Wassermann, L. M. | bioRxiv Science

bioRxiv2024

Monitoring the Coating of Single DNA Origami Nanostructures with a Molecular Fluorescence Lifetime Sensor

The high functionality of DNA nanostructures makes them a promising tool for biomedical applications, their intrinsic instability under application-relevant conditions, still remains challenging. Protective coating of DNA nanostructures with materials like silica or cationic polymers has evolved as a simple, yet powerful strategy to improve their stability even under extreme conditions. While over time, various materials and protocols have been developed, the characterization and quality assessment of the coating is either time consuming, highly invasive or lacks detailed insights on single nanostructures. Here, we introduce a cyanine dye based molecular sensor designed to non-invasively probe the coating of DNA origami by either a cationic polymer or by silica, in real-time and on a single nanostructure level. The cyanine dye reports changes in its local environment upon coating via increased fluorescence lifetime induced by steric restriction and water exclusion. Exploiting the addressability of DNA origami, the molecular sensor can be placed at selected positions to probe the coating layer with nanometer precision. We demonstrate the reversibility of the sensor and use it to study the stability of the different coatings in degrading conditions. To showcase the potential for correlative studies, we combine the molecular fluorescence lifetime sensor with DNA PAINT super-resolution imaging to investigate coating and structural integrity as well as preserved addressability of DNA nanostructures. The reported sensor presents a valuable tool to probe the coating of DNA nanodevices in complex biochemical environments in real-time and at the single nanosensor level and aids the development of novel stabilization strategies.

biophysics↗

bioRxiv2022

Full site-specific addressability in DNA origami-templated silica nanostructures

DNA nanotechnology allows for the fabrication of nano-meter-sized objects with high precision and selective addressability as a result of the programmable hybridization of complementary DNA strands. Such structures can template the formation of other materials, including metals and complex silica nanostructures, where the silica shell simultaneously acts to protect the DNA from external detrimental factors. However, the formation of silica nanostructures with site-specific addressability has thus far not been explored. Here we show that silica nanostructures templated by DNA origami remain addressable for post silicification modification with guest molecules even if the silica shell measures several nm in thickness. We used the conjugation of fluorescently labelled oligonucleotides to different silicified DNA origami structures carrying a complementary ssDNA handle as well as DNA PAINT super-resolution imaging to show that ssDNA handles remain unsilicified and thus ensure retained addressability. We also demonstrate that not only handles, but also ssDNA scaffold segments within a DNA origami nanostructure remain accessible, allowing for the formation of dynamic silica nanostructures. Finally we demonstrate the power of this approach by forming 3D DNA origami crystals from silicified monomers. Our results thus present a fully site-specifically addressable silica nanostructure with complete control over size and shape.

biophysics↗