Clog and Release, and Reverse Motions of DNA in a Nanopore

Abstract

Motions of circular and linear DNA molecules of various lengths near a nanopore of 100 or 200 nm diameter were experimentally observed and investigated by fluorescence microscopy. The movement of DNA molecules through nanopores, known as translocation, is mainly driven by electric fields near and inside the pores. We found significant clogging of nanopores by DNA molecules, particularly by circular DNA and linear T4 DNA (165.65 kbp). Here, the probabilities of DNA clogging events, depending on the DNA length and shape-linear or circular-were determined. Furthermore, two distinct DNA motions were observed: clog and release by linear T4 DNA, and a reverse direction motion at the pore entrance by circular DNA, after which both molecules moved away from the pore. Finite element method-based numerical simulations were performed. The results indicated that DNA molecules with pores 100⁻200 nm in diameter were strongly influenced by opposing hydrodynamic streaming flow, which was further enhanced by bulky DNA configurations.

Keywords: DNA; electro-osmosis; nanopore; translocation.

Figure 1

Clog probability vs. DNA length and its shape-linear (squares) or circular (circles) for pore diameters of 100 nm (red) and 200 nm (blue). Error bars represent the standard deviation of each data point. The solid line is the analytical prediction of knot formation, 1−exp(−N/N₀), where N₀ = 143 ± 5 kbp by Plesa et al. [27].

Figure 2

Release of T4 DNA to the cis side after clogging a pore. (a) Time-resolved fluorescence images of T4 DNA molecules near a nanopore. Scale bar is 5 μm. Images were extracted at t = 0.00, 0.21, 0.29, 1.14, and 2.21 s from a sequence of 21 frames recorded at 14 Hz. A DNA approaches a nanopore located at the center in each image marked by a yellow circle. The DNA enters the pore at t = 0.29 s, stays clogged at t = 1.14 s, and then leaves the pore to the cis side. While the DNA clogs the pore, it appears to be stretched out toward the cis side at t = 1.14 s. An external bias voltage of 0.3 V was applied to force the DNA to translocate towards the trans side during observation (see also Video S1 Releases of T4 DNA). (b) Probability of such releasing events after clogging in 100 and 200 nm diameter pores. (c) Life-time of clogging for released T4 DNA in 100 and 200 nm diameter pores. The bin size is 0.14 s (2 frames).

Figure 3

U turn of circular DNA at 100 nm diameter nanopore. (a) Two sequences of fluorescence images of a DNA molecule of 10 kbp and phi X174 near nanopore recorded at 14 Hz. Scale bar is 5 μm. A circular DNA molecule moves toward a nanopore within the region inside the capture radius (yellow circle) by t = 0.07 s, approaches further at t = 0.14 s, and then moves away from the pore after t = 0.21 s. An external bias voltage was 0.3 V (see also Video S3, S4 U turn of circular DNA). The images for 10 kbp and phi X174 were acquired with different CCD cameras, altering the pixel counts/scale bar of 5μm (see Materials and Methods). (b) Proportion of translocation, clog, and U turn of circular DNA, phi X174 and 10 kbp for 100 and 200 nm diameter pores. Nearly half of DNA U turns. (c) Representative trajectories of U turn DNA of 10 kbp. Dotted line shows trajectories moving away from a pore. These DNA molecules mostly turned around at the nanopore. (d) A schematic drawing of an angle of deflection at a pore. (e) Distributions of the angles of circular DNA for 100 and 200 nm diameter pores.

Figure 4

Numerical simulations of DNA motions with the presence of DNA at a nanopore with its diameter, 100 nm (a) and 200 nm (b). Upper: Zoomed images show the estimated DNA velocities (red arrows) at pore entrance. White arrows represent the velocities of bulk fluidic flow, while green arrows display the velocities by electrophoresis. Middle: Estimated velocities inside a pore. Lower: Schematics indicate the locations of a rod and/or a ring resembling to DNA inside a pore. In the left images simulating no DNA, the red arrows point down towards the trans side of expected translocation. In the other images hosting rod and/or ring, the |v_z| of the red arrows decreased not only inside the pore but also at the pore entrances as the oppositely flow velocities (white arrows) increased.