Biology subjects

Baptist, A. V.

Publications and source records attributed to Baptist, A. V..

4 recordsLinked to original sources

bioRxiv2025

Customizable silicification of DNA origami nanostructures

The silicification of DNA origami nanostructures offers a powerful strategy for enhancing their mechanical stability and resistivity against detrimental environmental conditions. In the past years, several studies have investigated different aspects of the silica coating procedure, leading to several different silicification protocols. Until now, the silica coating generally served as a protective layer or as the base for the further deposition of inorganic materials. However, it did not carry any additional functionality itself. Here, we present two different approaches for the customization of the silica coating on DNA origami nanostructures. Firstly, we developed a custom synthesized silica precursor carrying a fluorescein molecule to endow the silica coating of both DNA origami monomers and crystals with fluorescence and show the applicability of this novel silicification for the stabilization and tracking of DNA origami nanostructures intracellularly. Secondly, we employ a silica precursor containing a disulfide bridge to develop a silica coating that is dissolvable in a reducing environment. We anticipate that the results presented in this study will expand the toolbox of silicification in DNA nanotechnology and will further pave the way towards applications in drug delivery and material science.

biophysics↗

bioRxiv2024

Compliant DNA Origami Nanoactuators as Size-Selective Nanopores

Biological nanopores crucially control the import and export of biomolecules across lipid membranes in cells. They have found widespread use in biophysics and biotechnology, where their typically narrow, fixed diameters enable selective transport of ions and small molecules as well as DNA and peptides for sequencing applications. Yet, due to their small channel sizes, they preclude the passage of large macromolecules, e.g., therapeutics. Here, we harness the unique combined properties of DNA origami nanotechnology, machine-inspired design, and synthetic biology, to present a structurally reconfigurable DNA origami MechanoPore (MP) that features a lumen that is tuneable in size through molecular triggers. Controllable switching of MPs between three stable states is confirmed by 3D-DNA-PAINT super-resolution imaging and through dye-influx assays, after reconstitution of the large MPs in the membrane of liposomes via an inverted-emulsion cDICE technique. Confocal imaging of transmembrane transport shows size-selective behaviour with adjustable thresholds. Importantly, the conformational changes are fully reversible, attesting to the robust mechanical switching that overcomes pressure from the surrounding lipid molecules. These MPs advance nanopore technology, offering functional nanostructures that can be tuned on-demand - thereby impacting fields as diverse as drug-delivery, biomolecule sorting and sensing, as well as bottom-up synthetic biology.

biophysics↗

bioRxiv2024

Determination of Absolute Intramolecular Distances in Proteins by Anomalous X-ray Scattering Interferometry

Biomolecular structures are typically determined using frozen or crystalline samples. Measurement of intramolecular distances in solution can provide additional insights into conformational heterogeneity and dynamics of biological macromolecules and their complexes. The established molecular ruler techniques used for this (NMR, FRET, and EPR) are, however, limited in their dynamic range and require model assumptions to determine absolute distance (distributions). Here, we introduce anomalous X-ray scattering interferometry (AXSI) for intramolecular distance measurements in proteins, which are labeled at two sites with small gold nanoparticles of 0.7 nm radius. We apply AXSI to two different cysteine-variants of maltose binding protein in the presence and absence of its ligand maltose and find distances in quantitative agreement with single-molecule FRET experiments. Our study shows that AXSI enables determination of absolute intramolecular distance distributions under virtually arbitrary solution conditions and we anticipate its broad use to characterize protein conformational ensembles and dynamics.

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↗