Nucleic acids (DNA and RNA) are widely used to construct nanoscale structures with ever increasing complexity1–14 for possible applications in fields as diverse as structural biology, biophysics, synthetic biology and photonics. The nanostructures are formed through one-pot self-assembly, with early examples typically containing on the order of 10 unique DNA strands. The introduction of DNA origami4, which uses many staple strands to fold one long scaffold strand into a desired structure, gave access to kilo- to mega-dalton nanostructures containing about 102 unique DNA strands6,7,10,13 . Aiming for even larger DNA origami structures is in principle possible15,16, but faces the challenge of having to manufacture and route an increasingly long scaffold strand. An alternative and in principle more readily scalable approach uses DNA brick assembly8,9, which doesn’t need a scaffold and instead uses hundreds of short DNA brick strands that self-assemble according to specific inter-brick interactions. First-generation bricks used to create 3D structures are 32-nt long with four 8-nt binding domains that directed 102 distinct bricks into well-formed assemblies, but attempts to create larger structures encountered practical challenges and had limited success.9 Here we show that a new generation of DNA bricks with longer binding domains makes it possible to self-assemble 0.1 – 1 giga-dalton three-dimensional nanostructures from 104 unique components, including a 0.5 giga-dalton cuboid containing 30,000 unique bricks and a 1 giga-dalton rotationally symmetric tetramer. We also assemble a cuboid containing 10,000 bricks and 20,000 uniquely addressable ‘nano-voxels’ that serves as a molecular canvas for three-dimensional sculpting, with introduction of sophisticated user-prescribed 3D cavities yielding structures such as letters, a complex helicoid and a teddy bear. We anticipate that, with further optimization, even larger assemblies might be accessible and prove useful as scaffolds or for positioning functional components.

Using DNA as programmable, sequence specific ‘glues’, shape-controlled hydrogel units are self-assembled into prescribed structures. Here we report that aggregates are produced using hydrogel cubes with edge length ranging from 30 micrometers to 1 millimeter, demonstrating assembly across scales. In a simple one-pot agitation reaction, 25 dimers are constructed in parallel from 50 distinct hydrogel cube species, demonstrating highly multiplexed assembly. Using hydrogel cuboids displaying face-specific DNA glues, diverse structures are achieved in aqueous and in interfacial agitation systems. These include dimers, extended chains, and open network structures in an aqueous system; and dimers, chains of fixed length, T-junctions, and square shapes in the interfacial system, demonstrating the versatility of the assembly system.

Information technologies enable programmers and engineers to design and synthesize systems of startling complexity that nonetheless behave as intended. This mastery of complexity is made possible by a hierarchy of formal abstractions that span from high-level programming languages down to low-level implementation specifications, with rigorous connections between the levels. DNA nanotechnology presents us with a new molecular information technology whose potential has not yet been fully unlocked in this way. Developing an effective hierarchy of abstractions may be critical for increasing the complexity of programmable DNA systems. Here, we build on prior practice to provide a new formalization of ‘domain-level’ representations of DNA strand displacement systems that has a natural connection to nucleic acid biophysics while still being suitable for formal analysis. Enumeration of unimolecular and bimolecular reactions provides a semantics for programmable molecular interactions, with kinetics given by an approximate biophysical model. Reaction condensation provides a tractable simplification of the detailed reactions that respects overall kinetic properties. The applicability and accuracy of the model is evaluated across a wide range of engineered DNA strand displacement systems. Thus, our work can serve as an interface between lower-level DNA models that operate at the nucleotide sequence level, and high-level chemical reaction network models that operate at the level of interactions between abstract species.

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.