Polyethylene glycol (PEG) precipitation is another widely implemented method for DNA nanostructure purification1. PEG precipitation works by excluded volume depletion. Briefly, PEG, a large and hydrophilic polymer, occupies significant solution volume and sterically excludes large DNA nanostructures from the same space as the PEG. This exclusion creates an osmotic pressure that concentrates the large nanostructures into a smaller volume and can precipitate them out of solution. DNA staples and salts are small enough such that they are not excluded by PEG and can occupy the same space, therefore they do not precipitate. Choosing the correct molecular weight PEG can affect your purification efficiency. PEG8k is a typical starting point for scaffolded origami around 4.5 MDa. Similar to ultrafiltration, PEG purification will not remove aggregates or misassembled origami similar in size to the intended structure2.
When implementing PEG purification for your origami there are several variables that can be tuned to optimize purification. Adjusting the concentration of PEG, the centrifugation time, the molecular weight of PEG, and the number of cycles all affect the recovery yield and purification efficacy. Briefly:
- Increasing the concentration of PEG too high can improve origami retention but also cause co-precipitation of staples with origami. Decreasing the concentration of PEG can lead to reduced origami retention but more complete removal of excess staples.
- Reducing centrifugation time can cause poor recovery yield due to incomplete precipitation of the nanostructures. Increasing centrifugation time can cause aggregation3.
- Increasing the number of cycles leads to a reduction in yield but an increase in purity1.
Another challenge of PEG purification is the difficulty in removing residual PEG. Residual PEG can be found in your final sample and can have downstream effects in subsequent experiments.
References
- Stahl, E., Martin, T. G., Praetorius, F. & Dietz, H. Facile and Scalable Preparation of Pure and Dense DNA Origami Solutions. Angew Chem Int Ed Engl 53, 12735–12740 (2014).
- Sheheade, B. et al. Self-Assembly of DNA Origami Heterodimers in High Yields and Analysis of the Involved Mechanisms. Small 15, 1902979 (2019).
- Baptist, A. V. & Heuer-Jungemann, A. Lyophilization Reduces Aggregation of Three-Dimensional DNA Origami at High Concentrations. ACS Omega 8, 18225–18233 (2023).