Programmability of Dynamic DNA Origami Structures Examined at the Single-Molecule Level

DNA nanotechnology has revolutionized molecular engineering, enabling the construction of dynamic, programmable architectures with nanometer precision. Among these, DNA origami structures stand out as molecular rulers─offering unparalleled control over spatial organization at the nanoscale. By strategically positioning fluorophores within these scaffolds, Förster resonance energy transfer (FRET) can be used to probe molecular distances, conformational changes and dynamics of the origami and attached molecular components, as well as hybridization dynamics with single-molecule sensitivity, and can even be tuned to respond to external triggers. However, despite the inherent programmability of DNA origami structures, kinetic and structural uncertainties arise due to sequence (dis-)placement, environmental factors, and dye–DNA interactions. Here, we employ single-molecule FRET (smFRET) in solution and on surface-immobilized molecules to benchmark the spatial and temporal precision of DNA origami structures as kinetic rulers. We systematically investigated the binding dynamics of single-stranded DNA (ssDNA) tethers to designed docking sites along an L-shaped DNA origami scaffold. We altered the docking site position, hybridization length, and the type or presence of the fluorophores used. While DNA origami provides remarkable tunability and reproducibility at the nanoscale, we find that local effects─including fluorophore charge and the nanoenvironment around the docking site─can modulate hybridization kinetics by up to a factor of 2. These insights refine the use of dynamic DNA origami as molecular benchmarks for fluorescence-based assays and highlights design parameters that must be considered when engineering DNA-based nanodevices for precision and sensing applications.