Third Generation DNA Sequencing with a Nanopore - Nanopores: Sensing and Fundamental Biological Interactions - page 300

the DNA in the pore shown in the insets to Fig. 12.5b reveal the molecular structure
with atomic detail, indicating that the methylated DNA is more ordered and stiffer
[ 61 ]. This is also evident in the root-mean-square (rms) deviation in the helix
diameter. At 4 V, the interior segments of methylated and unmethylated DNA
(shown in yellow in the insets to Fig. 12.5b ) have an rms-deviation 0.29 and
0.49 nm, respectively.
When

l

-DNA is injected into the electrolyte at the negative (cis) electrode and a
voltage is applied across the membrane 15.0 2.2 nm thick membrane with a pore

2.5 0.2 nm cross-section - smaller than the DNA double helix - in it like

that shown in Fig. 12.6a , current transients such as those shown in Fig. 12.6b are
observed [ 21 ]. The current transients occur randomly as a function of time as
illustrated in the figure, but the inter-arrival time decreases with increasing concen-
tration of DNA. Such blockades ostensibly represent the reduction of the electro-
lytic pore current due to the translocation of DNA.
Figure 12.6b illustrates threshold behavior presumably due to stretching as
alluded to above, showing a dearth of transients found in a current trace measured
at 200 mV compared to an 800 mV trace. Figure 12.6c summarizes the voltage
dependence of the frequency of blockade events over the range from 100 mV to
1 V. Generally, we observe an abrupt rise in the number of blockades over a range
of ~200 mV near the critical threshold. If we assume each blockade corresponds to
dsDNA permeating the pore, then the permeation rate can be described by the
transition-state relation of the Kramers type: R ¼ R 0 V/ ( 1 + exp [ q* ( U-V ) /kT ],
where R 0 is a frequency factor, q*U is the effective barrier height, q*V is the
reduction in the energy barrier due to the applied potential, and kT is the thermal
energy. Using this relation, the data was fit and the results were overlaid on the
scatter plot in Fig. 12.6c . We deduce a threshold of U ¼

2.0

0.46 0.02 V with

q * 0.2e, which presumably corresponds to the force required to stretch

the leading nucleotides in the pore.

¼ 0.8

Fig. 12.6 (a) A TEM image of a 2.5 2.0 nm cross-section pore in a silicon nitride membrane
15 nm thick. (b) Electrolytic current measured in 100 mM KCl at 800 mV ( top ) and 200 mV
( bottom ) through the pore shown in (a) as a function of time. The frequency of blockades decreases
dramatically with voltage; at 0.2 V practically no transients are observed. (c) The frequency of
blockades observed with the 2.5 nm pore as a function of membrane voltage, illustrating
the frequency drop as voltage decreases below 0.5 V. The dotted line represents a fit to the data.
(d) Distributions illustrating the frequency as a function of the duration of a current blockade, t D ,
above threshold at 1.0 V ( left ), 800 mV ( center ) and 700 mV ( right ). The distribution depends
sensitively on the voltage. Inset : The reciprocal of the duration, t D 1 , as a function of the applied
voltage. Adapted from reference [ 21 ]