Observation of DNA Knots Using Solid-State Nanopores

Abstract

Long DNA molecules can self-entangle into knots. Experimental techniques to observe such DNA knots (primarily gel electrophoresis) are limited to bulk methods and circular molecules below 10 kbp in length. Here we show that solid-state nanopores can be used to directly observe individual DNA knots in both linear and circular single molecules of arbitrary length. DNA knots are observed as short spikes in the nanopore current traces of traversing DNA molecules. The observation of knots is dependent on sufficiently high measurement resolution, which can be achieved using high-concentration LiCl buffers. We study the percentage of DNA molecules with knots for different DNA molecules, up to 166 kbp in length. We find that the knotting probability rises strongly with length, and compare our experimental data to 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 small sizes below 100 nm. In the case of linear molecules, we observe that knots are able to slide out upon applying high driving forces (voltage). Our results demonstrate that the solid-state nanopore technique can provide a wealth of information about the position and the size of knots, including the number of DNA strands inside DNA knots.