Biological materials are self-assembled with near-atomic precision in living cells, whereas synthetic 3D structures generally lack such precision and controllability. Recently, DNA nanotechnology, especially DNA origami technology, has been useful in the bottom-up fabrication of well-defined nanostructures ranging from tens of nanometres to sub-micrometres. In this Primer, we summarize the methodologies of DNA origami technology, including origami design, synthesis, functionalization and characterization. We highlight applications of origami structures in nanofabrication, nanophotonics and nanoelectronics, catalysis, computation, molecular machines, bioimaging, drug delivery and biophysics. We identify challenges for the field, including size limits, stability issues and the scale of production, and discuss their possible solutions. We further provide an outlook on next-generation DNA origami techniques that will allow in vivo synthesis and multiscale manufacturing.

We demonstrate here the rational design of purely entropic domains as a versatile approach to achieve control of the input/output response of synthetic molecular receptors. To do so and to highlight the versatility and generality of this approach, we have rationally re-engineered two model DNA-based receptors: a clamp-like DNA-based switch that recognizes a specific DNA sequence and an ATPbinding aptamer. We show that, by varying the length of the linker domain that connects the two recognition portions of these receptors, it is possible to finely control their affinity for their specific ligand. Through mathematical modeling and thermodynamic characterization, we also demonstrate for both systems that entropy changes associated with changes in linker length are responsible for affinity modulation and that the linker we have designed behaves as a disordered random-coil polymer. The approach also allows us to regulate the ligand concentration range at which the receptors respond and show optimal specificity. Given these attributes, the use of purely entropic domains appears as a versatile and general approach to finely control the activity of synthetic receptors in a highly predictable and controlled fashion.

Scaffold proteins regulate cell signalling by promoting the proximity of putative interaction partners. Although they are frequently applied in cellular settings, fundamental understanding of them in terms of, amongst other factors, quantitative parameters has been lagging behind. Here we present a scaffold protein platform that is based on the native 14‐3‐3 dimeric protein and is controllable through the action of a small‐molecule compound, thus permitting study in an in vitro setting and mathematical description. Robust small‐molecule regulation of caspase‐9 activity through induced dimerisation on the 14‐3‐3 scaffold was demonstrated. The individual parameters of this system were precisely determined and used to develop a mathematical model of the scaffolding concept. This model was used to elucidate the strong cooperativity of the enzyme activation mediated by the 14‐3‐3 scaffold. This work provides an entry point for the long‐needed quantitative insights into scaffold protein functioning and paves the way for the optimal use of reengineered 14‐3‐3 proteins as chemically inducible scaffolds in synthetic systems.

The rational regulation of the pK a of an ionizable group in a synthetic device could be achieved by controlling the entropy of the linker connecting the hydrogen bond forming domains. We demonstrate this by designing a set of pH-responsive synthetic DNA-based nanoswitches that share the same hydrogen bond forming domains but differ in the length of the linker. The observed acidic constant (pK a) of these pH-dependent nanoswitches is linearly dependent on the entropic cost associated with loop formation and is gradually shifted to more basic pH values when the length of the linker domain is reduced. Through mathematical modeling and thermodynamic characterization we demonstrate that the modulation of the observed pK a is due to a purely entropic contribution. This approach represents a very versatile strategy to rationally modulate the pK a of synthetic devices in a highly predictable and accurate way.

In recent years, several antibody drug conjugates (ADC) have been accepted by the FDA as therapeutics against cancer. It is well‐known that control of drug‐to‐antibody ratio (DAR) is vital for the success of an ADC, which inspires the advancement of better and simpler methods for tight control of DAR. We present the development of an antibody DNA wireframe cube conjugate for precise control of DAR. The DNA wireframe cube consists of four single strands, which when folded present eight single stranded domains. One domain is bound to a monofunctionalized antibody DNA conjugate, and the seven others are attached to DNA functionalized with the potent tubulin inhibitor MMAE, thereby preparing an ADC with a DAR of precisely seven. The formation of the ADC is investigated by gel electrophoresis and atomic force microscopy. Lastly, the developed MMAE loaded ADC was used for targeted drug delivery in vitro.

The compaction and organization of genomic DNA is a central mechanism in eukaryotic cells, but engineered architectural control over double‐stranded DNA (dsDNA) is notably challenging. Here, long dsDNA templates are folded into designed shapes via triplex‐mediated self‐assembly. Triplex‐forming oligonucleotides (TFOs) bind purines in dsDNA via normal or reverse Hoogsteen interactions. In the triplex origami methodology, these non‐canonical interactions are programmed to compact dsDNA (linear or plasmid) into well‐defined objects, which demonstrate a variety of structural features: hollow and raster‐filled, single‐ and multi‐layered, with custom curvatures and geometries, and featuring lattice‐free, square‐, or honeycomb‐pleated internal arrangements. Surprisingly, the length of integrated and free‐standing dsDNA loops can be modulated with near‐perfect efficiency; from hundreds down to only six bp (2 nm). The inherent rigidity of dsDNA promotes structural robustness and non‐periodic structures of almost 25.000 nt are therefore formed with fewer unique starting materials, compared to other DNA‐based self‐assembly methods. Densely triplexed structures also resist degradation by DNase I. Triplex‐mediated dsDNA folding is methodologically straightforward and orthogonal to Watson‐Crick‐based methods. Moreover, it enables unprecedented spatial control over dsDNA templates.

DNA-templated chemical reactions have found wide applications in drug discovery, programmed multistep synthesis, nucleic acid detection and targeted drug delivery. The control of these reactions has, however, been limited to nucleic acid hybridization as a means to direct the proximity between reactants.In this work we introduce a system capable of translating protein-protein binding events into a DNAtemplated reaction which leads to the covalent formation of a product. We achieve protein-templated reactions by employing two DNA-antibody conjugates that are both able to recognize the same target protein and to colocalize a pair of reactant DNA strands able to undergo a click reaction. We engineered two individual systems each responsive to human serum albumin (HSA) and human IgG and we demonstrated that, while no reaction occurs in the absence of proteins, both protein-templated reactions can occur simultaneously in the same solution without any inter-system crosstalk.

Homogeneous assays for determining the concentration of small molecules in biological fluids are of importance for monitoring blood levels of critical drugs in patients. We have developed a strand displacement competition assay for the drugs dabigatran, methotrexate, and linezolid, which allows detection and determination of the concentration of the drugs in plasma; however, a surprising kinetic behavior of the assay was observed with an initial rapid change in apparent FRET values. We found that protein-induced fluorescent enhancement or quenching (PIFE/Q) caused the initial change in fluorescence within the first minute after addition of protein, which could be exploited to construct assays for concentration determination within minutes in the low nanomolar range in plasma. A kinetic model for the assay was established, and when taking the new finding into account, the in silico simulations were in good agreement with the experimentally observed results. Utilizing these findings, a simpler assay was constructed for detection of dabigatran, which allowed for detection within minutes without any time dependencies.