Biology subjects

Caneva, S.

Publications and source records attributed to Caneva, S..

3 recordsLinked to original sources

bioRxiv2026

Nanophotonic DyeCycling delivers single-molecule FRET beyond photobleaching to identify heterogenous dynamics in DNA and protein systems

With its unique spatiotemporal resolution at the single-molecule level, Forster resonance energy transfer (FRET) is a powerful tool for investigating biomolecular conformational dynamics and function. However, early photobleaching remains a key limitation restricting the achievable observation time and therefore the information gain and application range of single-molecule FRET. Here, we establish nanophotonic DyeCycling, which overcomes photobleaching by reversible fluorophore binding and efficient background suppression using zero-mode waveguides. Nanoscale conformational changes are sensitively resolved from milliseconds up to the hour range, enabling robust kinetic analyses at the single-molecule level. As a result, outlier molecules reflecting static heterogeneity and time-dependent kinetics within individual molecules reflecting dynamic heterogeneity are reliably resolved. The versatility of DyeCycling is demonstrated using DNA and protein systems. Altogether, nanophotonic DyeCycling extends smFRET observations beyond the conventional photobleaching limit, revealing previously inaccessible kinetic effects and providing deeper insight into biomolecular systems with multiple states and rates.

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↗

bioRxiv2023

Diffusion of DNA on Atomically Flat 2D Material Surfaces

Accurate localization of biomolecules is pivotal for understanding biological processes. Utilizing the atomically flat surface of 2D materials offers a promising route to achieve this without the need for tethering or constraining. Here we comprehensively investigate the binding and diffusion of DNA on hexagonal boron nitride (hBN) surfaces. Our findings reveal non-specific binding of DNA to pristine hBN, with subsequent diffusion and confinement within the 2D plane. Through single-molecule experiments and computational techniques, we explore DNA dynamics, and the effects of defects, step edges and domain boundaries on the motion, which gives insights on the interactions between solid-state surfaces and biomolecules. By engineering a narrow hBN ribbon structure, we enhance confinement, demonstrating its potential in nanofluidic guiding of biomolecules. Our 2D platform serves as a proving ground for next generation high-throughput single-molecule manipulation techniques for enabling applications in biotechnology and nanotechnology.

molecular biology↗