[O1]. Design of the origami library-inspired virus with different sizes, shapes, and ligand density.
A new, efficient purification method for DNA origami nanostructures was developed based on size exclusion chromatography using the HiTrap Capto Core 700 column, which, to our knowledge, had not been previously applied to DNA origami purification. This method allows high-yield purification and production of sufficient quantities of DNA origamis for further studies. DLS and STEM confirmed the formation of well-defined DNA origami structures using this method.
Additionally, protocols for DNA origami–protein conjugation and purification were established. The DNA origami ring was successfully modified with albumin through click-chemistry, and the resulting conjugates were isolated and characterised. This conjugation strategy provides a versatile platform for the functionalisation of DNA origamis with other biomolecules, such as enzymes or targeting proteins, paving the way for future applications in nanobiotechnology and drug delivery.
[O2]. Biomolecular corona characterisation on origami library.
DNA origami nanostructures exhibited remarkable stability in buffers with progressively decreasing magnesium concentrations, with some maintaining their integrity even in pure water. Furthermore, buffers containing alternative stabilising cations, such as ethylenediamine, were found to support the formation of well-defined origamis. This buffer was subsequently used for biocorona studies.
Before corona isolation, stability tests in human serum revealed that only the ring-shaped origami exhibited partial degradation after one hour of incubation. This observation highlighting the importance of verifying stability prior to corona isolation, a step often overlooked in published studies.
Origami-biocorona complexes were sucessfully isolated using size exclusion chromatography. SDS-PAGE analysis of the biocorona components revealed few differences among the three origamis. In turn, mass spectrometry analysis revealed an unexpected background of proteins originating from the serum control sample, preventing clear conclusions regarding differences between the samples. Further optimisation and follow-up studies will be required to clarify these findings and improve the robustness of biocorona analysis in DNA origami systems.
[O3]. Study of the effect of the biomolecular corona on the cell response.
DNA origamis were successfully labelled with two commonly used fluorescent DNA-binding dyes, Hoechst and GelGreen, using both pre-assembly (during folding) and post-assembly (after folding) approaches. This work demonstrated that fluorescent labelling can be achieved without compromising structural integrity, as confirmed by DLS and STEM, offering a straightforward and affordable method for visualising DNA nanostructures when precise positional control of dyes is unnecessary.
Fluorescent DNA origamis (ring, cuboid, and gear) were then incubated with A549 epithelial cells. After 24 hours, cell viability exceeded 90% under all conditions. Internalisation studies revealed time-dependent uptake, with GelGreen-labelled origamis accumulating preferentially in mitochondria, while Hoechst-labelled origamis showed partial colocalisation with endocytosis markers, suggesting uptake through endocytic pathways.
The complement activation assays demonstrated a concentration-dependent response, where cuboid and gear structures induced strong activation levels comparable to, or even higher than, the positive control at the highest tested concentrations. Finally, experiments with phagocytic cells revealed a preferential uptake of the ring structure in comparison with the more rigid cuboid and gear designs.
