Single-Molecule Nucleic Acid Detection with a Reconfigurable Rotating DNA Origami Nanodevice

Dynamic DNA nanodevices, inspired by macroscopic machines, provide a versatile platform for constructing nanoscale mechanisms with specialized functionalities. In this study, we developed a DNA origami-based rotating nanodevice for continuous nucleic acid sensing, achieving a detection limit in the low nanomolar range. The nanodevice exhibits reversibility, enabling multiple rounds of target detection through toehold-mediated strand displacement. This regeneration capability was demonstrated at both the ensemble and single-molecule levels, with the latter offering high-resolution insights into dynamic conformational changes. By exploiting the spatial precision and programmability of DNA origami, we designed two distinct systems: a single-mode device that generates a Förster Resonance Energy Transfer (FRET) signal, and a dual-mode device capable of producing either a FRET or quenched signal, depending on the target detected, and enabling multiplexed measurements. Using these setups both at the ensemble and single-molecule level, we systematically explored various parameters to enhance binding efficiency, providing insights for optimizing and fine-tuning future designs.