Purification of DNA Origami Nanostructures Using Capillary Electrophoresis

Abstract

DNA origami are nanostructures designed based on Watson-Crick base-pairing that fold a scaffold into non-arbitrary morphologies using an excess of linear single-stranded DNA "staples". As an engineered nanomaterial (ENM) with great customizability, DNA origami also enjoys the benefit of naturally encoded and well-studied structural and functional properties. The flexibility of different folding patterns allows for construction of a wide variety of shapes and sizes of DNA origami, showing potential applications in fields such as medicine, biocomputing, biomedical engineering , and measurement science. Despite the successes as a functional nanomaterial, a major barrier to the applicability of DNA origami rests in the lack of pure, well-folded structures. As such, the development of different purification techniques is essential to support the rapid development of the material toward a vast scope of applications. Current techniques to purify DNA origami from excess precursors (staples), misfolded structures and other impurities have shown low yields, low scalability, tendency for aggregated samples, and lack optimization for automation. Capillary electrophoresis (CE) has previously shown effective separation of single-stranded DNA based on differences of size and charge in a manner similar to gel electrophoresis, but with the added benefit of automation and more substantial control and detection throughout the separation. The development of CE as a purification technique for DNA origami is investigated in this study, where a highly reproducible separation between folded DNA origami from excess DNA staples was achieved by manipulating and understanding the effect of buffer conditions , capillary specifications , and injection parameters on the electropherogram profile. Specifically , CE was investigated under both capillary zone electrophoresis (CZE) and capillary transient isotachophoresis (ctlTP) modes, and optimization of both systems yielded baseline resolved separations of DNA origami from the staple strands. The ctlTP system demonstrated superior performance in terms of decreasing band broadening, improving resolution, and improving the Gaussian character of migration peaks. Further, the optimized ctlTP separation was used in a fraction collection procedure, where the resulting fractions were imaged by atomic force microscopy (AFM) for offline validation of purified structures. However, issues with the intercalating dye and origami aggregation were suspected to impede the imaging process. The reproducibility of the fraction collection procedure was validated to show a highly linear relationship between the peak area of a reinjection of pooled sample and the number of pooled fractions. An approach to calculating the percent yield of CE-based purification was attempt ed but requires further validation. Continued exploration and analysis of CE for the purification of DNA origami could thus lead to a novel , promising, and efficient tool to advance the field as a whole.