Nanopore Force Spectroscopy: Insights from Molecular Dynamics Simulations - Nanopores: Sensing and Fundamental Biological Interactions - page 337

are filled with an electrolyte solution. A pore having a diameter from one to tens of
nanometers has been drilled in the membrane. Solutes injected on one side of the
chamber can only reach the other side by passing through the nanopore. In
Fig. 14.1a , force is applied to a molecule of double-stranded DNA (dsDNA),
which threads through the pore. A relatively bulky enzyme is bound to the DNA
which has too large a cross-section to pass through the pore. The walls of the pore
apply a force to the enzyme opposite to that applied to DNA, stressing the
intermolecular bond between the two. Thus, in NFS we can determine the behavior
of molecular assemblies under force, revealing the thermodynamic and kinetic
properties of the intermolecular bond [ 10 - 12 ] as well as structural changes neces-
sary for biological functionality and technological applications.

14.1.1 Nanopore Force Spectroscopy Experiments

In NFS experiments, the size of the nanopore can be tailored to the problem of
interest. To probe enzyme-DNA interactions, such as those binding the complex
shown in Fig. 14.1a , a pore having a diameter of 3 nm is sufficiently large to permit
unfettered passage of dsDNA (which has a diameter of 2.5 nm), but too small to

accommodate the enzyme. Similarly, to probe interactions between DNA strands, a
smaller pore (

2.0 nm) can be used that permits passage of unstructured DNA but
excludes the passage of portions of the molecule having bulky secondary structure,
such as double helices. Using focused beams of charged particles, nanopores having
diameters as large as 20 nm and as small as 1.0 nm are routinely fabricated [ 13 ],
permitting NFS to be applied to a broad range of molecular assemblies.
A majority of NFS experiments have involved DNA in some way, due to the
importance of the molecule in biology and biotechnology. In addition to the
extensive use of NFS for probing the forces involved in unfolding of single DNA
molecules [ 14 - 20 ], protein-DNA interactions have also been explored [ 21 - 23 ].
DNA is an especially good subject for NFS because it is highly negatively charged
( e per nucleotide at physiological pH) and, therefore, can easily be captured by
the electric field of the nanopore. Despite this bias toward DNA, Goodrich et al. [ 24 ]
used NFS to study unfolding of peptide molecules, while experiments by Siwy and
colleagues [ 25 ], in which chemically modified nanopores were used to detect
specific proteins, suggest the prospect of studying protein-protein interactions
with NFS. Indeed, NFS is an extremely general method that can be used to probe
many types of interactions at the single-molecule level when at least one of the
analytes carries a nonzero charge.
A reader unfamiliar with NFS might be left with several questions: (i) How does
one load an analyte into the nanopore? (ii) How does one apply force to the analyte
once it gets there? (iii) How does one observe the effect of the force on the analyte?
Questions (i) and (ii) have the same answer: the nanopore geometry focuses an
applied electric field, capturing charged analytes and allowing sustained forces to
be applied. Electrodes are placed on both sides of the pore, which allow an