# Mechanical Design of DNA Origami in the Classroom
## 3 Results  
### The Compliant Hinge Joint Design  
The DO design process for nanomechanical DNA structures generally consists of (1) determining a desired geometry based on the intended function; (2) dividing the target geometry into sub-components (if necessary); (3) assigning a cross-section to each sub-component (or assigning ssDNA component composition); (4) determining scaffold routing to satisfy the desired geometry and component design; (5) determining the staple routing. Since the design process for nanomechanical devices requires significant expertise, here, we bypass the design process by using a previously established compliant DO structure. As a brief note, over the last 15 years, the most widely used design tool is caDNAno [36]. More recently, a number of additional design tools have been developed with varying levels of automation [39, 40, 41, 42, 43]. An overview of design tools is provided in a recent review article [44], and many tutorials can be found online (see Table S3 in Supplemental Information), if educators wish to include lessons on DO design. The CHJ was developed by [PERSON] et al. [35] as a compliant nanostructure similar to a torsional spring with tunable equilibrium angle and flexibility. The design is comprised of two arms that are \(\sim\)55 nm long from the vertex to ends of arms and made up of 18-helix bundles each organized in a 6 \(\times\) 3 hexagonal lattice cross-section (Figure 2A). The arms are connected at the joint end by six flexible ssDNA linkers that span across the joint connecting the bottom layer of helices on the two arms, and the top layer of six double-stranded DNA (dsDNA) helices connects the two arms directly across the joint. The six-helix dsDNA layer deforms in bending due to forces generated by the entropic elasticity of the ssDNA linkers. Hence, the top layer is the compliant component that deforms to modify the overall structure geometry. Here, the ssDNA linkers function as entropic springs that apply forces causing the dsDNA compliant layer to bend. The geometry of loading and the anisotropic bending stiffness of the six-helix layer ensures that bending occurs primarily in a single direction, leading to a well-defined joint angle (deformations previously studied in detail by [PERSON] et al. [45]). The two outer arms are stiff and remain basically straight [35]. The CHJ was originally designed in caDNAno [36] with additional details provided by [PERSON] et al. [35]. Figure 2B shows the CHJ structure configuration predicted from oxDNA simulations conducted in the oxView web interface [37,38] and visualized using VMD software (version 1.9.4a53) [46]. The deformation of the compliant joint, and hence the overall conformation of the CHJ, can be engineered by modifying the length of the ssDNA linker (Figure 2C). Shorter ssDNA linkers will generate a larger force, inducing stronger bending of the compliant joint. The CHJ design allows for easy tuning of the hinge configuration by introducing a scaffold reservoir near the joint (blue loops protruding from right arm in Figure 2C), so the length of the linkers can be easily adjusted by substituting a subset of staples near the joint that shift scaffold length between the reservoir and linkers. We focus here on three different configurations: high force (HF), medium force (MF), and low force (LF). Figure 2D shows the three configurations of the CHJ analyzed by gel electrophoresis, demonstrating a small shift between the HF and MF configurations and a larger shift between the MF and LF configurations. For all the structures, we observed some aggregation, indicated by structures stuck in the well; for MF and LF structures, we observed a clear slower running band, which is likely indicative of a dimer population. We focused the rest of our analysis on the fastest running bands, which are the well-folded DO structures as confirmed by TEM images (Supporting Figure S2). Figure 2: Design and folding of the CHJ.