Biomedical Engineering Reference
In-Depth Information
the molecule forward into the pore while the positively charged ion cloud
surrounding it is driven back. There are also electrostatic and nonpolar (van der
Waals) interactions with the pore walls, and finally a drag force associated with the
movement of the DNA polymer in solution.
When pore diameter is large (~10 nm),
dsDNA
may translocate through the pore
in folded configuration making single base pair analysis impracticable [
18
,
56
].
Therefore, pores with diameters comparable to a double helix (
<
3 nm) are of
special interest. The smaller the pore diameter; the larger the blockade current
and therefore the larger the signal. However, the electromechanics involved in a
translocation are very different in pores with smaller diameters (
3 nm) because of
the viscosity of water, the screening and the size of the DNA. While only a small
voltage is required to force
dsDNA
through a pore
<
3 nm in diameter, when the
pore diameter is smaller than the double helix the leading edge of the
dsDNA
penetrates into a constriction to a diameter of about ~2.5 nm and stalls there.
Applying a voltage bias above a critical threshold provides enough differential
force, exceeding that required to stretch
dsDNA
(~60 pN) [
48
,
49
], and the molecule
is pulled towards the center of and eventually through the membrane. The two
strands comprising the double helix do not pass through pores with diameters
1.6
>
2.5 nm in the same way as they do through larger pores [
57
,
58
]. The
confinement of the smaller pores causes the base-pairs to tilt. Due to the activation
energy required to begin this stretching transition, DNA will not be able to
translocate below the threshold. The threshold depends on the pH, the composition
of the strand and the methylation profile [
49
,
59
].
We have thoroughly characterized the electromechanics of
dsDNA
in a
nanopore [
48
,
49
,
57
-
59
]. As illustrated in Fig.
12.5a
,wefindthata2.0nm
pore in a nominally 10 nm thick silicon nitride membrane exhibits a threshold
U ¼
<
d
<
2.9 V for permeation of
dsDNA
, while a 2.0 nm pore in the 20 nm
membrane shows a threshold voltage
U
5.5 V because of the change in the
field profile associated with the thickness of the membrane. The estimated
differential tensile force on the leading nucleotides in the strand is
F ¼ qE >
60
pN where
E
is the electric field in the pore, which is large enough to stretch
dsDNA
. It must be that the origin of the sharp field threshold for permeation is
due to the stretching transition.
Recently, we established that the voltage threshold for permeation of
dsDNA
through a nanopore with a diameter smaller than the double helix, depends on the
methylation level and pattern too, and it can be substantially smaller than for an
unmethylated variant of the same DNA [
58
]. These observations could have a
bearing on sequencing the epigenome with a nanopore without bi-sulfite treatment
of the DNA first. We investigated the permeability of MS3 with different methyla-
tion levels and profiles through two pores with similar (~1.8 nm) diameters as show
in Fig.
12.5b, c
. Figure
12.5b
represents the results of three
qPCR
analyses - one for
unmethylated, one for hemi- and another for fully methylated DNA - showing the
number of DNA copies permeating the pore as a function of the applied potential.
The threshold voltages,
U
, for fully- and hemi-methylated MS3 are easily resolved
and fall below that observed for the unmethylated variant. For example, the
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