Membrane Remodeling by DNA Origami Nanorods: Experiments Exploring the Parameter Space for Vesicle Remodeling.

Inspired by the ability of cell membranes to alter their shape in response to bound particles, we report an experimental study of long, slender nanorods binding to lipid bilayer vesicles and altering the membrane shape. Our work illuminates the role of particle concentration, adhesion strength, and membrane tension in determining the membrane morphology. We combined giant unilamellar vesicles with oppositely charged nanorods, carefully tuning the adhesion strength, membrane tension, and particle concentration. With increasing adhesion strength, the primary behaviors observed were membrane deformation, vesicle-vesicle adhesion, and vesicle rupture. These behaviors were observed in well-defined regions in the parameter space with sharp transitions between them. We observed the deformation of the membrane resulting in tubulation, textured surfaces, and small and large lipid-particle aggregates. These responses are robust and repeatable and provide a new physical understanding of the dependence on the shape, binding affinity, and particle concentration in membrane remodeling. The design principles derived from these experiments may lead to new bioinspired membrane-based materials.

Time-Resolved Small-Angle X-ray Scattering Reveals Millisecond Transitions of a DNA Origami Switch.

Self-assembled DNA structures enable creation of specific shapes at the nanometer-micrometer 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 MgCl2 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.

Reversible Covalent Stabilization of Stacking Contacts in DNA Assemblies.

Stacking bonds formed between two blunt-ended DNA double helices can be used to reversibly stabilize higher-order complexes that are assembled from rigid DNA components. Typically, at low cation concentrations, stacking bonds break and thus higher-order complexes disassemble. Herein, we present a site-specific photochemical mechanism for the reversible covalent stabilization of stacking bonds in DNA assemblies. To this end, we modified one blunt end with the 3-cyanovinylcarbazole (cnv K) moiety and positioned a thymine residue (T) at the other blunt end. In the bound state, the two blunt-ended helices are stacked together, resulting in a co-localization of cnv K and T. Such a configuration induces the formation of a covalent bond across the stacking contact upon irradiation with 365 nm light. This bond can also be cleaved upon irradiation with 310 nm light, allowing repeated formation and cleavage of the same covalent bond on the timescale of seconds. Our system will expand the range of conditions under which stacking-bond-stabilized objects may be utilized.

How We Make DNA Origami.

DNA origami has attracted substantial attention since its invention ten years ago, due to the seemingly infinite possibilities that it affords for creating customized nanoscale objects. Although the basic concept of DNA origami is easy to understand, using custom DNA origami in practical applications requires detailed know-how for designing and producing the particles with sufficient quality and for preparing them at appropriate concentrations with the necessary degree of purity in custom environments. Such know-how is not readily available for newcomers to the field, thus slowing down the rate at which new applications outside the field of DNA nanotechnology may emerge. To foster faster progress, we share in this article the experience in making and preparing DNA origami that we have accumulated over recent years. We discuss design solutions for creating advanced structural motifs including corners and various types of hinges that expand the design space for the more rigid multilayer DNA origami and provide guidelines for preventing undesired aggregation and on how to induce specific oligomerization of multiple DNA origami building blocks. In addition, we provide detailed protocols and discuss the expected results for five key methods that allow efficient and damage-free preparation of DNA origami. These methods are agarose-gel purification, filtration through molecular cut-off membranes, PEG precipitation, size-exclusion chromatography, and ultracentrifugation-based sedimentation. The guide for creating advanced design motifs and the detailed protocols with their experimental characterization that we describe here should lower the barrier for researchers to accomplish the full DNA origami production workflow.

Conformational Changes and Flexibility of DNA Devices Observed by Small-Angle X-ray Scattering.

Self-assembled DNA origami nanostructures enable the creation of precisely defined shapes at the molecular scale. Dynamic DNA devices that are capable of switching between defined conformations could afford completely novel functionalities for diagnostic, therapeutic, or engineering applications. Developing such objects benefits strongly from experimental feedback about conformational changes and 3D structures, ideally in solution, free of potential biases from surface attachment or labeling. Here, we demonstrate that small-angle X-ray scattering (SAXS) can quantitatively resolve the conformational changes of a DNA origami two-state switch device as a function of the ionic strength of the solution. In addition, we show how SAXS data allow for refinement of the predicted idealized three-dimensional structure of the DNA object using a normal mode approach based on an elastic network model. The results reveal deviations from the idealized design geometries that are otherwise difficult to resolve. Our results establish SAXS as a powerful tool to investigate conformational changes and solution structures of DNA origami and we anticipate our methodology to be broadly applicable to increasingly complex DNA and RNA devices.

Revealing the structures of megadalton-scale DNA complexes with nucleotide resolution.

The methods of DNA nanotechnology enable the rational design of custom shapes that self-assemble in solution from sets of DNA molecules. DNA origami, in which a long template DNA single strand is folded by many short DNA oligonucleotides, can be employed to make objects comprising hundreds of unique DNA strands and thousands of base pairs, thus in principle providing many degrees of freedom for modelling complex objects of defined 3D shapes and sizes. Here, we address the problem of accurate structural validation of DNA objects in solution with cryo-EM based methodologies. By taking into account structural fluctuations, we can determine structures with improved detail compared to previous work. To interpret the experimental cryo-EM maps, we present molecular-dynamics-based methods for building pseudo-atomic models in a semi-automated fashion. Among other features, our data allows discerning details such as helical grooves, single-strand versus double-strand crossovers, backbone phosphate positions, and single-strand breaks. Obtaining this higher level of detail is a step forward that now allows designers to inspect and refine their designs with base-pair level interventions.

Custom-Size, Functional, and Durable DNA Origami with Design-Specific Scaffolds.

DNA origami nano-objects are usually designed around generic single-stranded "scaffolds". Many properties of the target object are determined by details of those generic scaffold sequences. Here, we enable designers to fully specify the target structure not only in terms of desired 3D shape but also in terms of the sequences used. To this end, we built design tools to construct scaffold sequences de novo based on strand diagrams, and we developed scalable production methods for creating design-specific scaffold strands with fully user-defined sequences. We used 17 custom scaffolds having different lengths and sequence properties to study the influence of sequence redundancy and sequence composition on multilayer DNA origami assembly and to realize efficient one-pot assembly of multiscaffold DNA origami objects. Furthermore, as examples for functionalized scaffolds, we created a scaffold that enables direct, covalent cross-linking of DNA origami via UV irradiation, and we built DNAzyme-containing scaffolds that allow postfolding DNA origami domain separation.

Sequence-programmable covalent bonding of designed DNA assemblies.

Bottom-up fabrication of custom nanostructures using the methods of DNA nanotechnology has great potential for applications in many areas of science and technology. One obstacle to applications concerns the constrained environmental conditions at which DNA objects retain their structure. We present a general, site-selective, and scalable method for creating additional covalent bonds that increase the structural stability of DNA nanostructures. Placement of thymidines in close proximity within DNA nanostructures allows the rational creation of sites for covalent cyclobutane pyrimidine dimer (CPD) bonds induced via ultraviolet irradiation. The additional covalent bonds may be used in a sequence-programmable fashion to link free strand termini, to bridge strand breaks at crossover sites, and to create additional interhelical connections. Thus designed multilayer DNA origami objects can remain stable at temperatures up to 90°C and in pure double-distilled water with no additional cations present. In addition, these objects show enhanced resistance against nuclease activity. Cryo-electron microscopy (cryo-EM) structural analysis of non-cross-linked and cross-linked objects indicated that the global shape and the internal network of crossovers are preserved after irradiation. A cryo-EM map of a CPD-stabilized multilayer DNA origami object determined at physiological ionic strength reveals a substantial swelling behavior, presumably caused by repulsive electrostatic forces that, without covalent stabilization, would cause disassembly at low ionic strength. Our method opens new avenues for applications of DNA nanostructures in a wider range of conditions.

Dynamic DNA devices and assemblies formed by shape-complementary, non-base pairing 3D components.

We demonstrate that discrete three-dimensional (3D) DNA components can specifically self-assemble in solution on the basis of shape-complementarity and without base pairing. Using this principle, we produced homo- and heteromultimeric objects, including micrometer-scale one- and two-stranded filaments and lattices, as well as reconfigurable devices, including an actuator, a switchable gear, an unfoldable nanobook, and a nanorobot. These multidomain assemblies were stabilized via short-ranged nucleobase stacking bonds that compete against electrostatic repulsion between the components' interfaces. Using imaging by electron microscopy, ensemble and single-molecule fluorescence resonance energy transfer spectroscopy, and electrophoretic mobility analysis, we show that the balance between attractive and repulsive interactions, and thus the conformation of the assemblies, may be finely controlled by global parameters such as cation concentration or temperature and by an allosteric mechanism based on strand-displacement reactions.

Rapid folding of DNA into nanoscale shapes at constant temperature.

We demonstrate that, at constant temperature, hundreds of DNA strands can cooperatively fold a long template DNA strand within minutes into complex nanoscale objects. Folding occurred out of equilibrium along nucleation-driven pathways at temperatures that could be influenced by the choice of sequences, strand lengths, and chain topology. Unfolding occurred in apparent equilibrium at higher temperatures than those for folding. Folding at optimized constant temperatures enabled the rapid production of three-dimensional DNA objects with yields that approached 100%. The results point to similarities with protein folding in spite of chemical and structural differences. The possibility for rapid and high-yield assembly will enable DNA nanotechnology for practical applications.