Folding of an origami is a critical step. Besides the question whether a designed structure folds at all, the yield and the fraction of misfolded structures are also important. The yield determines which concentrations could be applied for further experiments. If significant amounts of misfoldings occur, thorough purification is needed, which in turn corresponds to reduced yield due to purification losses. For these reasons, it was not only aimed to design a structure which can be expected to fold easily, but some effort was also dedicated to testing different thermal folding ramps and check the efficiency of folding.
For more detailed information, please consult the instruction in the labbook.
Several hundreds of staples have to be mixed for a folding batch. Those staples, which are frequently used together for different variants of the structure, were mixed in prestocks. Later on, for small modifications of the structure, only the appropriate prestock needs to be remixed, while all other parts could be used again. Prestocks may vary in the number of included staples, but in one prestock, all staples are equally concentrated (usually 10µl 100µM staple are mixed).
For every structure variant, one working stock is mixed. Here, all needed prestocks are mixed in such a way, that all staples finally have the same concentration, 500nM.
The folding batch contains FOB20, 20nM scaffold and 200nM of each staple. Dye-labeled oligonucleotides are added extra at this step.
Three different ramps were tested, which proceed at different velocities during the critical steps of folding.
This shortest and most simple folding ramp starts at 65°C. Every 15 minutes the temperature is decreased by 1°C, to a final temperature of 25°C. After finishing this ramp, temperature is set to 4°C.
Here again, the starting temperature is 65°C. Every 15 minutes the temperature is decreased by 1°C, to a final temperature of 57°C. The next part of the ramp, down to 26°C, proceeds with a cooling velocitiy of 1°C per 1:30 hours. After finishing this ramp, temperature is set to 4°C.
For this longest ramp, again the first part is cooling down from 65°C to 57°C by -1°C per 15 minutes. Then, the batch is cooled down by 1°C per 3 hours. Finally, temperature is set to 4°C.
Validation of folding efficiency
Success of folding was evaluated with agarose gel electrophoresis. Thereby, it could be investigated, whether additional bands besides the main structure do occur. Subsequently, these bands could be analyzed at the TEM.
Faltung erklären, also Vorgehensweise, welche Rampen, belegen warum.
Links zu Results und Labbook.