Nanopore Force Spectroscopy: Insights from Molecular Dynamics Simulations - Nanopores: Sensing and Fundamental Biological Interactions - page 346

studied by NFS [ 14 , 16 , 18 - 20 , 48 - 50 ]. Interest in the transition between helical
secondary structure and an unstructured coil conformation (or helix-coil transition)
is important for both biological and technological applications. For example, during
transcription of genomic DNA, RNA polymerase must unwind and dissociate the
DNA double helix so that the RNA transcript can be synthesized [ 51 ]. Also, at the
start of DNA replication, initiator proteins must dissociate the double helix at
particular stretches along the genomic DNA referred to as replication origins. To
facilitate the helix-coil transition, these replication origins often contain high
fractions of A-T basepairs, which are less strongly bound than G-C basepairs [ 51 ].
Technological interest in the helix-coil transition is motivated by the develop-
ment of advanced DNA sequencing methods. Inexpensive DNA sequencing
methods promise to have an enormous impact on both personal health care and
basic science [ 52 , 53 ], and methods based on reading the DNA sequence as it passes
through a nanopore have been actively developed [ 54 - 57 ]. The mechanics of DNA
translocation through nanopores plays an important role in so-called nanopore
sequencing methods and is a natural fit for NFS studies. Furthermore, research
has shown potential for sequencing based on NFS itself. In a particularly dramatic
demonstration of the power of NFS, Nakane et al. [ 48 ], collected hundreds of
individual DNA duplex dissociation events, allowing the distribution of dissocia-
tion times under different applied forces to be determined and permitting statistical
discrimination of sequences differing by single nucleotides. A number of other
experimental protocols have shown the ability of NFS to discriminate DNA
sequences by statistical analysis of the dissociation time of duplex DNA under
force [ 17 , 19 ].
As with enzyme-DNA complexes, experiments have shown a sharp dependence
of the translocation probability of hairpin DNA on the transmembrane voltage
[ 18 , 20 ], which manifests itself as a relatively well defined threshold voltage for
translocation of hairpin DNA through synthetic nanopores having diameters 1.0 nm
<d< 2.5 nm. However, these experiments arrived at a seemingly counter-intuitive

result: pores having minimum diameters 1.2 nm showed much lower threshold

voltages than pores having minimum diameters 1.4 nm [ 18 , 20 ], i.e. more force

was apparently required to drive the helix through larger pores than through smaller
pores. This led to the suggestion that there was some fundamental change in the way
by which translocation occurred between the larger and smaller pores. Here was a
problem for which MD could give insight available by no other method: it was
possible to visualize the translocation process for different pore sizes.
As shown in Fig. 14.4 , a clear difference was observed in the mode of transloca-
tion between small pores and large pores in SMD simulations in which the coil of
the hairpin DNA was displaced at a constant rate through the pore [ 18 ]. The largest
constriction was 1.6 nm; thus, in all cases the double helix of the hairpin DNA
(having a diameter of

2.5 nm) had to be distorted to allow passage. For the pores
having minimum diameters of 1.0 and 1.3 nm (Fig. 14.4a, b ), the basepairs of the
DNA were unzipped one by one and the DNA passed through the constriction as a
single strand. However, for the pore having a minimum diameter of 1.6 nm, both
strands passed through the pore simultaneously and some of the basepairs were