Nanopore Force Spectroscopy: Insights from Molecular Dynamics Simulations - Nanopores: Sensing and Fundamental Biological Interactions - page 341

which are proteins that bind to double-stranded DNA at specific sequences and
cleave the molecule in two, are indispensable tools in recombinant DNA technol-
ogy [ 38 ] as well as many DNA sequencing and genotyping methods [ 39 - 41 ].
The type II restriction enzyme Eco RI binds to dsDNA and is thought to diffuse
linearly, pausing near sequences resembling the cognate sequence GAATTC [ 42 ].
In the presence of Mg 2+ , the Eco RI cleaves both DNA strands at particular
points along the cognate sequence with high specificity. In the absence of Mg 2+ ,
Eco RI does not cleave the DNA, but still binds strongly to sites having the cognate
sequence.
The extraordinary specificity of type II restriction enzymes for their respective
cognate sequences combined with the high throughput potential of NFS has been
proposed as the basis of a method of genotyping genomic DNA [ 22 ]. In short, a
solution of DNA molecules decorated with bound enzymes ( Eco RI or similar
enzymes such as Bam HI) can be introduced into the chamber on one side of the
membrane. The DNA is captured by the pore and threads through it until the enzyme
reaches the pore opening as illustrated in Fig. 14.2a . If the pore is sufficiently small
(having a diameter

5 nm) and the transmembrane voltage is sufficiently low,
the enzyme will block the pore and halt translocation of the DNA. However, for
larger transmembrane voltages, the force on the enzyme-DNA bond dramatically
increases the probability for the rupture of the complex. Rupture of the complex
permits the DNA to continue threading through the pore until the next enzyme is
encountered. The value of the transmembrane voltage at which rupture occurs shows
a strong dependence on the sequence to which the enzyme is bound, being nearly zero
for nonspecific binding and a few volts for the cognate sequence. Therefore, by
measuring the transmembrane voltage required to dissociate an enzyme from DNA,
one can identify particular subsequences within a given DNA molecule.
Quantitative polymerase chain reaction has been used to count the number of
DNA molecules passing through the pore at various transmembrane voltages [ 22 ].
When enzymes are specifically bound to the DNA molecules, this number increases
sharply over a small range of transmembrane voltage. For example, in a nanopore
having a minimum elliptical cross section of 3.4 nm

<

4.5 nm, the molecule count
increases from near zero to 10 8 over a range between 1.8 and 2.0 V [ 22 ]. One can
therefore define a threshold value of the transmembrane voltage above which nearly
all enzymes reaching the pore dissociate, and below which the mean time until
rupture is much greater than the timescale of the experiment. When Eco RI was
bound to its cognate sequence GAATTC, the complex was shown to have a much
higher threshold voltage than when it was bound to a sequence that differed by one
basepair [ 22 , 23 ]. The sequence GGATCC, which differs from the cognate sequence
by two basepairs, showed no threshold at all. Even the basepairs just next to the
cognate sequence were shown to measurably affect the threshold voltage [ 23 ].
Furthermore, it was shown that another restriction enzyme ( Bam HI) bound to its
cognate sequence (GGATCC) could be distinguished from Eco RI bound to its cognate
sequence due to the difference in the threshold voltage [ 22 ].
Despite the many insights yielded by the experiments, there were a number of
questions about the behavior of the complex in the pore. How deeply did the enzyme