When synthesizing a nanostructure, DNA staples and scaffold are heated up (often to 80 °C) before being slowly cooled to room temperature. The maximum temperature and the rate of cooling are two parameters to be considered. The maximum temperature should be chosen high enough such that all oligos' secondary structure will be melted as will any DNA duplexes. Physically, after high-temperature incubation all staples and scaffold are single-stranded.
Now as the temperature is slowly cooled, sequential origami folding occurs. At high temperatures, only staples with high melting temperatures will bind stably to the scaffold. These staples then constrain the scaffold geometry before staples with lower melting temperatures bind to the scaffold and continue the folding process. For typical designs, the folding occurs primarily between 40 °C and 60 °C. For your specific design, you can utilize tools like NUPACK to predict your staples' melting temperatures to better optimize your ramp.
Within this temperature range, the cooling rate is most important as that is when most of the folding will occur. If the ramp is too fast, staples may not find their correct binding sites and non-optimal binding can become irreversibly stable at lower temperatures. This can manifest as a reduction in yield or greater frequency of misfolded structures with improper staple incorporations. If the ramp is too slow, the optimal yield will likely be achieved but the synthesis time can become impractically long and reduce the rate at which experiments can be performed. For some origami, slow long anneals can increase aggregation and the presence of undesired multimers1.
Generally, focused ramps are recommended as the most efficient yet productive ramps12. Focused ramps cool the sample quickly outside the productive folding range and then cool the sample slowly within the primary folding range (around the staple melting temperatures).
References
- Dey, A. et al. Effect of Temperature Ramp in Rapid Folding of 3D DNA Origami Structures. Small Structures 7, e202500682 (2026).
- Mohammadi-Zerankeshi, M., Houston, J., Elisha-Wigwe, O. K. U., Sachi, A. & Marras, A. E. Unraveling the Folding Dynamics of DNA Origami Structures. Small 21, e04855 (2025).