantner or A. Radenovic.

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Competing interests

A.R., G.E.F., S.M.L. and V.N. filed a patent application PCT/IB2022/055136 titled ‘Nanopore-based scanning system and method’, Switzerland.

Peer review

Peer review information

Nature Nanotechnology thanks Paolo Actis, Manoj Varma and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–16.

Source data

Source Data Fig. 1b

Source data containing the conductance signal as a function of distance for Fig. 1b.

Source Data Figs. 1c and 2b

Source data containing the conductance signal as a function of distance for Figs. 1c and 2b.

Source Data Fig. 2c

Source data containing the conductance signal as a function of distance for four different velocities for Fig. 2c.

Source Data Fig. 2d

SNR values for the detection of markers at different translocation velocities.

Source Data Fig. 2e

Values of amplitude and distance of the ruler’s markers in free translocations and controlled translocations.

Source Data Fig. 2f

Source data containing the conductance signal as a function of distance for Fig. 2f.

Source Data Fig. 2g

Source data containing the conductance signal as a function of distance for Fig. 2g.

Source Data Fig. 3c

Source data containing the conductance signal as a function of distance for Fig. 3c.

Source Data Fig. 3d

Source data containing the conductance values at a corresponding distance used to plot the density map in Fig. 3d.

Source Data Fig. 3e

Source data containing the conductance values at a corresponding distance used to plot the density map in Fig. 3e.

Source Data Fig. 4b top

Source data containing the conductance values at a corresponding distance used to plot the density map in Fig. 4b (top).

Source Data Fig. 4b bottom

Source data containing the conductance values at a corresponding distance used to plot the density map in Fig. 4b (bottom).

Source Data Fig. 4d

Source data containing the conductance values at a corresponding distance used to plot the density map in Fig. 4d.

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Leitao, S.M., Navikas, V., Miljkovic, H. et al. Spatially multiplexed single-molecule translocations through a nanopore at controlled speeds. Nat. Nanotechnol. 18, 1078–1084 (2023). https://doi.org/10.1038/s41565-023-01412-4

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