When we are designing new DNA nanostructures here are some useful tips and tricks we suggest you keep in mind.
Before even beginning to design your DNA nanostructure it is important to keep in mind your final objective for the DNA nanostructure. DNA nanotechnology encompasses many different design frameworks: Scaffolded helical bundles, scaffolded wireframes, scaffold-free wireframes, DNA Bricks, DNA Slats, and small multi-DNA complexes.
When deciding which of these methods to use, we ask ourselves several questions: (1) What size-range do we want for our structure, (2) Do we need a rigid or flexible structure, (3) Do we need high-yield, (4) What is our budget, (5) Do we value addressability?
These are in addition to functional questions like: (1) Are we interfacing with living materials, (2) What buffer conditions do we require stability in, (3) Will these function in solution or on surfaces, and many more questions to consider unique to your application.
Once you have decided on what class of nanostructure you will design, many existing software have been developed to improve the ease of design.
For all softwares, we recommend using Nupack to minimize the secondary structure of all oligos and SeqWalkto maximize sequence orthogonality. However, for scaffolded approaches it’s important to note the designer has much less control over oligo sequences since you’re limited to the complement of the chosen scaffold. For scaffolded origami approaches we recommend two additional tools for improving your design: dnafold and pyOrigamiBreak. Finally, for all approaches we recommend simulating your nanostructure with oxDNA to better understand its stability and equilibrium structure.
Now that you’ve designed your nanostructure, it is time to order the oligos, synthesize, and characterize successful formation. Supplies like Integrated DNA Nanotechnologies (IDT), Twist Biosciences, and Eurofins Genomics all sell high-purity oligos.
Once your oligos have arrived it is time to test the formation of your nanostructure and optimize its assembly. Typically, there are three main experimental variables we tune when optimizing assembly: (1) Thermal annealing ramp, (2) Buffer salt concentrations, and (3) Staple : Scaffold ratio for scaffolded structures. Recommendations for each can be found at the links above!
To first determine if a structure has folded and in what yield we use gel electrophoresis, either Polyacrylamide gel electrophoresis (PAGE) for small structures or agarose gel electrophoresis (AGE) for larger structures. For both techniques, we recommend always including a DNA ladder and for scaffolded origami, include a scaffold only control well. Without the scaffold only control well, it is easy to mistake an unfolded DNA scaffold for a DNA nanostructure band.
Depending on your application, you may need your nanostructure to be purified from excess oligos or aggregates. Purification methods include ultrafiltration, Polyethylene glycol (PEG) precipitation, gel extraction, magnetic-bead purification, size-exclusion chromatography, among many others. For simplicity and general effectiveness for megadalton sized nanostructures, we typically use spin filtration, although the optimal method will depend on your nanostructure. Thus, we recommend trying multiple purification methods for each origami depending on the equipment and expertise within your lab. For more information on the benefits and drawbacks of each approach please click the links above.
Following purification, it is important to assess the recovery yield, the purity, and the impact of the purification on the structure. To achieve this we recommend again running gel electrophoresis with the purified samples. From the gel, you can determine if aggregates were removed, excess staples were removed, and if the purification damaged your structure or induced aggregation.
The next step is to perform structural verification using high resolution imaging methods like atomic force microscopy or transmission electron microscopy. For the highest information content we recommend AFM for 2D structures and TEM for 3D structures. It is important to keep in mind that both of these methods are imaging nanostructures adsorbed onto a surface. Therefore, the imaged structures may differ from their solution-state form.
For more in-depth tutorials and information, we highly recommend the following resources:
- Overview of Experimental Procedure: K. F. Wagenbauer, F. A. S. Engelhardt, E. Stahl, V. K. Hechtl, P. Stömmer, F. Seebacher, L. Meregalli, P. Ketterer, T. Gerling, H. Dietz, ChemBioChem 2017, 18, 1873.
- Original Implementation of DNA Origami: Rothemund, P. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006). https://doi.org/10.1038/nature04586
- Overview of designing, synthesizing, and characterizing DNA origami: Castro, C., Kilchherr, F., Kim, DN. et al. A primer to scaffolded DNA origami. Nat Methods 8, 221–229 (2011). https://doi.org/10.1038/nmeth.1570
- DNA Origami and its Applications: Dey, S., Fan, C., Gothelf, K.V. et al. DNA origami. Nat Rev Methods Primers 1, 13 (2021). https://doi.org/10.1038/s43586-020-00009-8