Orientation discrimination of single-stranded DNA inside the alpha-hemolysin membrane channel - PubMed (original) (raw)

Orientation discrimination of single-stranded DNA inside the alpha-hemolysin membrane channel

Jérôme Mathé et al. Proc Natl Acad Sci U S A. 2005.

Abstract

We characterize the voltage-driven motion and the free motion of single-stranded DNA (ssDNA) molecules captured inside the approximately 1.5-nm alpha-hemolysin pore, and show that the DNA-channel interactions depend strongly on the orientation of the ssDNA molecules with respect to the pore. Remarkably, the voltage-free diffusion of the 3'-threaded DNA (in the trans to cis direction) is two times slower than the corresponding 5'-threaded DNA having the same poly(dA) sequence. Moreover, the ion currents flowing through the blocked pore with either a 3'-threaded DNA or 5' DNA differ by approximately 30%. All-atom molecular dynamics simulations of our system reveal a microscopic mechanism for the asymmetric behavior. In a confining pore, the ssDNA straightens and its bases tilt toward the 5' end, assuming an asymmetric conformation. As a result, the bases of a 5'-threaded DNA experience larger effective friction and forced reorientation that favors co-passing of ions. Our results imply that the translocation process through a narrow pore is more complicated than previously believed and involves base tilting and stretching of ssDNA molecules inside the confining pore.

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Figures

Fig. 1.

Fig. 1.

DNA hairpins can be used to facilitate translocation and diffusion measurements as a function of their orientation. (a) With ssDNA, 3′ and 5′ entries to the nanopore are mixed. (Inset) A typical translocation event measured with V = 120 mV. (b) The escape probability of DNA hairpins, with long single-stranded overhang, can be measured as a function of _t_off by using dynamic voltage control (see text). (Inset) The applied voltage and the corresponding current traces for two events. Note that at the end of the _t_off period only one of two options can exist: an empty pore or a blocked pore, either of which are readily detected by applying a small probing voltage (13).

Fig. 2.

Fig. 2.

Comparison between the distributions of the blocked ion current from ssDNA and ssDNA with terminal hairpins, threaded into the α-HL pore. (a) Mean translocation current distribution for a ssDNA [poly(dA60)]. Although the solution contains only one, monodispersed population of DNA molecules, the current distribution displays two peaks, a primary peak at 0.09 and a minor peak at 0.12. (b) Histogram of mean blockade current for DNA hairpin HP3′ (gray bars) and HP5′ (white bars). The curves are Gaussian fits yielding peak values of 0.09 ± 0.02 and 0.12 ± 0.03 for HP3′ and HP5′, respectively. Data were accumulated for the same fixed period for the two hairpins, under identical conditions. In these distributions, each molecule was counted once.

Fig. 3.

Fig. 3.

The probability that a molecule will stay in the pore, _P_stay, as a function of the waiting time, _t_off, after threading of the single-stranded overhang into the pore (_t_d = 750 μs) for HP3′ molecules (filled symbols) and HP5′ (open symbols). Solid lines represent monoexponential fits to the data at t > 100 μs. Dashed lines are fits to the data according to the model discussed in the text. Each data point in this graph is a compilation of nearly 1,500 events (over 60,000 molecules were individually analyzed for this graph).

Fig. 4.

Fig. 4.

_P_stay of each hairpin as a function of the driving time in the pore. (Left) HP3′.(Right) HP5′. Filled circles, _t_d = 750 μs; open circles, _t_d = 300 μs; squares, _t_d = 100 μs. Each data point in the graphs is a compilation of nearly 1,500 separate molecules.

Fig. 5.

Fig. 5.

Measured probability distributions of staying in the pore of the fully threaded hairpins after a negative voltage _V_p is applied, as a function of time. Results for HP3′ (filled circles) and HP5′ (open circles) are shown in the main figure for _V_p =–80 mV. (Inset) The characteristic escape velocity measured as a function of _V_p (see text). The escape velocity is higher for the HP5′ molecules as compared with the HP3′ for all values of _V_p by a factor of 1.16.

Fig. 6.

Fig. 6.

Alignment of DNA bases in confined geometry. (a) The average angle between the base and the backbone of a nucleotide in a poly(dA11) strand confined inside a cylindrical pore, from a 1.2-ns MD simulation. Snapshots (at 1.0, 1.25, and 3.0 nm, from left to right) illustrate DNA conformations during this simulation when the diameter of the pore was reduced from 3.0 to 1.0 nm. The horizontal dashed line indicates the most probable base–backbone angle in an unconstrained strand. Inside small pores (≤1.5 nm) the DNA bases are tilted toward the 5′ end of the strand. The vertical dashed line at 1.3 nm corresponds to the narrow constriction in the α-HL. The tilt of the DNA bases inside the α-HL pore depends on the global orientation of the strand: In the (cis)5′-dA58-3′(trans) strand (b) bases are tilted upward, whereas in the (cis)3′-dA58-5′(trans) strand (c) bases are tilted downward.

Fig. 7.

Fig. 7.

MD simulation of DNA electrophoresis through the transmembrane pore of α-HL. (a) Center of mass of two sequence-wise identical but global orientation-wise different poly(dA58) strands vs. time. Both strands translocate through the pore of α-HL driven by a 1.2-V bias. The poly(dA58) strand is observed to move further toward the trans side with its 3′ end forward. (b) Simulated cumulative currents of ions through the α-HL channel when it is blocked by two sequence-wise identical but global orientation-wise different poly(dA58) strands. The global orientation of a DNA strand is characterized by the location of its 3′ and 5′ ends; (cis)5′-dA58-3′(trans) data are plotted as squares, whereas (cis)3′-dA58-5′(trans) data are plotted as circles. (Inset) A 356,065-atom microscopic model of the experimental system comprising of one α-HL channel (maroon), DPPC lipid bilayer (brown), a 58-nt DNA strand (lime), water, and ions (not shown).

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