high-speed-atomic-force-microscopy-imaging-dna-three-point-star-motif

High-Speed Atomic Force Microscopy Imaging of DNA Three-Point-Star Motif Self Assembly Using Photothermal Off-Resonance Tapping

High-speed atomic force microscopy (HS-AFM) is a popular molecular imaging technique for visualizing single-molecule biological processes in real-time due ...

Ecole Poly-technique Fédérale Lausanne

The extracellular matrix (ECM) and cells have a reciprocal relationship, one shapes the other and vice versa. One of the main challenges of synthetic material systems for developmental cell culturing, organoid and stem cell work includes the implementation of this reciprocal nature. The largest hurdle to achieve true cell-instructive materials in biomaterials engineering is a lack of spatial and temporal control over material properties and the display of bioactive signals compared to the natural cell environment. ECM-mimicking hydrogels have been developed using a wide range of polymers, assembly and cross-linking strategies. While our synthetic toolbox is larger than nature, often our systems underperform when compared to ECM systems with natural components like Matrigel. Material properties and three-dimensional structure ill-represent the three-dimensional ECM reciprocal nature and ligand presentation is an oversimplified version of the complexity found in nature. We hypothesize that the lack of programmable control in properties and ligand presentation forms the basis of this mismatch in performance and analyze the presence of control in current state of the art ECM-mimicking systems based on covalent, supramolecular and recombinant polymers. We conclude that through combining the dynamics of supramolecular materials, robustness from covalent systems and the programmable spatial control of bio-activation in recombinant ECM materials, the optimal synthetic artificial ECM could be assembled.

Programmable Control in Extracellular Matrix-mimicking Polymer Hydrogels.

DNA as a biomaterial has evoked great interest as a potential platform for therapeutics and diagnostics and as hydrogel scaffolds due to the relative ease of programming its robust and uniform shape, site-specific functionality and controlled responsive behavior. However, for a stable self-assembled product, a relatively high cation concentration is required to prevent denaturation. Physiological and cell-culture conditions do not match these concentrations and present additional nucleases that cause a serious threat to the integrity of DNA-based materials. For the translation of this promising technology towards bioengineering challenges, stability needs to be guaranteed. Over the past years, various methods have been developed addressing the stability-related weaknesses of DNA-origami. This mini-review explains the common stability issues and compares the stabilization strategies recently developed. We present a detailed overview of each method in order to ease the selection process on which method to use for future users of DNA-origami as a biomaterial.

Engineering a stable future for DNA-origami as a biomaterial.

Most vaccines developed today include only the antigens that best stimulate the immune system rather than the entire virus or microbe, which makes vaccine production and use safer and easier, though they lack potency to induce acceptable immunity and long-term protection. The incorporation of additional immune stimulating components, named adjuvants, is required to generate a strong protective immune response. Nucleic acids (DNA and RNA) and their synthetic analogs are promising candidates as vaccine adjuvants activating Toll-like receptors (TLRs). Additionally, in the last few years several nanocarriers have emerged as platforms for targeted co-delivery of antigens and adjuvants. In this review, we focus on the recent developments in polymer nanomaterials presenting nucleic acids as vaccine adjuvants. We aim to compare the effectiveness of the various classes of polymers in immune modulating materials (nanoparticles, dendrimers, single-chain particles, nanogels, polymersomes and DNA-based architectures). In particular, we address the critical role of parameters such as size, shape, complexation and release of TLR ligands, cellular uptake, stability, toxicity and potential importance of spatial control in ligand presentation.

Nucleic acids presenting polymer nanomaterials as vaccine adjuvants.

Nucleic acids and lipids function in close proximity in biological processes, as well as in nanoengineered constructs for therapeutic applications. As both molecules carry a rich charge profile, and frequently coexist in complex ionic solutions, the electrostatics surely play a pivotal role in interactions between them. Here we discuss how each component of a DNA/ion/lipid system determines its electrostatic attachment. We examine membrane binding of a library of DNA molecules varying from nanoengineered DNA origami through plasmids to short DNA domains, demonstrating the interplay between the molecular structure of the nucleic acid and the phase of lipid bilayers. Furthermore, the magnitude of DNA/lipid interactions is tuned by varying the concentration of magnesium ions in the physiologically relevant range. Notably, we observe that the structural and mechanical properties of DNA are critical in determining its attachment to lipid bilayers and demonstrate that binding is correlated positively with the size, and negatively with the flexibility of the nucleic acid. The findings are utilized in a proof-of-concept comparison of membrane interactions of two DNA origami designs - potential nanotherapeutic platforms - showing how the results can have a direct impact on the choice of DNA geometry for biotechnological applications.

Interplay of the mechanical and structural properties of DNA nanostructures determines their electrostatic interactions with lipid membranes.

This chapter discusses the methods involved in achieving and analyzing cellular uptake of DNA origami. While cells naturally internalize substances from their surroundings, more than a simple addition of DNA origami in the surrounding cell medium is necessary to ensure DNA origami particles successfully enter the intracellular environment. Starting with the folding of the DNA, careful handling of sterile buffers and tools is essential, as well as the use of an endotoxin free scaffold. We explain how DNA origami needs a certain form of stabilization or protection to survive the degrading low-salt and high-nuclease environment of common cell culture media. Depending on the preferred method of post-uptake analysis (confocal), microscopy, or flow cytometry, we elaborate on the full protocols and crucial steps to prepare cell uptake experiments. Finally, notes are added on the intracellular fate (see Notes 14 and 15), and cellular retention of DNA origami (see Note 16) is discussed.

Article	Year
Programmable Control in Extracellular Matrix-mimicking Polymer Hydrogels. Chimia\| PubMed ID: 28662736	2017
Engineering a stable future for DNA-origami as a biomaterial. Biomaterials science\| PubMed ID: 30534709	2019
Nucleic acids presenting polymer nanomaterials as vaccine adjuvants. Journal of materials chemistry. B\| PubMed ID: 31460563	2019
Interplay of the mechanical and structural properties of DNA nanostructures determines their electrostatic interactions with lipid membranes. Nanoscale\| PubMed ID: 36688792	2023
Cellular Uptake of DNA Origami. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 37166720	2023

Maartje Bastings

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