p9344-BurrH scaffold routings and initial Goniometer designs were made using Cadnano2.2
23 Optimization of staples (i.e. staple auto-breaking) and generation of pipetting instructions for the staple stocks were performed using a custom software toolkit (manuscript in preparation) developed in the Douglas Lab. Staple stocks for the goniometers were prepared using Labcyte
Echo 525 (Labcyte, CA, USA). Folding reactions were performed using Bio-Rad
MJ Research PTC-240 Tetrad thermal cycler. The temperature annealing ramp for the DNA Origami Goniometers was:
Incubate at 65°C for 00:10:00
Incubate at 60°C for 01:00:00
Decrease by 1.0°C every cycle
Cycle to step 2 for 20 more times
Folding was performed in 1XFOB20 (5 mM Tris-Base, 1 mM EDTA, 5 mM NaCl, 20 mM MgCl
2 at pH 8.0). In the folding reaction, p9344-BurrH scaffold concentration was 20 nM and each staple concentration was 200 nM. The Goniometers were purified via PEG precipitation as described previously
24 . Briefly, 15% PEG-8000 solution (15% w/v PEG-8000, 5 mM Tris-Base, 1 mM EDTA, 500 mM NaCl, 20 mM MgCl
2 at pH 8.0) were mixed with the folding reaction at a 1:1 ratio. The mix was centrifuged at 16,000× rcf for 25 minutes at room temperature. The supernatant was discarded, and the pellet resuspended in 1XFOB20. PEG precipitation was repeated one more time and the final pellet was resuspended in 1XFOB20 and stored at 4°C.
Aksel T., Yu Z., Cheng Y, & Douglas S.M. (2020). Molecular goniometers for single-particle cryo-electron microscopy of DNA-binding proteins. Nature biotechnology, 39(3), 378-386.
