Ultrafiltration is one of the most widely used purification methods for isolating DNA origami and large DNA nanostructures1. Ultrafiltration uses a semipermeable membrane with a defined molecular weight cut off (MWCO) to separate molecules above and below the size threshold. Molecules larger than the MWCO are retained while molecules smaller than the MWCO flow through. For DNA origami, the objective of ultrafiltration is to separate intact DNA nanostructures from excess staples which are small and pass through the filter. Typically, 100 kDa MWCO filters are used. Several rounds of ultrafiltration can be performed to increase purity, however recovery yield also decreases as successive rounds of filtration and washing are performed.
When performing ultrafiltration, researchers run the risk of inducing aggregation2. While ultrafiltration is being performed, buffers including MgCl2 and Tris flow through and your final volume is less than your initial volume. This results in an increase in concentration of your origami which makes inter-origami interactions more favorable. It is also important to keep in mind that ultrafiltration will not remove free scaffold, aggregated origami, or dimerized origami as these all lie above the MWCO and will be retained. When high molar excess of staples are added, the concentration of free scaffold is typically assumed to be negligible. For single origami nanostructures, mechanical shearing is highly unlikely. However, for multi-component assemblies, shearing becomes a larger concern and ultrafiltration may not be compatible with these larger complexes.
Ultrafiltration can also be used for buffer exchange. The Tris, EDTA, MgCl2, and/or other salts in your buffer will pass through the filter. By reconstituting the retentate in a new buffer and performing successive washing and reconstitution steps, the original buffer and its contents can be diluted to negligible amounts. This can be useful when the optimal buffer for origami assembly is incompatible with downstream functional applications of the nanostructure.
After filtration it is important to once again characterize your sample. AGE can inform you whether your sample has aggregated, whether you have effectively removed excess staples, and whether you have retained your origami.
References
- Wagenbauer, K. F. et al. How We Make DNA Origami. ChemBioChem 18, 1873–1885 (2017).
- Dey, S. et al. DNA origami. Nat Rev Methods Primers 1, 13 (2021).