The collective behavior of spring-like motifs tethered to a DNA origami nanostructure.

Dynamic DNA nanotechnology relies on the integration of small switchable motifs at suitable positions of DNA nanostructures, thus enabling the manipulation of matter with nanometer spatial accuracy in a trigger-dependent fashion. Typical examples of such motifs are hairpins, whose elongation into duplexes can be used to perform long-range, translational movements. In this work, we used temperature-dependent FRET spectroscopy to determine the thermal stabilities of distinct sets of hairpins integrated into the central seam of a DNA origami structure. We then developed a hybrid spring model to describe the energy landscape of the tethered hairpins, combining the thermodynamic nearest-neighbor energy of duplex DNA with the entropic free energy of single-stranded DNA estimated using a worm-like chain approximation. We show that the organized scaffolding of multiple hairpins enhances the thermal stability of the device and that the coordinated action of the tethered motors can be used to mechanically unfold a G-quadruplex motif bound to the inner cavity of the origami structure, thus surpassing the operational capabilities of freely diffusing motors. Finally, we increased the complexity of device functionality through the insertion of two sets of parallel hairpins, resulting in four distinct states and in the reversible localization of desired molecules within the reconfigurable regions of the origami architecture.