24 research outputs found
Molecular robots guided by prescriptive landscapes
Traditional robots rely for their function on computing, to store internal representations of their goals and environment and to coordinate sensing and any actuation of components required in response. Moving robotics to the single-molecule level is possible in principle, but requires facing the limited ability of individual molecules to store complex information and programs. One strategy to overcome this problem is to use systems that can obtain complex behaviour from the interaction of simple robots with their environment. A first step in this direction was the development of DNA walkers, which have developed from being non-autonomous, to being capable of directed but brief motion on one-dimensional tracks. Here we demonstrate that previously developed random walkersâso-called molecular spiders that comprise a streptavidin molecule as an inert âbodyâ and three deoxyribozymes as catalytic âlegsââshow elementary robotic behaviour when interacting with a precisely defined environment. Single-molecule microscopy observations confirm that such walkers achieve directional movement by sensing and modifying tracks of substrate molecules laid out on a two-dimensional DNA origami landscape. When using appropriately designed DNA origami, the molecular spiders autonomously carry out sequences of actions such as âstartâ, âfollowâ, âturnâ and âstopâ. We anticipate that this strategy will result in more complex robotic behaviour at the molecular level if additional control mechanisms are incorporated. One example might be interactions between multiple molecular robots leading to collective behaviour; another might be the ability to read and transform secondary cues on the DNA origami landscape as a means of implementing Turing-universal algorithmic behaviour
Reconfigurable DNA Origami to Generate Quasifractal Patterns
The specificity of WatsonâCrick base pairing,
unique mechanical
properties of DNA, and intrinsic stability of DNA double helices makes
DNA an ideal material for the construction of dynamic nanodevices.
Rationally designed strand displacement reactions can be used to produce
dynamic reconfiguration of DNA nanostructures postassembly. Here we
describe a âfoldâreleaseâfoldâ strategy
of multiple strand displacement and hybridization reactions to reconfigure
a simple DNA origami structure into a complex, quasifractal pattern,
demonstrating a complex transformation of DNA nanoarchitectures
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
Super-Resolution Fingerprinting Detects Chemical Reactions and Idiosyncrasies of Single DNA Pegboards
We employ the single-particle fluorescence nanoscopy technique points accumulation for imaging in nanoscale topography (PAINT) using site-specific DNA probes to acquire two-dimensional density maps of specific features patterned on nanoscale DNA origami pegboards. We show that PAINT has a localization accuracy of âŒ10 nm that is sufficient to reliably distinguish dense (>10[superscript 4] features ÎŒm[superscript â2]) sub-100 nm patterns of oligonucleotide features. We employ two-color PAINT to follow enzyme-catalyzed modification of features on individual origami and to show that single nanopegboards exhibit stable, spatially heterogeneous probe-binding patterns, or âfingerprints.â Finally, we present experimental and modeling evidence suggesting that these fingerprints may arise from feature spacing variations that locally modulate the probe binding kinetics. Our study highlights the power of fluorescence nanoscopy to perform quality control on individual soft nanodevices that interact with and position reagents in solution.National Science Foundation (U.S.) (Collaborative Research Award CCF-0829579)United States. Multidisciplinary University Research Initiative (W911NF-12-1-0420
DNA Origami with Double-Stranded DNA As a Unified Scaffold
Scaffolded DNA origami is a widely used technology for self-assembling precisely structured nanoscale objects that contain a large number of addressable features. Typical scaffolds are long, single strands of DNA (ssDNA) that are folded into distinct shapes through the action of many, short ssDNA staples that are complementary to several different domains of the scaffold. However, sources of long single-stranded DNA are scarce, limiting the size and complexity of structures that can be assembled. Here we demonstrated that dsDNA (double-stranded DNA) scaffolds can be directly used to fabricate integrated DNA origami structures that incorporate both of the constituent ssDNA molecules. Two basic principles were employed in the design of scaffold folding paths: folding path asymmetry and periodic convergence of the two ssDNA scaffold strands. Asymmetry in the folding path minimizes unwanted complementarity between staples, and incorporating an offset between the folding paths of each ssDNA scaffold strand reduces the number of times that complementary portions of the strands are brought into close proximity with one another, both of which decrease the likelihood of dsDNA scaffold recovery. Meanwhile, the folding paths of the two ssDNA scaffold strands were designed to periodically converge to promote the assembly of a single, unified structure rather than two individual ones. Our results reveal that this basic strategy can be used to reliably assemble integrated DNA nanostructures from dsDNA scaffolds
A Replicable Tetrahedral Nanostructure Self-Assembled from a Single DNA Strand
We report the design and construction of a nanometer-sized tetrahedron from a single strand of DNA that is 286 nucleotides long. The formation of the tetrahedron was verified by restriction enzyme digestion, Ferguson analysis, and atomic force microscopy (AFM) imaging. We further demonstrate that synthesis of the tetrahedron can be easily scaled up through in vivo replication using standard molecular cloning techniques. We found that the in vivo replication efficiency of the tetrahedron is significantly higher in comparison to in vitro replication using rolling-circle amplification (RCA). Our results suggest that it is now possible to design and replicate increasingly complex, single-stranded DNA nanostructures in vivo
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
Robust DNA-Functionalized Core/Shell Quantum Dots with Fluorescent Emission Spanning from UVâvis to Near-IR and Compatible with DNA-Directed Self-Assembly
The assembly and isolation of DNA oligonucleotide-functionalized
gold nanoparticles (AuNPs) has become a well-developed technology
that is based on the strong bonding interactions between gold and
thiolated DNA. However, achieving DNA-functionalized semiconductor
quantum dots (QDs) that are robust enough to withstand precipitation
at high temperature and ionic strength through simple attachment of
modified DNA to the QD surface remains a challenge. We report the
synthesis of stable core/shell (1â20 monolayers) QDâDNA
conjugates in which the end of the phosphorothiolated oligonucleotide
(5â10 nucleotides) is âembeddedâ within the shell
of the QD. These reliable QDâDNA conjugates exhibit excellent
chemical and photonic stability, colloidal stability over a wide pH
range (4â12) and at high salt concentrations (>100 mM Na<sup>+</sup> or Mg<sup>2+</sup>), bright fluorescence emission with quantum
yields of up to 70%, and broad spectral tunability with emission ranging
from the UV to the NIR (360â800 nm)