Molecular Detection and Force Spectroscopy in Solid-State Nanopores with Integrated Optical Tweezers - Nanopores: Sensing and Fundamental Biological Interactions - page 45

2.6 Measurements on Protein-Coated DNA Molecules

Experiments are also performed by forming a second type of bead-conjugated
target molecules: RecA-coated dsDNA [ 13 ]. RecA is a protein with an important
role in homologous recombination and dsDNA break repair [ 20 ]. For the present
experiments it was chosen for two main reasons. First, it is able to bind coopera-
tively along an entire molecule of dsDNA [ 21 ], resulting in an uninterrupted
coating of protein that can be formed under non-dissociable conditions (upon
using ATP-gS). Second, the attachment of this protein coating changes the physical
properties of the molecule, the most relevant changes being an increase in diameter
(from 2.2 nm for bare dsDNA to ~7 nm for RecA coated dsDNA [ 22 ]) and the
overall charge (RecA monomers carry a net negative charge).
When nanopore capture experiments are performed on this nucleoprotein
filament, the measured change in conductance upon insertion of a molecule is
found to differ significantly from that of bare dsDNA under the same experimental
conditions. Examples of typical conductance blockades for both kinds of molecule
taken in 1 M KCl are shown in Fig. 2.9a . At 1 M KCl, the RecA-dsDNA is found
to cause a much larger conductance blockade of about 7 nS, similar to transloca-
tion measurements on the same type of molecule [ 23 ]. This also agrees qualita-
tively with the description of how the presence of a molecule affects the measured
nanopore conductance [ 14 ], wherein the conductance blockade DG can be
expressed as

1
L pore pa 2

:

ðm cat þ m an Þn tot e þ m counter q l

D G ¼ (2.2)

Here, L pore represents the length of the pore, m cat and m an are the electrophoretic

mobilities of the cation and anion, respectively, n tot is the total number density of

ions in solution, e is the elementary charge, m counter is the mobility of counterions

near the surface of the molecule (which we adopt to equal the bulk mobility), and q l
is the net charge per unit length of carriers surrounding the molecule, which to first
order equals the effective line charge density Q eff of the molecule [ 13 ]. This
expression shows that when the ionic concentration is high ( n tot is large), the first
term, which scales with the square of the diameter a of the molecule, is dominant,
and thus the conductance change is essentially a measure of molecular size. Since
RecA-coated dsDNA has a larger diameter than bare dsDNA, it makes sense that
the conductance change for the former is greater than the latter.
Amore complete comparison can be made by examining the conductance change
for each molecule under various salt concentrations (Fig. 2.9 ). In both cases,
measurements indicate a transition from conductance blockades at high ionic
strength to conductance enhancement at low ionic strength. A fit to the data [ 13 ]
for bare dsDNA yields an apparent charge of 0.22 nC/m which compares reason-

ably with translocation measurements [ 14 ] on the same molecule which yielded