Biomedical Engineering Reference
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to MD, but might be a relevant mechanism for hairpin DNA translocation in
experiments.
Another puzzling result of NFS experiments involving hairpin DNA was the
wide range of ion current values measured. In experiments, ion currents ranging
from
2
I
0
, where
I
0
was the current in the absence of DNA, were
observed under identical bulk conditions and were attributed to the microscopic
arrangement of the DNA within the pore [
18
,
20
]. However, was the microscopic
arrangement sufficient to produce such large swings in current? Did the explanation
of such swings require interactions between multiple hairpin DNA molecules
or the existence of impurities in the system? If indeed the range of current values
could be associated with arrangement of the nanopore-DNA system, could the value
of the current at a particular time be used to predict the arrangement at that time?
First, our MD simulations demonstrated that the range of current values
observed in experiment could be explained by different arrangements of a single
hairpin DNA molecules within the nanopore. Next, these simulations also sug-
gested interpretations of the lowest and highest current values observed. For
instance, the greatest reductions of the current were observed for the loop-first
orientation (Fig.
14.5c
) when the loop occupied the constriction of the pore [
20
].
However, the largest values of current (2.17
I
0
) were seen when the DNA had passed
through the constriction and accumulated on the far side of the pore, just beyond the
constriction. It also was possible to distinguish the translocation mode by the
current values: because only one strand occupied the constriction during unzipping
(Fig.
14.5a
) rather than two during the translocation by the stretching/distortion
pathway (Fig.
14.5b
or
14.5c
), the relative ion current (
I=I
0
) was significantly
different.
While the simulations discussed above involved DNA translocation through
synthetic nanopores, MD can also assist NFS experiments employing protein
pores. Before synthetic nanopores were popular for nanopore experiments, the
protein
>
<
0.1
I
0
to
a
-hemolysin was the nanopore of choice and is still widely used. Just as
with synthetic nanopores, MD can be used to simulate
-hemolysin systems [
59
];
however, the time required for translocation can be much longer than in synthetic
nanopores. This is principally because
a
-hemolysin has a very small minimum
diameter (
a
1.4 nm [
59
]) and because the protein pore is suspended in a lipid
bilayer, which is unable to withstand transmembrane voltages
>
1.0
V
. Thus,
experiments employing
-hemolysin typically involve smaller transmembrane
voltages and consequently much lower dissociation rates than those employing
synthetic nanopores. Therefore, we cannot simulate hairpin DNA translocation by
simply applying the transmembrane voltage used in experiment, because hundreds
of microseconds might be required to observe unfolding of the hairpin DNA. Using
a larger transmembrane voltage would disrupt the bilayer and could lead to unreal-
istic conformational changes in the protein.
SMD presents a means to simulate translocation on timescales accessible to MD
[
44
,
45
]. However, one problem with conventional SMD simulations is that the
force is applied to the molecule in an unrealistic way. Usually, the center of mass of
some group of atoms is pulled at a constant velocity, resulting in unrealistic
a
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