# Mechanical Design of DNA Origami in the Classroom
## 3 Results
### Folding the CHI with Classroom-Friendly Protocols  
The previous folding was carried out using laboratory thermocyclers, which are not practical for use in most classrooms. Enabling folding of DO in a science or engineering class or lab requires two key steps: (1) identifying a suitably short-duration and constant-temperature annealing protocol, and (2) ensuring that the protocol can be carried out with low-cost equipment (i.e., hot plates). Standard folding procedures include annealing for long times over many precisely controlled temperatures, which is why a thermocycler is typically used. However, previous work has shown that DO structures can be folded at constant temperature over shorter periods of time [32], which is conducive to folding with low-cost equipment in a reasonable classroom period time. Figure 3A shows the thermal ramp comparison between laboratory (top curve) and classroom (bottom curve) folding approaches. Both consist of three phases: melting, annealing, and quenching; however, for the classroom approach, we could achieve constant temperature annealing over short times by using a heating element consisting of a water bath heated by a hot plate. To first explore short annealing times, we tested the three different versions (HF, MF, and LF) with different annealing times of 10 min, 20 min, and 30 min at a constant annealing temperature of 52 \({}^{\circ}\)C in the thermal cycler. We previously showed that 52 \({}^{\circ}\)C works well for the constant temperature annealing of many different DO structures [33]. All folding reactions still included a melting phase of 5 min at 65 \({}^{\circ}\)C and a quenching phase of 5 min at 4 \({}^{\circ}\)C. After folding, structures were evaluated via agarose gel electrophoresis (with the research laboratory electrophoresis setup). Figure 3B shows the gel results for the three versions of the CHJ, illustrating a similar pattern of gel shifts to the control structures (Figure 2D). For all annealing times, MF ran slightly faster than HF, and LF exhibited a larger shift with respect to MF. TEM for all versions and annealing times confirmed well-folded structures (Supporting Figure S2). These results demonstrated that the 10 min annealing time is suitable for illustrating the gel shifts that are indicative of distinct deformed configurations. Interestingly, we also found that there is much less aggregation in the shorter folding time in comparison with the 2.5-day protocol. This is consistent with prior work showing that shorter annealing times may help avoid aggregate formation for DO structures [32]. Since we aimed to fold the CHJ using low-cost equipment, which would lead to less precise control over the folding temperatures, we also tested i  
Figure 3: Protocol optimization for class. (**A**) Schematic of annealing ramp during folding process between laboratory and class. (**B**) Folding time optimization and temperature sensitivity test. (**C**) Temperature tested in 55, 54.7, 54.2, 53.2, 52, 51, 50.3, 50 \({}^{\circ}\)C for structure annealing. sensitive to the exact annealing temperature. We conducted a screen of constant annealing temperatures from 55 \({}^{\circ}\)C to 50 \({}^{\circ}\)C in a thermal cycler with the same folding protocol (5 min melting at 65 \({}^{\circ}\)C, 10 min at set annealing temperature, 5 min quenching at 4 \({}^{\circ}\)C). Figure 4C shows the results of this temperature screen for MF (LF, HF shown in Supplement Figure S3). For all annealing temperatures, the folded structure bands exhibited a similar mobility to the previously tested structures, with proper folding confirmed by TEM. These results indicate that the folding quality is mostly insensitive to annealing temperature over the range of 50-55 \({}^{\circ}\)C. That said, for higher temperatures in this range, we observed that the yields were slightly lower, as indicated by a more smeared structure band and some intensity in the wells.