Stretching DNA using the electric field in a synthetic nanopore

Abstract

The mechanical properties of DNA over segments comparable to the size of a protein-binding site (3-10 nm) are examined using an electric-field-induced translocation of single molecules through a nanometer diameter pore. DNA, immersed in an electrolyte, is forced through synthetic pores ranging from 0.5 to 1.5 nm in radius in a 10 nm thick Si(3)N(4) membrane using an electric field. To account for the stretching and bending, we use molecular dynamics to simulate the translocation. We have found a threshold for translocation that depends on both the dimensions of the pore and the applied transmembrane bias. The voltage threshold coincides with the stretching transition that occurs in double-stranded DNA near 60 pN.

Figure 1

(Top) TEM images of pores with apparent radii of 0.5 ± 0.1 (a), 0.95 ± 0.1 (b), and 1.5 ± 0.1 nm (c) in a 10 nm thick Si₃N₄ membrane at 0° tilt. (Middle) Gel arrays showing five horizontal lanes with fluorescent bands that identify 58-mer ssDNA and 58bp dsDNA at the positive (+) and negative (−) electrodes separated by 0.5nm (e), 1.0nm (f), and 1.5nm (g) pores (with 100bp ladder as a reference). The applied voltage was 200 mV. (Bottom) I-V characteristics in 1M KCl solution of the same three pores. Line fits yield slopes of 0.63 ± 0.03 nS, 1.09 ± 0.03 nS, and 1.24 ± 0.03 nS for the three pores, respectively.

Figure 2

(a) 1.3 V was applied to drive dsDNA into 0.7-nm-radius (circles) and 1.0-nm-radius (squares) pores. After about 4.5 ns, the translocation of DNA halted in both. The snapshots show the conformations of DNA at the end of these simulations. The inset to (a) shows the radius of the pore cross-section near the first base inside the 0.7nm and 1.0nm pores. The translocation of dsDNA halts when the pore narrows to 1.25±0.1nm. (b) Translocations of 58bp dsDNA through 0.7nm(circle), 1.0nm(square), and 1.5nm(triangle) radius pores. Black (1.3V) and red (6.5V) symbols reflect the applied bias. The snapshot on the left illustrates dsDNA permeating the 1.0-nm radius pore at 6.5V bias. This pore was not observed to conduct dsDNA at 1.3V bias. The middle snapshot shows the final conformation of dsDNA after passing through the narrowest part of the 1.5nm radius pore at 1.3 V. The right illustrates the unzipping of the dsDNA inside the 0.7nm pore at 6.5 V. The snapshots are each of different scale. (c) dsDNA permeates a 1.0nm radius pore for voltages >3.2V. The snapshot illustrates the conformation of dsDNA at 3.2 V after 12ns.

Figure 3

(a) Gel arrays containing 3 horizontal lanes with fluorescent bands indicating 58bp dsDNA found at the negative (−) and positive (+) electrodes in a bi-cell with a 1nm radius pore (along with a 100bp ladder.) The 58bp permeates the pore only for V>2.75V. (b) Similar to (a) but instead using a 1.1nm radius pore. The 58bp permeates this pore only for V>2.25V. (c) qPCR results obtained for the same pores showing the copy number versus voltage. Upper left insets TEM micrograph taken at 0° tilt for 1nm radius (blue) and 1.1nm radius(red). 622bp dsDNA permeates the 1nm pore for V>2.5V, and the 1.1nm pore for V>2.25V. Lower right inset Expanded view of the data near threshold.

Figure 4

(a) The force versus distance from the center of the pore. Inset Expanded view of the same data near threshold. The vertical bands indicated the location of the leading DNA base pair. (b) Electrostatic potential contours inside a 1.0nm-radius pore at 2.6 V.