8 research outputs found
DNAâCholesterol Barges as Programmable Membrane-Exploring Agents
DNA nanotechnology enables the precise construction of nanoscale devices that mimic aspects of natural biomolecular systems yet exhibit robustly programmable behavior. While many important biological processes involve dynamic interactions between components associated with phospholipid membranes, little progress has been made toward creating synthetic mimics of such interfacial systems. We report the assembly and characterization of cholesterol-labeled DNA origami âbargesâ capable of reversible association with and lateral diffusion on supported lipid bilayers. Using single-particle fluorescence microscopy, we show that these DNA barges rapidly and stably embed in lipid bilayers and exhibit Brownian diffusion in a manner dependent on both cholesterol labeling and bilayer composition. Tracking of individual barges rapidly generates super-resolution maps of the contiguous regions of a membrane. Addition of appropriate command oligonucleotides enables membrane-associated barges to reversibly exchange fluorescent cargo with bulk solution, dissociate from the membrane, or form oligomers within the membrane, opening up new possibilities for programmable membrane-bound molecular devices
Mapping the Thermal Behavior of DNA Origami Nanostructures
Understanding
the thermodynamic properties of complex DNA nanostructures,
including rationally designed two- and three-dimensional (2D and 3D,
respectively) DNA origami, facilitates more accurate spatiotemporal
control and effective functionalization of the structures by other
elements. In this work fluorescein and tetramethylrhodamine (TAMRA),
a FoÌrster resonance energy transfer (FRET) dye pair, were incorporated
into selected staples within various 2D and 3D DNA origami structures.
We monitored the temperature-dependent changes in FRET efficiency
that occurred as the dye-labeled structures were annealed and melted
and subsequently extracted information about the associative and dissociative
behavior of the origami. In particular, we examined the effects of
local and long-range structural defects (omitted staple strands) on
the thermal stability of common DNA origami structures. The results
revealed a significant decrease in thermal stability of the structures
in the vicinity of the defects, in contrast to the negligible long-range
effects that were observed. Furthermore, we probed the global assembly
and disassembly processes by comparing the thermal behavior of the
FRET pair at several different positions. We demonstrated that the
staple strands located in different areas of the structure all exhibit
highly cooperative hybridization but have distinguishable melting
temperatures depending on their positions. This work underscores the
importance of understanding fundamental aspects of the self-assembly
of DNA nanostructures and can be used to guide the design of more
complicated DNA nanostructures, to optimize annealing protocol and
manipulate functionalized DNA nanostructures
Steric Crowding and the Kinetics of DNA Hybridization within a DNA Nanostructure System
The ability to generate precisely designed molecular networks and modulate the surrounding environment is vital for fundamental studies of chemical reactions. DNA nanotechnology simultaneously affords versatility and modularity for the construction of tailored molecular environments. We systematically studied the effects of steric crowding on the hybridization of a 20 nucleotide (nt) single-stranded DNA (ssDNA) target to a complementary probe strand extended from a rectangular six-helix tile, where the number and character of the surrounding strands influence the molecular environment of the hybridization site. The hybridization events were monitored through an increase in the quantum yield of a single reporter fluorophore (5-carboxyfluorescein) upon hybridization of the 20-nt ssDNA, an effect previously undocumented in similar systems. We observed that as the hybridization site moved from outer to inner positions along the DNA tile, the hybridization rate constant decreased. A similar rate decrease was observed when noncomplementary single- and double-stranded DNA flanked the hybridization site. However, base-pairing interactions between the hybridization site of the probe and the surrounding DNA resulted in a reduction in the reaction kinetics. The decreases in the hybridization rate constants can be explained by the reduced probability of successful nucleation of the invading ssDNA target to the complementary probe
DNAzyme-Based Logic Gate-Mediated DNA Self-Assembly
Controlling DNA self-assembly processes using rationally
designed logic gates is a major goal of DNA-based nanotechnology and
programming. Such controls could facilitate the hierarchical engineering
of complex nanopatterns responding to various molecular triggers or
inputs. Here, we demonstrate the use of a series of DNAzyme-based
logic gates to control DNA tile self-assembly onto a prescribed DNA
origami frame. Logic systems such as âYES,â âOR,â
âAND,â and âlogic switchâ are implemented
based on DNAzyme-mediated tile recognition with the DNA origami frame.
DNAzyme is designed to play two roles: (1) as an intermediate messenger
to motivate downstream reactions and (2) as a final trigger to report
fluorescent signals, enabling information relay between the DNA origami-framed
tile assembly and fluorescent signaling. The results of this study
demonstrate the plausibility of DNAzyme-mediated hierarchical self-assembly
and provide new tools for generating dynamic and responsive self-assembly
systems
Multifactorial Modulation of Binding and Dissociation Kinetics on Two-Dimensional DNA Nanostructures
We use single-particle fluorescence
resonance energy transfer (FRET)
to show that organizing oligonucleotide probes into patterned two-dimensional
arrays on DNA origami nanopegboards significantly alters the kinetics
and thermodynamics of their hybridization with complementary targets
in solution. By systematically varying the spacing of probes, we demonstrate
that the rate of dissociation of a target is reduced by an order of
magnitude in the densest probe arrays. The rate of target binding
is reduced less dramatically, but to a greater extent than reported
previously for one-dimensional probe arrays. By additionally varying
target sequence and buffer composition, we provide evidence for two
distinct mechanisms for the markedly slowed dissociation: direct hopping
of targets between adjacent sequence-matched probes and nonsequence-specific,
salt-bridged, and thus attractive electrostatic interactions with
the DNA origami pegboard. This kinetic behavior varies little between
individual copies of a given array design and will have significant
impact on hybridization measurements and overall performance of DNA
nanodevices as well as microarrays
Understanding the Elementary Steps in DNA Tile-Based Self-Assembly
Although
many models have been developed to guide the design and
implementation of DNA tile-based self-assembly systems with increasing
complexity, the fundamental assumptions of the models have not been
thoroughly tested. To expand the quantitative understanding of DNA
tile-based self-assembly and to test the fundamental assumptions of
self-assembly models, we investigated DNA tile attachment to preformed
âmulti-tileâ arrays in real time and obtained the thermodynamic
and kinetic parameters of single tile attachment in various sticky
end association scenarios. With more sticky ends, tile attachment
becomes more thermostable with an approximately linear decrease in
the free energy change (more negative). The total binding free energy
of sticky ends is partially compromised by a sequence-independent
energy penalty when tile attachment forms a constrained configuration:
âloopâ. The minimal loop is a 2 Ă 2 tetramer (Loop4).
The energy penalty of loops of 4, 6, and 8 tiles was analyzed with
the independent loop model assuming no interloop tension, which is
generalizable to arbitrary tile configurations. More sticky ends also
contribute to a faster on-rate under isothermal conditions when nucleation
is the rate-limiting step. Incorrect sticky end contributes to neither
the thermostability nor the kinetics. The thermodynamic and kinetic
parameters of DNA tile attachment elucidated here will contribute
to the future improvement and optimization of tile assembly modeling,
precise control of experimental conditions, and structural design
for error-free self-assembly
Redox Engineering of Cytochrome c using DNA Nanostructure-Based Charged Encapsulation and Spatial Control
Three-dimensional
(3D) DNA nanostructures facilitate the directed self-assembly of various
objects with designed patterns with nanometer scale addressability.
Here, we report the enhancement of cytochrome c (cyt c) redox activity
by using a designed 3D DNA nanostructure attached to a gold electrode
to spatially control the position of cyt c within the tetrahedral
framework. Charged encapsulation and spatial control result in the
significantly increased redox potential and enhanced electron transfer
of this redox protein when compared to cyt c directly adsorbed on
the gold surface. Two different protein attachment sites on one double
stranded edge of a DNA tetrahedron were used to position cyt c inside
and outside of the cage. Cyt c at both binding sites show similar
redox potential shift and only slight difference in the electron transfer
rate, both orders of magnitude faster than the cases when the protein
was directly deposited on the gold electrode, likely due to an effective
electron transfer pathway provided by the stabilization effect of
the protein created by the DNA framework. This study shows great potential
of using structural DNA nanotechnology for spatial control of protein
positioning on electrode, which opens new routes to engineer redox
proteins and interface microelectronic devices with biological function
Electron Microscopic Visualization of Protein Assemblies on Flattened DNA Origami
DNA provides an ideal substrate for the engineering of versatile nanostructures due to its reliable WatsonâCrick base pairing and well-characterized conformation. One of the most promising applications of DNA nanostructures arises from the site-directed spatial arrangement with nanometer precision of guest components such as proteins, metal nanoparticles, and small molecules. Two-dimensional DNA origami architectures, in particular, offer a simple design, high yield of assembly, and large surface area for use as a nanoplatform. However, such single-layer DNA origami were recently found to be structurally polymorphous due to their high flexibility, leading to the development of conformationally restrained multilayered origami that lack some of the advantages of the single-layer designs. Here we monitored single-layer DNA origami by transmission electron microscopy (EM) and discovered that their conformational heterogeneity is dramatically reduced in the presence of a low concentration of dimethyl sulfoxide, allowing for an efficient flattening onto the carbon support of an EM grid. We further demonstrated that streptavidin and a biotinylated target protein (cocaine esterase, CocE) can be captured at predesignated sites on these flattened origami while maintaining their functional integrity. Our demonstration that protein assemblies can be constructed with high spatial precision (within âŒ2 nm of their predicted position on the platforms) by using strategically flattened single-layer origami paves the way for exploiting well-defined guest molecule assemblies for biochemistry and nanotechnology applications