Controlled translocation of individual DNA molecules through protein nanopores with engineered molecular brakes

Abstract

Protein nanopores may provide a cheap and fast technology to sequence individual DNA molecules. However, the electrophoretic translocation of ssDNA molecules through protein nanopores has been too rapid for base identification. Here, we show that the translocation of DNA molecules through the α-hemolysin protein nanopore can be slowed controllably by introducing positive charges into the lumen of the pore by site directed mutagenesis. Although the residual ionic current during DNA translocation is insufficient for direct base identification, we propose that the engineered pores might be used to slow down DNA in hybrid systems, for example, in combination with solid-state nanopores.

Figure 1

Section through a 7R₇ nanopore. The amino acids at positions 113, 115, 117, 119, 121, 123 and 125 were replaced in the WT₇ nanopore (PDB:7AHL) by arginine by using PyMOL software (DeLano Scientific LLC, v1.0). 7R₇ is in the RL2 background, in which lysine 8 is replaced by alanine as shown here. Negatively charged residues are colored in red and positively charged residues in blue.

Figure 2

DNA translocation through 7R₇ nanopores. a) Single-channel recordings of RL2₇ (top) and 7R₇ nanopores (bottom) at +120 mV after the addition of 1.0 μM 92-mer ssDNA to the cis compartment. b) Event histogram showing the translocation time distribution at +120 mV through a 7R₇ pore upon addition of 0.7 μM ssDNA (92-mer) to the cis compartment. Events of < 10 ms are attributed to the transient gating of the 7R₇ nanopore and are ignored (Figure S1). The solid line shows a fit to the histogram of a Gaussian followed by a single exponential. The most likely translocation time (t_P) is defined by the peak of the Gaussian fit. c) Dependence on the applied voltage of the most likely translocation time per base (t_b = t_P / 92) of ssDNA (92 mer) through 7R₇ nanopores. The red line shows a single exponential fit. Experiments were performed in 1 M KCl, 25 mM Tris.HCl, containing 100 μM EDTA, at pH 8.0.

Figure 3

Residual current (I_RES) through αHL nanopores during ssDNA translocation at +120 mV. Errors are expressed as standard deviations. All nanopores, except WT₇ and NN₇, are in the RL2 background. Nanopores with additional positive charge compared to the WT₇ and RL2₇ pores are in blue. Experiments were performed in 1 M KCl, 25 mM Tris.HCl, containing 100 μM EDTA, at pH 8.0.

Figure 4

Dependence of the mean most likely translocation time per base (t_b) at +120 mV on the number of arginine residues in the barrel of RL2 pores. Homo- and hetero-heptamers are shown in open circles and blue triangles, respectively. t_b is expressed on a logarithmic scale and the data are fitted to a linear regression. Hetero-heptamers were obtained by mixing RL2- and 7R- monomers. Experiments were performed in 1 M KCl, 25 mM Tris.HCl, containing 100 μM EDTA, at pH 8.

Figure 5

Potential uses of 7R₇ nanopores. a) A protein nanopore (e.g. NN₇, green) is used to detect base-specific ionic current differences in a DNA strand, while a synthetic protein barrel (red) containing positive charges is employed to control the speed of DNA translocation. b) A protein nanopore with positive internal charge (e.g. 7R₇, red) is paired with a solid-state nanopore (gray) equipped with a tunnelling probe integrated into the device with atomic precision. The speed at which DNA is translocated through the solid-state nanopore is controlled by the applied potential and the number of positive charges in the barrel of the protein nanopore. Each base will be read by monitoring the tunnelling current, providing that the bases translocate at a constant speed and arrive with the same orientation at the tunnelling probe.