Lyophilization Reduces Aggregation of Three-Dimensional DNA Origami at High Concentrations

Abstract

Although for many purposes, low concentrations of DNA origami are sufficient, certain applications such as cryo electron microscopy, measurements involving small-angle X-ray scattering, or in vivo applications require high DNA origami concentrations of >200 nM. This is achievable by ultrafiltration or polyethylene glycol precipitation but often at the expense of increasing structural aggregation due to prolonged centrifugation and final redispersion in low buffer volumes. Here, we show that lyophilization and subsequent redispersion in low buffer volumes can achieve high concentrations of DNA origami while drastically reducing aggregation due to initially very low DNA origami concentrations in low salt buffers. We demonstrate this for four structurally different types of three-dimensional DNA origami. All of these structures exhibit different aggregation behaviors at high concentrations (tip-to-tip stacking, side-to-side binding, or structural interlocking), which can be drastically reduced by dispersion in larger volumes of a low salt buffer and subsequent lyophilization. Finally, we show that this procedure can also be applied to silicified DNA origami to achieve high concentrations with low aggregation. We thus find that lyophilization is not only a tool for long-term storage of biomolecules but also an excellent way for up-concentrating while maintaining well-dispersed solutions of DNA origami.

Dominant Analytical Techniques in DNA Nanotechnology for Various Applications

“…In the dye-modified construct, ultrafiltration was used to give a resulting yield of around 82%, but in the two protein constructs, the recovery was unable to be measured due to the limited separation between the residual proteins and nanostructure itself; for reference, Alexa 488 is about 45 kDa, and IgG and Ferritin have molecular weights of 150 and 480 kDa, respectively . Additionally, this method is ideal for small working volumes, as the filter columns are only able to hold a maximum of 500 μL at a time . There are 15 mL alternatives available, but the loss in good product is correlated with the surface area of the cellulose membrane within the column, thereby giving poorer performance on scale-up.…”

“…There are 15 mL alternatives available, but the loss in good product is correlated with the surface area of the cellulose membrane within the column, thereby giving poorer performance on scale-up. In regards to design specification, this method is the least effective for rod-like constructs because they are able to pass through the filter unit if oriented favorably, leading to significant sample loss and, in turn, low yield . To combat low yield, this purification technique demands a multifold excess starting material.…”

“…Emerging purification techniques in DNA nanostructure analysis. (A) TEM image comparison for DNA origami nanostructures purified and concentrated via lyophilization and PEG precipitation . (B) Schematic representative of how to isolate single-stranded scaffold DNA from a double-stranded DNA template using magnetic bead separation .…”

“…One can overcome the challenge of aggregation by storing the DNA nanostructure at lower salts and lyophilization. Prior to applying the DNA nanostructure, simply reconstituting the sample in water was shown to pose no aggregation issues . Lyophilization, commonly utilized for the long-term storage of biomolecules, is a technique in which the sample is shock-frozen in liquid nitrogen and, in turn, subjected to sublimation under vacuum.…”

“…This technique leaves behind solid components in a sample, including salts; thus, it is imperative that the initial buffer be of minimal salt concentration before subjecting a sample to this freeze-drying process (Figure A). Salt concentration is again dependent on nanostructure geometry; dense structures tend to require higher levels of salt for prolonged structural integrity . It is hypothesized that the rapid exclusion of salt and EDTA during shock-freezing reduces the DNA nanostructure-to-nanostructure aggregation.…”