The Rule of Thirds: Controlling Junction Chirality and Polarity in 3D DNA Tiles

The successful self-assembly of tensegrity triangle DNA crystals heralded the ability to programmably construct macroscopic crystalline nanomaterials from rationally-designed, nanoscale components. This 3D DNA tile owes its “tensegrity” nature to its three rotationally stacked double helices locked together by the tensile winding of a center strand segmented into 7 base pair (bp) inter-junction regions, corresponding to two-thirds of a helical turn of DNA. All reported tensegrity triangles to date have employed $\left(Z+2/3\right)$ turn inter-junction segments, yielding right-handed, antiparallel, “J1” junctions. Here a minimal DNA triangle motif consisting of 3-bp inter-junction segments, or one-third of a helical turn is reported. It is found that the minimal motif exhibits a reversed morphology with a left-handed tertiary structure mediated by a locally-parallel Holliday junction—the “L1” junction. This parallel junction yields a predicted helical groove matching pattern that breaks the pseudosymmetry between tile faces, and the junction morphology further suggests a folding mechanism. A Rule of Thirds by which supramolecular chirality can be programmed through inter-junction DNA segment length is identified. These results underscore the role that global topological forces play in determining local DNA architecture and ultimately point to an under-explored class of self-assembling, chiral nanomaterials for topological processes in biological systems.     

A Bipedal DNA Motor that Travels Back and Forth between Two DNA Origami Tiles

In this work, the successful operation of a dynamic DNA device constructed from two DNA origami building blocks is reported. The device includes a bipedal walker that strides back and forth between the two origami tiles. Two different DNA origami tiles are first prepared separately; they are then joined together in a controlled manner by a set of DNA strands to form a stable track in high yield as confirmed by single‐molecule fluorescence (SMF). Second, a bipedal DNA motor, initially attached to one of the two origami units and operated by sequential interaction with “fuel” and “antifuel” DNA strands, moves from one origami tile to another and then back again. The operational yield, measured by SMF, was similar to that of a motor operating on a similar track embedded in a single origami tile, confirming that the transfer across the junction from one tile to the other does not result in dissociation that is any more than that of steps on a single tile. These results demonstrate that moving parts can reliably travel from one origami unit to another, and it demonstrates the feasibility of dynamic DNA molecular machines that are made of more than a single origami building block. This study is a step toward the development of motors that can stride over micrometer distances.     

DNA nanodevices

The molecular recognition properties of DNA molecules combined with the distinct mechanical properties of single and double strands of DNA can be utilized for the construction of nanodevices, which can perform ever more tasks with possible applications ranging from nanoconstruction to intelligent drug delivery. With the help of DNA it is possible to construct machinelike devices that are capable of rotational motion, pulling and stretching, or even unidirectional motion. It is possible to devise autonomous nanodevices, to grab and release molecules, and also to perform simple information-processing tasks.     

Controlling the formation of DNA origami structures with external signals

Degradable Newkome-type and polylysine dendrons functionalized with spermine surface units are used to control the formation of DNA origami structures. The intact dendrons form polyelectrolyte complexes with the scaffold strands, therefore blocking the origami formation. Degradation of the dendron with an optical trigger or chemical reduction leads to the release of the DNA scaffold and efficient formation of the desired origami structure. These results provide new insights towards realizing responsive materials with DNA origami.     

Solution‐Controlled Conformational Switching of an Anchored Wireframe DNA Nanostructure

Self‐assembled DNA origami nanostructures have a high degree of programmable spatial control that enables nanoscale molecular manipulations. A surface‐tethered, flexible DNA nanomesh is reported herein which spontaneously undergoes sharp, dynamic conformational transitions under physiological conditions. The transitions occur between two major macrostates: a spread state dominated by the interaction between the DNA nanomesh and the BSA/streptavidin surface and a surface‐avoiding contracted state. Due to a slow rate of stochastic transition events on the order of tens of minutes, the dynamic conformations of individual structures can be detected in situ with DNA PAINT microscopy. Time series localization data with automated imaging processing to track the dynamically changing radial distribution of structural markers are combined. Conformational distributions of tethered structures in buffers with elevated pH exhibit a calcium‐dependent domination of the spread state. This is likely due to electrostatic interactions between the structures and immobilized surface proteins (BSA and streptavidin). An interaction is observed in solution under similar buffer conditions with dynamic light scattering. Exchanging between solutions that promote one or the other state leads to in situ sample‐wide transitions between the states. The technique herein can be a useful tool for dynamic control and observation of nanoscale interactions and spatial relationships.     

Self‐Assembly of DNA Origami Heterodimers in High Yields and Analysis of the Involved Mechanisms

Efficient fabrication of structurally and functionally diverse nanomolecular devices and machines by organizing separately prepared DNA origami building blocks into a larger structure is limited by origami attachment yields. A general method that enables attachment of origami building blocks using ‘sticky ends' at very high yields is demonstrated. Two different rectangular origami monomers are purified using agarose gel electrophoresis conducted in solute containing 100 × 10^?3 m NaCl, a treatment that facilitates the dissociation of most of the incorrectly hybridized origami structures that form through blunt‐end interactions during the thermal annealing process and removes these structures as well as excess strands that otherwise interfere with the desired heterodimerization reaction. Heterodimerization yields of gel‐purified monomers are between 98.6% and 99.6%, considerably higher than that of monomers purified using the polyethylene glycol (PEG) method (88.7–96.7%). Depending on the number of PEG purification rounds, these results correspond to about 4‐ to 25‐fold reduction in the number of incorrect structures observed by atomic force microscopy. Furthermore, the analyses of the incorrect structures observed before and after the heterodimerization reactions and comparison of the purification methods provide valuable information on the reaction mechanisms that interfere with heterodimerization.     

DNA Nanotechnology as an Emerging Tool to Study Mechanotransduction in Living Systems

The ease of tailoring DNA nanostructures with sub‐nanometer precision has enabled new and exciting in vivo applications in the areas of chemical sensing, imaging, and gene regulation. A new emerging paradigm in the field is that DNA nanostructures can be engineered to study molecular mechanics. This new development has transformed the repertoire of capabilities enabled by DNA to include detection of molecular forces in living cells and elucidating the fundamental mechanisms of mechanotransduction. This Review first describes fundamental aspects of force‐induced melting of DNA hairpins and duplexes. This is then followed by a survey of the currently available force sensing DNA probes and different fluorescence‐based force readout modes. Throughout the Review, applications of these probes in studying immune receptor signaling, including the T cell receptor and B cell receptor, as well as Notch and integrin signaling, are discussed.     

The Brownian and Flow‐Driven Rotational Dynamics of a Multicomponent DNA Origami‐Based Rotor

Nanomechanical devices are becoming increasingly popular due to the very diverse field of potential applications, including nanocomputing, robotics, and drug delivery. DNA is one of the most promising building materials to realize complex 3D structures at the nanoscale level. Several mechanical DNA origami structures have already been designed capable of simple operations such as a DNA box with a controllable lid, bipedal walkers, and cargo sorting robots. However, the nanomechanical properties of mechanically interlinked DNA nanostructures that are in general highly deformable have yet to be extensively experimentally evaluated. In this work, a multicomponent DNA origami‐based rotor is created and fully characterized by electron microscopy under negative stain and cryo preparations. The nanodevice is further immobilized on a microfluidic chamber and its Brownian and flow‐driven rotational behaviors are analyzed in real time by single‐molecule fluorescence microscopy. The rotation in previous DNA rotors based either on strand displacement, electric field or Brownian motion. This study is the first to attempt to manipulate the dynamics of an artificial nanodevice with fluidic flow as a natural force.     

DyNAMiC Workbench: an integrated development environment for dynamic DNA nanotechnology

Dynamic DNA nanotechnology provides a promising avenue for implementing sophisticated assembly processes, mechanical behaviours, sensing and computation at the nanoscale. However, design of these systems is complex and error-prone, because the need to control the kinetic pathway of a system greatly increases the number of design constraints and possible failure modes for the system. Previous tools have automated some parts of the design workflow, but an integrated solution is lacking. Here, we present software implementing a three ‘tier’ design process: a high-level visual programming language is used to describe systems, a molecular compiler builds a DNA implementation and nucleotide sequences are generated and optimized. Additionally, our software includes tools for analysing and ‘debugging’ the designs in silico, and for importing/exporting designs to other commonly used software systems. The software we present is built on many existing pieces of software, but is integrated into a single package—accessible using a Web-based interface at http://molecular-systems.net/workbench. We hope that the deep integration between tools and the flexibility of this design process will lead to better experimental results, fewer experimental design iterations and the development of more complex DNA nanosystems.     

DNA Origami‐Enabled Engineering of Ligand–Drug Conjugates for Targeted Drug Delivery

Effective drug delivery systems that can systematically and selectively transport payloads to disease cells remain a challenge. Here, a targeting ligand‐modified DNA origami nanostructure (DON) as an antibody–drug conjugate (ADC)‐like carrier for targeted prostate cancer therapy is reported. Specifically, DON of six helical bundles is modified with a ligand 2‐[3‐(1,3‐dicarboxy propyl)‐ureido] pentanedioic acid (DUPA) against prostate‐specific membrane antigen (PSMA), to serve as the antibody for drug conjugation in ADC. Doxorubicin (Dox) is then loaded to DON through intercalation to dsDNA. This platform features in spatially controllable organization of targeting ligands and high drug loading capacity. With this nanocomposite, selective delivery of Dox to the PSMA+ cancer cell line LNCaP is readily achieved. The consequent therapeutic efficacy is critically dependent on the numbers of targeting ligand assembled on DON. This target‐specific and biocompatible drug delivery platform with high maximum tolerated doses shows immense potential for developing novel nanomedicine.     

The transition mechanism of DNA overstretching: a microscopic view using molecular dynamics

The overstretching transition in torsionally unconstrained DNA is studied by means of atomistic molecular dynamics simulations. The free-energy profile as a function of the length of the molecule is determined through the umbrella sampling technique providing both a thermodynamic and a structural characterization of the transition pathway. The zero-force free-energy profile is monotonic but, in accordance with recent experimental evidence, becomes two-state at high forces. A number of experimental results are satisfactorily predicted: (i) the entropic and enthalpic contributions to the free-energy difference between the basic (B) state and the extended (S) state; (ii) the longitudinal extension of the transition state and (iii) the enthalpic contribution to the transition barrier. A structural explanation of the experimental finding that overstretching is a cooperative reaction characterized by elementary units of approximately 22 base pairs is found in the average distance between adenine/thymine-rich regions along the molecule. The overstretched DNA adopts a highly dynamical and structurally disordered double-stranded conformation which is characterized by residual base pairing, formation of non-native intra-strand hydrogen bonds and effective hydrophobic screening of apolar regions.