Periodic Reporting for period 1 - ORBIT (Elucidating the nano-biointeractions on DNA origami)
Período documentado: 2023-09-15 hasta 2025-09-14
The primary objective of this project was to investigate the biointeractions of DNA origamis with varying sizes, geometries, and ligand densities in biological environments. Aiming to elucidate potential correlations between the structural properties of these nanostructures and their interactions with serum proteins (biocorona), their cellular behaviour, and/or their ability to trigger immune responses.
The specific scientific objectives of this project were:
[O1]. Design of the origami library-inspired virus with different sizes, shapes, and ligand density.
[O2]. Biomolecular corona characterisation on origami library.
[O2]. Biomolecular corona characterisation on origami library.
The specific scientific objectives of this project were:
[O1]. Design of the origami library-inspired virus with different sizes, shapes, and ligand density.
[O2]. Biomolecular corona characterisation on origami library.
[O2]. Biomolecular corona characterisation on origami library.
[O1]. Design of the origami library-inspired virus with different sizes, shapes, and ligand density.
Three DNA origami structures with distinct sizes and geometries were used to establish a small “virus-inspired” origami library: a DNA origami ring, cuboid, and gear. The DNA origami ring was designed in the laboratory and incorporated three modifiable staples on the outer part to enable protein conjugation. The cuboid and gear origami structures were commercially sourced and proved instrumental in establishing and optimising the purification protocol. Purification was performed using size exclusion chromatography (SEC). Two different columns were evaluated, Superose 6 and HiTrap Capto Core 700. Both columns allowed the purification of DNA nanostructures, which were characterised using agarose gel electrophoresis, scanning transmission electron microscopy (STEM), and dynamic light scattering (DLS), confirming the formation of well-defined DNA origami structures. The HiTrap Capto Core 700 column was selected for its superior speed and efficiency.
Subsequently, the DNA origami ring was conjugated with bovine serum albumin (BSA) as a model protein. The DNA origami conjugates were successfully separated from excess protein using SEC. The resulting conjugates were characterised by agarose gel electrophoresis, UV-Vis spectroscopy, and transmission electron microscopy (TEM).
[O2]. Biomolecular corona characterisation on origami library.
The stability of DNA origamis was systematically evaluated in different buffer compositions to identify conditions compatible with biological assays.
Additionally, the stability of DNA origamis in human serum was evaluated prior the isolation of the origami-biocorona complex. For biocorona isolation, two approaches were tested: centrifugation and SEC. Due to the low density of DNA nanostructures, SEC yielded the most reliable results. Origami-biocorona complexes were isolated and characterised by SDS-PAGE and mass spectrometry.
[O3]. Study of the effect of the biomolecular corona on the cell response.
To enable cell tracking of DNA origamis, the nanostructures were labelled with DNA-binding fluorescent dyes, providing a simple and cost-effective method for fluorescence labelling when spatial control of dye placement is not required.
Cell viability and internalisation assays were performed using A549 lung carcinoma epithelial cells. Cell viability remained consistently above 90%, indicating that the DNA origamis and their fluorescent labels were non-toxic under the tested conditions. Internalisation of the nanostructures was found to be time-dependent.
Finally, the immune response to DNA origamis was examined through complement activation assays and interaction studies with a phagocytic cell line. Complement activation was concentration-dependent, and uptake efficiency varied among the different DNA origami geometries studied.
Three DNA origami structures with distinct sizes and geometries were used to establish a small “virus-inspired” origami library: a DNA origami ring, cuboid, and gear. The DNA origami ring was designed in the laboratory and incorporated three modifiable staples on the outer part to enable protein conjugation. The cuboid and gear origami structures were commercially sourced and proved instrumental in establishing and optimising the purification protocol. Purification was performed using size exclusion chromatography (SEC). Two different columns were evaluated, Superose 6 and HiTrap Capto Core 700. Both columns allowed the purification of DNA nanostructures, which were characterised using agarose gel electrophoresis, scanning transmission electron microscopy (STEM), and dynamic light scattering (DLS), confirming the formation of well-defined DNA origami structures. The HiTrap Capto Core 700 column was selected for its superior speed and efficiency.
Subsequently, the DNA origami ring was conjugated with bovine serum albumin (BSA) as a model protein. The DNA origami conjugates were successfully separated from excess protein using SEC. The resulting conjugates were characterised by agarose gel electrophoresis, UV-Vis spectroscopy, and transmission electron microscopy (TEM).
[O2]. Biomolecular corona characterisation on origami library.
The stability of DNA origamis was systematically evaluated in different buffer compositions to identify conditions compatible with biological assays.
Additionally, the stability of DNA origamis in human serum was evaluated prior the isolation of the origami-biocorona complex. For biocorona isolation, two approaches were tested: centrifugation and SEC. Due to the low density of DNA nanostructures, SEC yielded the most reliable results. Origami-biocorona complexes were isolated and characterised by SDS-PAGE and mass spectrometry.
[O3]. Study of the effect of the biomolecular corona on the cell response.
To enable cell tracking of DNA origamis, the nanostructures were labelled with DNA-binding fluorescent dyes, providing a simple and cost-effective method for fluorescence labelling when spatial control of dye placement is not required.
Cell viability and internalisation assays were performed using A549 lung carcinoma epithelial cells. Cell viability remained consistently above 90%, indicating that the DNA origamis and their fluorescent labels were non-toxic under the tested conditions. Internalisation of the nanostructures was found to be time-dependent.
Finally, the immune response to DNA origamis was examined through complement activation assays and interaction studies with a phagocytic cell line. Complement activation was concentration-dependent, and uptake efficiency varied among the different DNA origami geometries studied.
[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.
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.