7 research outputs found
Efficient Production of Single-Stranded Phage DNA as Scaffolds for DNA Origami
Scaffolded DNA origami enables the
fabrication of a variety of complex nanostructures that promise utility
in diverse fields of application, ranging from biosensing over advanced
therapeutics to metamaterials. The broad applicability of DNA origami
as a material beyond the level of proof-of-concept studies critically
depends, among other factors, on the availability of large amounts
of pure single-stranded scaffold DNA. Here, we present a method for
the efficient production of M13 bacteriophage-derived genomic DNA
using high-cell-density fermentation of <i>Escherichia coli</i> in stirred-tank bioreactors. We achieve phage titers of up to 1.6
Ă 10<sup>14</sup> plaque-forming units per mL. Downstream processing
yields up to 410 mg of high-quality single-stranded DNA per one liter
reaction volume, thus upgrading DNA origami-based nanotechnology from
the milligram to the gram scale
Exploring Nucleosome Unwrapping Using DNA Origami
We
establish a DNA origami based tool for quantifying conformational
equilibria of biomolecular assemblies as a function of environmental
conditions. As first application, we employed the tool to study the
salt-induced disassembly of nucleosome core particles. To extract
binding constants and energetic penalties, we integrated nucleosomes
in the spectrometer such that unwrapping of the nucleosomal template
DNA, leading from bent to more extended states was directly coupled
to the conformation of the spectrometer. Nucleosome unwrapping was
induced by increasing the ionic strength. The corresponding shifts
in conformation equilibrium of the spectrometer were followed by direct
conformation imaging using negative staining TEM and by FRET read
out after gel electrophoretic separation of conformations. We find
nucleosome dissociation constants in the picomolar range at low ionic
strength (11 mM MgCl<sub>2</sub>), in the nanomolar range at intermediate
ionic strength (11 mM MgCl<sub>2</sub> with 0.5â1 M NaCl) and
in the micromolar range at larger ionic strength (11 mM MgCl<sub>2</sub> with â„1.5 M NaCl). Integration of up to four nucleosomes
stacked side-by-side, as it might occur within chromatin fibers, did
not appear to affect the salt-induced unwrapping of nucleosomes. Presumably,
such stacking interactions are already effectively screened at the
nucleosome unwrapping conditions. Our spectrometer provides a modular
platform with a direct read out to study conformational equilibria
for targets from small biomolecules up to large macromolecular assemblies
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
Time-Resolved Small-Angle Xâray Scattering Reveals Millisecond Transitions of a DNA Origami Switch
Self-assembled DNA
structures enable creation of specific shapes
at the nanometerâmicrometer scale with molecular resolution.
The construction of functional DNA assemblies will likely require
dynamic structures that can undergo controllable conformational changes.
DNA devices based on shape complementary stacking interactions have
been demonstrated to undergo reversible conformational changes triggered
by changes in ionic environment or temperature. An experimentally
unexplored aspect is how quickly conformational transitions of large
synthetic DNA origami structures can actually occur. Here, we use
time-resolved small-angle X-ray scattering to monitor large-scale
conformational transitions of a two-state DNA origami switch in free
solution. We show that the DNA device switches from its open to its
closed conformation upon addition of MgCl<sub>2</sub> in milliseconds,
which is close to the theoretical diffusive speed limit. In contrast,
measurements of the dimerization of DNA origami bricks reveal much
slower and concentration-dependent assembly kinetics. DNA brick dimerization
occurs on a time scale of minutes to hours suggesting that the kinetics
depend on local concentration and molecular alignment
Impact of Heterogeneity and Lattice Bond Strength on DNA Triangle Crystal Growth
One key goal of DNA nanotechnology
is the bottom-up construction
of macroscopic crystalline materials. Beyond applications in fields
such as photonics or plasmonics, DNA-based crystal matrices could
possibly facilitate the diffraction-based structural analysis of guest
molecules. Seeman and co-workers reported in 2009 the first designed
crystal matrices based on a 38 kDa DNA triangle that was composed
of seven chains. The crystal lattice was stabilized, unprecedentedly,
by WatsonâCrick base pairing. However, 3D crystallization of
larger designed DNA objects that include more chains such as DNA origami
remains an unsolved problem. Larger objects would offer more degrees
of freedom and design options with respect to tailoring lattice geometry
and for positioning other objects within a crystal lattice. The greater
rigidity of multilayer DNA origami could also positively influence
the diffractive properties of crystals composed of such particles.
Here, we rationally explore the role of heterogeneity and WatsonâCrick
interaction strengths in crystal growth using 40 variants of the original
DNA triangle as model multichain objects. Crystal growth of the triangle
was remarkably robust despite massive chemical, geometrical, and thermodynamical
sample heterogeneity that we introduced, but the crystal growth sensitively
depended on the sequences of base pairs next to the WatsonâCrick
sticky ends of the triangle. Our results point to weak lattice interactions
and high concentrations as decisive factors for achieving productive
crystallization, while sample heterogeneity and impurities played
a minor role
Characterization of Lipid-Based Hexosomes as Versatile Vaccine Carriers
Subunit vaccines
typically show insufficient immunogenicity. To
address this issue, we developed a novel self-adjuvanting particulate
carrier system based upon the lipids phytantriol (Phy) and mannide
monooleate (MaMo). Phy is a lipid known to form nonlamellar phases
in fully hydrated systems, whereas MaMo has been found to promote
immune responses in emulsion form. A bulk phase composition of Phy/MaMo
(14 wt %) showed hexagonal (HII) phase behavior over a practical temperature
range (including room and body temperature), and was therefore used
for particle development. Hexosomes stabilized with different concentrations
of either poloxamer 407, Myrj 59, or Pluronic F108 were successfully
prepared. To demonstrate the versatile nature of these systems, the
particles were further modified with either positively or negatively
charged lipids and loaded with model antigens, while maintaining the
HII structure. These hexosomes are structurally robust and amenable
to customization, rendering them suitable as antigen delivery carriers
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 currentâvoltage 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