4 research outputs found
Detection of Individual Proteins Bound along DNA Using Solid-State Nanopores
DNA
in cells is heavily covered with all types of proteins that regulate
its genetic activity. Detection of DNA-bound proteins is a challenge
that is well suited to solid-state nanopores as they provide a linear
readout of the DNA and DNA–protein volume in the pore constriction
along the entire length of a molecule. Here, we demonstrate that we
can realize the detection of even individual DNA-bound proteins at
the single-DNA-molecule level using solid-state nanopores. We introduce
and use a new model system of anti-DNA antibodies bound to lambda
phage DNA. This system provides several advantages since the antibodies
bind individually, tolerate high salt concentrations, and will, because
of their positive charge, not translocate through the pore unless
bound to the DNA. Translocation of DNA–antibody samples reveals
the presence of short 12 μs current spikes within the DNA traces,
with amplitudes that are about 4.5 times larger than that of dsDNA,
which are associated with individual antibodies. We conclude that
transient interactions between the pore and the antibodies are the
primary mechanism by which bound antibodies are observed. This work
provides a proof-of-concept for how nanopores could be used for future
sensing applications
Velocity of DNA during Translocation through a Solid-State Nanopore
While understanding translocation
of DNA through a solid-state nanopore is vital for exploiting its
potential for sensing and sequencing at the single-molecule level,
surprisingly little is known about the dynamics of the propagation
of DNA through the nanopore. Here we use linear double-stranded DNA
molecules, assembled by the DNA origami technique, with markers at
known positions in order to determine for the first time the local
velocity of different segments along the length of the molecule. We
observe large intramolecular velocity fluctuations, likely related
to changes in the drag force as the DNA blob unfolds. Furthermore,
we observe an increase in the local translocation velocity toward
the end of the translocation process, consistent with a speeding up
due to unfolding of the last part of the DNA blob. We use the velocity
profile to estimate the uncertainty in determining the position of
a feature along the DNA given its temporal location and demonstrate
the error introduced by assuming a constant translocation velocity
Fast Translocation of Proteins through Solid State Nanopores
Measurements on protein translocation through solid-state
nanopores
reveal anomalous (non-Smoluchowski) transport behavior, as evidenced
by extremely low detected event rates; that is, the capture rates
are orders of magnitude smaller than what is theoretically expected.
Systematic experimental measurements of the event rate dependence
on the diffusion constant are performed by translocating proteins
ranging in size from 6 to 660 kDa. The discrepancy is observed to
be significantly larger for smaller proteins, which move faster and
have a lower signal-to-noise ratio. This is further confirmed by measuring
the event rate dependence on the pore size and concentration for a
large 540 kDa protein and a small 37 kDa protein, where only the large
protein follows the expected behavior. We dismiss various possible
causes for this phenomenon and conclude that it is due to a combination
of the limited temporal resolution and low signal-to-noise ratio.
A one-dimensional first-passage time-distribution model supports this
and suggests that the bulk of the proteins translocate on time scales
faster than can be detected. We discuss the implications for protein
characterization using solid-state nanopores and highlight several
possible routes to address this problem
Ionic Permeability and Mechanical Properties of DNA Origami Nanoplates on Solid-State Nanopores
While DNA origami is a popular and versatile platform, its structural properties are still poorly understood. In this study we use solid-state nanopores to investigate the ionic permeability and mechanical properties of DNA origami nanoplates. DNA origami nanoplates of various designs are docked onto solid-state nanopores where we subsequently measure their ionic conductance. The ionic permeability is found to be high for all origami nanoplates. We observe the conductance of docked nanoplates, relative to the bare nanopore conductance, to increase as a function of pore diameter, as well as to increase upon lowering the ionic strength. The honeycomb lattice nanoplate is found to have slightly better overall performance over other plate designs. After docking, we often observe spontaneous discrete jumps in the current, a process which can be attributed to mechanical buckling. All nanoplates show a nonlinear current–voltage dependence with a lower conductance at higher applied voltages, which we attribute to a physical bending deformation of the nanoplates under the applied force. At sufficiently high voltage (force), the nanoplates are strongly deformed and can be pulled through the nanopore. These data show that DNA origami nanoplates are typically very permeable to ions and exhibit a number of unexpected mechanical properties, which are interesting in their own right, but also need to be considered in the future design of DNA origami nanostructures