Purification of DNA-origami nanostructures by rate-zonal centrifugation

Abstract

Most previously reported methods for purifying DNA-origami nanostructures rely on agarose-gel electrophoresis (AGE) for separation. Although AGE is routinely used to yield 0.1–1 µg purified DNA nanostructures, obtaining >100 µg of purified DNA-origami structure through AGE is typically laborious because of the post-electrophoresis extraction, desalting and concentration steps. Here, we present a readily scalable purification approach utilizing rate-zonal centrifugation, which provides comparable separation resolution as AGE. The DNA nanostructures remain in aqueous solution throughout the purification process. Therefore, the desired products are easily recovered with consistently high yield (40–80%) and without contaminants such as residual agarose gel or DNA intercalating dyes. Seven distinct three-dimensional DNA-origami constructs were purified at the scale of 0.1–100 µg (final yield) per centrifuge tube, showing the versatility of this method. Given the commercially available equipment for gradient mixing and fraction collection, this method should be amenable to automation and further scale up for preparation of larger amounts (e.g. milligram quantities) of DNA nanostructures.

Fractal assembly of micrometre-scale DNA origami arrays with arbitrary patterns

“…It has been shown that multi-origami structures can be successfully separated based on their sizes, for example using glycerol-gradient centrifugation or size-exclusion chromatography. 27,40 A possible concern for applying these methods to separate fractal arrays is that the interactions between origami tiles may be too weak to survive the separation process. To demonstrate that the origami arrays can be strengthened after fractal assembly, we added a 10-fold excess of a full set of 44 edge staples, that each have two stacking bonds, to the 4 by 4 arrays shown in Supplementary Fig.…”

DNA Nanostructures as Smart Drug-Delivery Vehicles and Molecular Devices

“…However, since the entire research field is constantly growing and developing, it is likely that scaling up of the production and simultaneous knockdown of the price will take place rapidly [3,64]. Very recent examples [65][66][67][68] promisingly show that production quantities of pure DNA origami objects are currently about to upgrade from micro-/milligram scale to gram scale. Today, 1 gram of scaffolded megadalton-sized DNA origamis costs approximately 100,000 $.…”

DNA Nanobiosensors: An Outlook on Signal Readout Strategies

“…Moreover, custom-tailored DNA scaffolds now allow the construction of DNA origami structures of different sizes and are not limited to the frequently used M13 single strand [80]. With the aid of suitable purification methods [81][82][83][84][85], these nanostructures can be prepared in pure forms that provide enhanced sensitivity. DNA, being a biomolecule, also provides an advantage of being biocompatible [86] and can be useful for biosensing in combination with biomimetic approaches.…”