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Direct observation of DNA knots using a solid-state nanopore

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Abstract

Long DNA molecules can self-entangle into knots. Experimental techniques for observing such DNA knots (primarily gel electrophoresis) are limited to bulk methods and circular molecules below 10 kilobase pairs in length. Here, we show that solid-state nanopores can be used to directly observe individual knots in both linear and circular single DNA molecules of arbitrary length. The DNA knots are observed as short spikes in the nanopore current traces of the traversing DNA molecules and their detection is dependent on a sufficiently high measurement resolution, which can be achieved using high-concentration LiCl buffers. We study the percentage of molecules with knots for DNA molecules of up to 166 kilobase pairs in length and find that the knotting occurrence rises with the length of the DNA molecule, consistent with a constant knotting probability per unit length. Our experimental data compare favourably with previous simulation-based predictions for long polymers. From the translocation time of the knot through the nanopore, we estimate that the majority of the DNA knots are tight, with remarkably small sizes below 100 nm. In the case of linear molecules, we also observe that knots are able to slide out on application of high driving forces (voltage).

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Figure 1: A schematic illustration of a DNA molecule with a knot translocating through a solid-state nanopore.
Figure 2: Six example events for lambda DNA translocating through a 10 nm pore at 200 mV in 2 M LiCl at 30 kHz bandwidth.
Figure 3: Percentage of events with knots as a function of DNA length.
Figure 4: Normalized centre position of knots observed in 20.7 kbp relaxed circular molecules.
Figure 5: Translocation duration of knots (τ) observed in 20.7 kbp linear molecules in 4 M LiCl at 100 mV.

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Acknowledgements

The authors would like to thank C. Micheletti, M. Di Stefano and P. Virnau for discussions, M.-Y. Wu for TEM drilling of nanopores and R. Joseph and S. W. Kowalczyk for early experiments. This work was supported by the Netherlands Organisation for Scientific Research (NWO/OCW), as part of the Frontiers of Nanoscience program, and by the European Research Council under research grant NanoforBio (no. 247072) and SynDiv (no. 669598), the Koninklijke Nederlandse Akademie van Wetenschappen (KNAW) Academy Assistants Program and by the Wenner-Gren Foundations. Y.R. and A.Y.G. would like to acknowledge support from the US–Israel Binational Science foundation.

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C.P., D.V., S.P., J.v.d.T., J.W.R, M.J.W. and M.P.J. carried out the measurements; C.P. and D.V. analysed experimental data; A.Y.G. and Y.R. provided theoretical interpretation; all authors discussed and interpreted results; C.P. and C.D. wrote the manuscript with input from all authors.

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Correspondence to Cees Dekker.

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Plesa, C., Verschueren, D., Pud, S. et al. Direct observation of DNA knots using a solid-state nanopore. Nature Nanotech 11, 1093–1097 (2016). https://doi.org/10.1038/nnano.2016.153

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