Self-assembly of three-dimensional prestressed tensegrity structures from DNA

Tensegrity, or tensional integrity, is a property of a structure indicating a reliance on a balance between components that are either in pure compression or pure tension for stability1,2. Tensegrity structures exhibit extremely high strength-toweight ratios and great resilience, and are therefore widely used in engineering, robotics and architecture3,4. Here, we report nanoscale, prestressed, three-dimensional tensegrity structures in which rigid bundles of DNA double helices resist compressive forces exerted by segments of singlestranded DNA that act as tension-bearing cables. Our DNA tensegrity structures can self-assemble against forces up to 14 pN, which is twice the stall force of powerful molecular motors such as kinesin or myosin5,6. The forces generated by this molecular prestressing mechanism can be used to bend the DNA bundles or to actuate the entire structure through enzymatic cleavage at specific sites. In addition to being building blocks for nanostructures, tensile structural elements made of single-stranded DNA could be used to study molecular forces, cellular mechanotransduction and other fundamental biological processes. Classic examples of tensegrity structures are the sculptures of Kenneth Snelson, which suspend isolated rigid columns in midair by interconnecting them with a continuous tensile cable network that prestresses the whole system (Fig. 1a)1, and the geodesic domes of Buckminster Fuller that use triangulation and minimal tensional paths between all pairs of neighbouring vertices to maintain their stability2. Prestressed tensegrity structures are found at all size scales in living systems7 and play a central role in cellular mechanotransduction8. A number of wireframe structures have been built from DNA9–13; however, these are relatively static shapes that do not display many of the novel mechanical features of prestressed tensegrity structures, such as the ability to globally reorient internal members and thereby strengthen in response to a local stress. Here, we set out to use the DNA-origami method14,15 to build prestressed tensegrity structures on the nanoscale that exhibit integrated mechanical responses similar to those displayed by living cellular systems. To accomplish this goal, we moved beyond the current DNAorigami methods used to create nanostructures, which rely only on paired bases to provide structural integrity. Instead, we incorporated stretched single-stranded DNA (ssDNA) segments as nanoconstruction elements that act in solution as entropic springs, the behaviour of which can be described over a wide range of loads using a modified freely jointed chain model (mFJC) that accounts for stretchable Kuhn segments16 (Supplementary Note S9). We designed and fabricated prestressed DNA tensegrity structures consisting of a 8,634-nucleotide (nt) M13mp18-based ‘scaffold strand’ and hundreds of oligodeoxyribonucleotide ‘staple strands’ that self-assemble into tensed structures despite kinetic barriers imposed by the prestress. The assembly process for prestressed origami objects is, as for DNA-origami objects in general, a onepot reaction where the scaffold strand, the staple strands and buffer containing Mg2þ ions are heated to 80 8C and then cooled down over the course of 72 h to room temperature to allow each staple strand to find its unique position on the scaffold sequence and hence achieve the correct assembly of the structure. The staple sequences were designed, using the software caDNAno (ref. 17), to promote self-assembly of rigid columns or struts composed of bundles of multiple DNA double helices. Importantly, this design differed from those used for previously reported DNAorigami structures because we also incorporated loops of hundreds of unpaired bases on the scaffold strand that connect the ends of multiple individual struts and act as the ssDNA springs. Two stretches of the scaffold DNA for which the sequences are prone to hairpin formation were incorporated into the rigid struts. A simplifying assumption of our model is that secondary-structure formation in the ssDNA springs can be ignored. Future experiments could use structure-free ssDNA (for example, scaffold segments programmed to consist primarily of the bases A, C and T) as the springs to make this assumption more valid. The energy necessary for tightening of these ssDNA springs is provided during the assembly process by base-pairing of the double helices that form the compression-resistant struts, thereby prestressing the entire integrated DNA structure. As a proof-of-principle for this strategy, we designed a threedimensional ‘tensegrity prism’ composed of three compressionresistant, 57-nm-long, 13-helix bundles held in place by nine tensed ssDNA springs (each 226 bases long) (Fig. 1b,c). Each strut was constructed from three segments of the scaffold that were far apart in the primary sequence, providing five, three and five of the 13 helices, respectively (Fig. 1d). Transmission electron microscopic (TEM) analysis of gel-purified structures self-assembled in this manner, and comparison of these two-dimensional images with predicted three-dimensional computer models, confirmed that this self-assembly process resulted in three-strut tensegrity prisms (Fig. 1e; see also Supplementary Fig. S1), and similar results were obtained by constructing another tensegrity prism using six-helix bundles as struts (Supplementary Fig. S2). With a view to investigating the influence of ssDNA spring length, and thus tension, on the structure and assembly success of DNA tensegrity structures, we created a two-dimensional, prestressed ‘kite’ structure (Fig. 2a). The ends of two 92-nm-long, 12-helix bundles were connected through four unpaired regions of