A DNA origami rotary ratchet motor.

To impart directionality to the motions of a molecular mechanism, one must overcome the random thermal forces that are ubiquitous on such small scales and in liquid solution at ambient temperature. In equilibrium without energy supply, directional motion cannot be sustained without violating the laws of thermodynamics. Under conditions away from thermodynamic equilibrium, directional motion may be achieved within the framework of Brownian ratchets, which are diffusive mechanisms that have broken inversion symmetry1-5. Ratcheting is thought to underpin the function of many natural biological motors, such as the F1F0-ATPase6-8, and it has been demonstrated experimentally in synthetic microscale systems (for example, to our knowledge, first in ref. 3) and also in artificial molecular motors created by organic chemical synthesis9-12. DNA nanotechnology13 has yielded a variety of nanoscale mechanisms, including pivots, hinges, crank sliders and rotary systems14-17, which can adopt different configurations, for example, triggered by strand-displacement reactions18,19 or by changing environmental parameters such as pH, ionic strength, temperature, external fields and by coupling their motions to those of natural motor proteins20-26. This previous work and considering low-Reynolds-number dynamics and inherent stochasticity27,28 led us to develop a nanoscale rotary motor built from DNA origami that is driven by ratcheting and whose mechanical capabilities approach those of biological motors such as F1F0-ATPase.

Hierarchical assembly is more robust than egalitarian assembly in synthetic capsids.

Self-assembly of complex and functional materials remains a grand challenge in soft material science. Efficient assembly depends on a delicate balance between thermodynamic and kinetic effects, requiring fine-tuning affinities and concentrations of subunits. By contrast, we introduce an assembly paradigm that allows large error-tolerance in the subunit affinity and helps avoid kinetic traps. Our combined experimental and computational approach uses a model system of triangular subunits programmed to assemble into T = 3 icosahedral capsids comprising 60 units. The experimental platform uses DNA origami to create monodisperse colloids whose three-dimensional geometry is controlled to nanometer precision, with two distinct bonds whose affinities are controlled to kBT precision, quantified in situ by static light scattering. The computational model uses a coarse-grained representation of subunits, short-ranged potentials, and Langevin dynamics. Experimental observations and modeling reveal that when the bond affinities are unequal, two distinct hierarchical assembly pathways occur, in which the subunits first form dimers in one case and pentamers in another. These hierarchical pathways produce complete capsids faster and are more robust against affinity variation than egalitarian pathways, in which all binding sites have equal strengths. This finding suggests that hierarchical assembly may be a general engineering principle for optimizing self-assembly of complex target structures.

Building machines with DNA molecules.

In nature, DNA molecules carry the hereditary information. But DNA has physical and chemical properties that make it attractive for uses beyond heredity. In this Review, we discuss the potential of DNA for creating machines that are both encoded by and built from DNA molecules. We review the main methods of DNA nanostructure assembly, describe recent advances in building increasingly complex molecular structures and discuss strategies for creating machine-like nanostructures that can be actuated and move. We highlight opportunities for applications of custom DNA nanostructures as scientific tools to address challenges across biology, chemistry and engineering.

Geometrically programmed self-limited assembly of tubules using DNA origami colloids.

Self-assembly is one of the most promising strategies for making functional materials at the nanoscale, yet new design principles for making self-limiting architectures, rather than spatially unlimited periodic lattice structures, are needed. To address this challenge, we explore the tradeoffs between addressable assembly and self-closing assembly of a specific class of self-limiting structures: cylindrical tubules. We make triangular subunits using DNA origami that have specific, valence-limited interactions and designed binding angles, and we study their assembly into tubules that have a self-limited width that is much larger than the size of an individual subunit. In the simplest case, the tubules are assembled from a single component by geometrically programming the dihedral angles between neighboring subunits. We show that the tubules can reach many micrometers in length and that their average width can be prescribed through the dihedral angles. We find that there is a distribution in the width and the chirality of the tubules, which we rationalize by developing a model that considers the finite bending rigidity of the assembled structure as well as the mechanism of self-closure. Finally, we demonstrate that the distributions of tubules can be further sculpted by increasing the number of subunit species, thereby increasing the assembly complexity, and demonstrate that using two subunit species successfully reduces the number of available end states by half. These results help to shed light on the roles of assembly complexity and geometry in self-limited assembly and could be extended to other self-limiting architectures, such as shells, toroids, or triply periodic frameworks.

Designing Rigid DNA Origami Templates for Molecular Visualization Using Cryo-EM.

DNA origami, a method for constructing nanostructures from DNA, offers potential for diverse scientific and technological applications due to its ability to integrate various molecular functionalities in a programmable manner. In this study, we examined the impact of internal crossover distribution and the compositional uniformity of staple strands on the structure of multilayer DNA origami using cryogenic electron microscopy (cryo-EM) single-particle analysis. A refined DNA object was utilized as an alignment framework in a host-guest model, where we successfully resolved an 8 kDa thrombin binding aptamer (TBA) linked to the host object. Our results broaden the spectrum of DNA in structural applications.

Active Nuclear Import of Mammalian Cell-Expressible DNA Origami.

DNA origami enables the creation of complex 3D shapes from genetic material. Future uses could include the delivery of genetic instructions to cells, but nuclear import remains a major barrier to gene delivery due to the impermeability of the nuclear membrane. Here we realize active nuclear import of DNA origami objects in dividing and chemically arrested mammalian cells. We developed a custom DNA origami single-strand scaffold featuring a mammalian-cell expressible reporter gene (mCherry) and multiple Simian virus 40 (SV40) derived DNA nuclear targeting sequences (DTS). Inclusion of the DTS within DNA origami rescued gene expression in arrested cells, indicating that active transport into the nucleus occurs. Our work successfully adapts mechanisms known from viruses to promote the cellular expression of genetic instructions encoded within DNA origami objects.

Gigadalton-scale shape-programmable DNA assemblies.

Natural biomolecular assemblies such as molecular motors, enzymes, viruses and subcellular structures often form by self-limiting hierarchical oligomerization of multiple subunits. Large structures can also assemble efficiently from a few components by combining hierarchical assembly and symmetry, a strategy exemplified by viral capsids. De novo protein design and RNA and DNA nanotechnology aim to mimic these capabilities, but the bottom-up construction of artificial structures with the dimensions and complexity of viruses and other subcellular components remains challenging. Here we show that natural assembly principles can be combined with the methods of DNA origami to produce gigadalton-scale structures with controlled sizes. DNA sequence information is used to encode the shapes of individual DNA origami building blocks, and the geometry and details of the interactions between these building blocks then control their copy numbers, positions and orientations within higher-order assemblies. We illustrate this strategy by creating planar rings of up to 350 nanometres in diameter and with atomic masses of up to 330 megadaltons, micrometre-long, thick tubes commensurate in size to some bacilli, and three-dimensional polyhedral assemblies with sizes of up to 1.2 gigadaltons and 450 nanometres in diameter. We achieve efficient assembly, with yields of up to 90 per cent, by using building blocks with validated structure and sufficient rigidity, and an accurate design with interaction motifs that ensure that hierarchical assembly is self-limiting and able to proceed in equilibrium to allow for error correction. We expect that our method, which enables the self-assembly of structures with sizes approaching that of viruses and cellular organelles, can readily be used to create a range of other complex structures with well defined sizes, by exploiting the modularity and high degree of addressability of the DNA origami building blocks used.

Biotechnological mass production of DNA origami.

DNA nanotechnology, in particular DNA origami, enables the bottom-up self-assembly of micrometre-scale, three-dimensional structures with nanometre-precise features. These structures are customizable in that they can be site-specifically functionalized or constructed to exhibit machine-like or logic-gating behaviour. Their use has been limited to applications that require only small amounts of material (of the order of micrograms), owing to the limitations of current production methods. But many proposed applications, for example as therapeutic agents or in complex materials, could be realized if more material could be used. In DNA origami, a nanostructure is assembled from a very long single-stranded scaffold molecule held in place by many short single-stranded staple oligonucleotides. Only the bacteriophage-derived scaffold molecules are amenable to scalable and efficient mass production; the shorter staple strands are obtained through costly solid-phase synthesis or enzymatic processes. Here we show that single strands of DNA of virtually arbitrary length and with virtually arbitrary sequences can be produced in a scalable and cost-efficient manner by using bacteriophages to generate single-stranded precursor DNA that contains target strand sequences interleaved with self-excising 'cassettes', with each cassette comprising two Zn2+-dependent DNA-cleaving DNA enzymes. We produce all of the necessary single strands of DNA for several DNA origami using shaker-flask cultures, and demonstrate end-to-end production of macroscopic amounts of a DNA origami nanorod in a litre-scale stirred-tank bioreactor. Our method is compatible with existing DNA origami design frameworks and retains the modularity and addressability of DNA origami objects that are necessary for implementing custom modifications using functional groups. With all of the production and purification steps amenable to scaling, we expect that our method will expand the scope of DNA nanotechnology in many areas of science and technology.

Regulating DNA-Hybridization Using a Chemically Fueled Reaction Cycle.

Molecular machines, such as ATPases or motor proteins, couple the catalysis of a chemical reaction, most commonly hydrolysis of nucleotide triphosphates, to their conformational change. In essence, they continuously convert a chemical fuel to drive their motion. An outstanding goal of nanotechnology remains to synthesize a nanomachine with similar functions, precision, and speed. The field of DNA nanotechnology has given rise to the engineering precision required for such a device. Simultaneously, the field of systems chemistry developed fast chemical reaction cycles that convert fuel to change the function of molecules. In this work, we thus combined a chemical reaction cycle with the precision of DNA nanotechnology to yield kinetic control over the conformational state of a DNA hairpin. Future work on such systems will result in out-of-equilibrium DNA nanodevices with precise functions.

Programmable icosahedral shell system for virus trapping.

Broad-spectrum antiviral platforms that can decrease or inhibit viral infection would alleviate many threats to global public health. Nonetheless, effective technologies of this kind are still not available. Here, we describe a programmable icosahedral canvas for the self-assembly of icosahedral shells that have viral trapping and antiviral properties. Programmable triangular building blocks constructed from DNA assemble with high yield into various shell objects with user-defined geometries and apertures. We have created shells with molecular masses ranging from 43 to 925 MDa (8 to 180 subunits) and with internal cavity diameters of up to 280 nm. The shell interior can be functionalized with virus-specific moieties in a modular fashion. We demonstrate this virus-trapping concept by engulfing hepatitis B virus core particles and adeno-associated viruses. We demonstrate the inhibition of hepatitis B virus core interactions with surfaces in vitro and the neutralization of infectious adeno-associated viruses exposed to human cells.

Phage-free production of artificial ssDNA with Escherichia coli.

Artificial single-stranded DNA (ssDNA) with user-defined sequences and lengths up to the kilobase range is increasingly needed in mass quantities to realize the potential of emerging technologies such as genome editing and DNA origami. However, currently available biotechnological approaches for mass-producing ssDNA require dedicated, and thus costly, fermentation infrastructure, because of the risk of cross-contaminating manufacturer plants with self-replicating phages. Here we overcome this problem with an efficient, scalable, and cross-contamination-free method for the phage-free biotechnological production of artificial ssDNA with Escherichia coli. Our system utilizes a designed phagemid and an optimized helper plasmid. The phagemid encodes one gene of the M13 phage genome and a freely chosen custom target sequence, while the helper plasmid encodes the other genes of the M13 phage. The phagemid particles produced with this method are not capable of self-replication in the absence of the helper plasmid. This enables cross-contamination-free biotechnological production of ssDNA at any contract manufacturer. Furthermore, we optimized the process parameters to reduce by-products and increased the maximal product concentration up to 83 mg L-1 of ssDNA in a stirred-tank bioreactor, thus realizing up to a 40-fold increase in maximal product concentration over previous scalable phage-free ssDNA production methods.

Nanoschalen aus DNA fangen und neutralisieren Viren.

The current pandemic has highlighted the need for new antiviral therapies, to respond to existing and emerging diseases transmitted by viral vectors. We developed a novel antiviral approach that is based on neutralizing viruses by trapping and encapsulation in artificial nano-shells. The surrounding shells prevent the interaction of viruses with host cells and thus interrupt an essential step in the lifecycle of most viruses.

Antigen-Triggered Logic-Gating of DNA Nanodevices.

Synthetic nanoscale devices that reconfigure dynamically in response to physiological stimuli could offer new avenues for diagnostics and therapy. Here, we report a strategy for controlling the state of DNA nanodevices based on sensing antigens with IgG antibodies. To this end, we use IgG antibodies as structural elements to kinetically trap reconfigurable DNA origami structures in metastable states. Addition of soluble antigens displace the IgGs from the objects and triggers reconfiguration. We demonstrate this mechanism by antigen-triggered disassembly of DNA origami shells for two different IgGs and their cognate antigens, and we determined the corresponding dose response curves. We also describe the logic-gated actuation of DNA objects with combinations of antigens, as demonstrated with AND-type shells that disassemble only when two different antigens are detected simultaneously. We apply our system for the antigen-triggered release of molecular payload as exemplified by the release of virus particles that we loaded into the DNA origami shells. We expect our approach to be applicable in many types of DNA nanostructures and with many other IgG-antigen combinations.

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.

Reversible membrane deformations by straight DNA origami filaments.

Membrane-active cytoskeletal elements, such as FtsZ, septin or actin, form filamentous polymers able to induce and stabilize curvature on cellular membranes. In order to emulate the characteristic dynamic self-assembly properties of cytoskeletal subunits in vitro, biomimetic synthetic scaffolds were here developed using DNA origami. In contrast to our earlier work with pre-curved scaffolds, we specifically assessed the potential of origami mimicking straight filaments, such as actin and microtubules, by origami presenting cholesteryl anchors for membrane binding and additional blunt end stacking interactions for controllable polymerization into linear filaments. By assessing the interaction of our DNA nanostructures with model membranes using fluorescence microscopy, we show that filaments can be formed, upon increasing MgCl2 in solution, for structures displaying blunt ends; and can subsequently depolymerize, by decreasing the concentration of MgCl2. Distinctive spike-like membrane protrusions were generated on giant unilamellar vesicles at high membrane-bound filament densities, and the presence of such deformations was reversible and shown to correlate with the MgCl2-triggered polymerization of DNA origami subunits into filamentous aggregates. In the end, our approach reveals the formation of membrane-bound filaments as a minimal requirement for membrane shaping by straight cytoskeletal-like objects.

Tethered multifluorophore motion reveals equilibrium transition kinetics of single DNA double helices.

We describe a tethered multifluorophore motion assay based on DNA origami for revealing bimolecular reaction kinetics on the single-molecule level. Molecular binding partners may be placed at user-defined positions and in user-defined stoichiometry; and binding states are read out by tracking the motion of quickly diffusing fluorescent reporter units. Multiple dyes per reporter unit enable singe-particle observation for more than 1 hour. We applied the system to study in equilibrium reversible hybridization and dissociation of complementary DNA single strands as a function of tether length, cation concentration, and sequence. We observed up to hundreds of hybridization and dissociation events per single reactant pair and could produce cumulative statistics with tens of thousands of binding and unbinding events. Because the binding partners per particle do not exchange, we could also detect subtle heterogeneity from molecule to molecule, which enabled separating data reflecting the actual target strand pair binding kinetics from falsifying influences stemming from chemically truncated oligonucleotides. Our data reflected that mainly DNA strand hybridization, but not strand dissociation, is affected by cation concentration, in agreement with previous results from different assays. We studied 8-bp-long DNA duplexes with virtually identical thermodynamic stability, but different sequences, and observed strongly differing hybridization kinetics. Complementary full-atom molecular-dynamics simulations indicated two opposing sequence-dependent phenomena: helical templating in purine-rich single strands and secondary structures. These two effects can increase or decrease, respectively, the fraction of strand collisions leading to successful nucleation events for duplex formation.

Reciprocal Coupling in Chemically Fueled Assembly: A Reaction Cycle Regulates Self-Assembly and Vice Versa.

In biology, self-assembly of proteins and energy-consuming reaction cycles are intricately coupled. For example, tubulin is activated and deactivated for assembly by a guanosine triphosphate (GTP)-driven reaction cycle, and the emerging microtubules catalyze this reaction cycle by changing the microenvironment of the activated tubulin. Recently, synthetic analogs of chemically fueled assemblies have emerged, but examples in which assembly and reaction cycles are reciprocally coupled remain rare. In this work, we report a peptide that can be activated and deactivated for self-assembly. The emerging assemblies change the microenvironment of their building blocks, which consequently accelerate the rates of building block deactivation and reactivation. We quantitatively understand the mechanisms at play, and we are thus able to tune the catalysis by molecular design of the peptide precursor.

Design of a molecular support for cryo-EM structure determination.

Despite the recent rapid progress in cryo-electron microscopy (cryo-EM), there still exist ample opportunities for improvement in sample preparation. Macromolecular complexes may disassociate or adopt nonrandom orientations against the extended air-water interface that exists for a short time before the sample is frozen. We designed a hollow support structure using 3D DNA origami to protect complexes from the detrimental effects of cryo-EM sample preparation. For a first proof-of-principle, we concentrated on the transcription factor p53, which binds to specific DNA sequences on double-stranded DNA. The support structures spontaneously form monolayers of preoriented particles in a thin film of water, and offer advantages in particle picking and sorting. By controlling the position of the binding sequence on a single helix that spans the hollow support structure, we also sought to control the orientation of individual p53 complexes. Although the latter did not yet yield the desired results, the support structures did provide partial information about the relative orientations of individual p53 complexes. We used this information to calculate a tomographic 3D reconstruction, and refined this structure to a final resolution of ∼15 Å. This structure settles an ongoing debate about the symmetry of the p53 tetramer bound to DNA.

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.