Long-range movement of large mechanically interlocked DNA nanostructures

Abstract

Interlocked molecules such as catenanes and rotaxanes, connected only via mechanical bonds have the ability to perform large-scale sliding and rotational movements, making them attractive components for the construction of artificial molecular machines and motors. We here demonstrate the realization of large, rigid rotaxane structures composed of DNA origami subunits. The structures can be easily modified to carry a molecular cargo or nanoparticles. By using multiple axle modules, rotaxane constructs are realized with axle lengths of up to 355 nm and a fuel/anti-fuel mechanism is employed to switch the rotaxanes between a mobile and a fixed state. We also create extended pseudo-rotaxanes, in which origami rings can slide along supramolecular DNA filaments over several hundreds of nanometres. The rings can be actively moved and tracked using atomic force microscopy.

Figure 1. Fabrication of DNA origami rotaxanes.

(a) A rotaxane is formed from an open ring (R1) with a flexible hinge and a dumbbell-shaped DNA origami structure (D1), which were prepared separately. The hinge of the ring consists of a series of strand crossovers into which additional thymines are inserted to provide higher flexibility. Ring and axis subunits are first connected and positioned with respect to each other using 18 nucleotide long, complementary sticky ends 33 nm away from the centre of the axis (blue regions). The ring is then closed around the dumbbell axis using closing strands (red), followed by the addition of release strands that separate dumbbell from ring via toehold-mediated strand displacement. (b) 3D models and corresponding averaged TEM images of the ring and dumbbell structure. Also shown are exemplary single-particle images. (c) TEM images of the completely assembled rotaxanes (R1D1). (d) 3D models, averaged and single-particle TEM images of R2 and D2, subunits of an alternative rotaxane design containing bent structural elements. The TEM images of the ring structure correspond to the closed (top) and open (bottom) configurations. (e) 3D representation and TEM images of the fully assembled R2D2 rotaxane. Origami models are generated using CanDo. Scale bar, 50 nm. See also Supplementary Figs 1–18 for additional TEM images, and Supplementary Fig. 19A for an AFM image of R1D1 rotaxanes. 3D, three dimensional.

Figure 2. Investigation of ring mobility using TEM.

(a) Images of the R1D1 rotaxane before (left) and after (right) the addition of release strands. (b) Histogram of the distances of the rings from the mean initial attachment position before and after release. The distributions can be fitted by a sum of two Gaussians. A broadening of the main peak and a larger fraction of rings in the second peak is observed after release. (c) Images of R1D1 structures labelled with 10 nm gold nanoparticles, which serve as markers for highlighting the rotational orientation of the components relative to each other. Images were taken without (left) and with (right) release strands. (d) Ratio of particles found in the second peak of the Histogram shown in b. The rings displaced from their starting position indicate translational mobility of the construct. (e) Analysis of the rotation of R1D1. Gold particle markers were mostly found in their cis starting configuration before the release of the rings. After release, a considerably larger fraction is found in the trans position. (f) R2D2 TEM images before (left) and after (right) release. (g) Particles are classified into two states—one in which R2 is in the middle of the axis, and another in which R2 sits on top of the stopper bars. After release of the rings, an increase of the fraction of rings in the remote ring position is observed. (h) Rotational movement of the rings was detected by comparing the number of particles with the marker block lying above or under the axis (initial attachment position) with those where the marker is found off-axis. Upon release the radial orientation of about 30% of all rings changed. Scale bar, 100 nm.

Figure 3. AuNP functionalization and bulk fluorescence experiments.

(a) R1D1 rotaxanes were modified with 10, 20 and 30 nm gold nanoparticles (from left to right), demonstrating the potential use of the rotaxanes as functional nanomechanical devices. Rotaxanes before addition of the release strands are shown in the first row, after addition of the release strand set in the second row. Scale bar, 100 nm. See also Supplementary Figs 15–19 for additional TEM and AFM images. (b) Scheme of the fuel/anitfuel mechanism used to switch the connection between R1 and D1. (c) Bulk spectroscopy experiments with R1D1 rotaxanes labelled with Cy3/Cy5 FRET pairs. Fuel and anti-fuel strands were added repeatedly (marked as blue and red triangles) with increasing concentrations (40 nM fuel, 40 nM anti-fuel, 160 nM fuel, 160 nM anti-fuel and 640 nM fuel). A decrease in the intensity of the FRET signal was observed upon release, as well as a recovery after the reattachment of the ring due to the displacement of the release strands. A moving average filter was applied and the intensities were normalized using the acceptor signal.

Figure 4. Elongated rotaxane structures composed of multiple components.

(a) 3D model, averaged and single TEM images of a ‘stopper module' (rotaxane axis with integrated stopper). (b) Schematic representation of a rotaxane chain created by polymerization of stopper modules with attached macrocycles. The corresponding TEM image shows the chain after the release of the macrocycles. The initial attachment positions are highlighted in the red frames. (c) Extended rotaxane constructed from two stopper modules separated by a 246-nm-long axis module. The ring was initially attached close to one of the bumper pieces. TEM images taken after the release of the rings demonstrate sliding mobility along the axis. Scale bars, 100 nm. 3D, three dimensional.

Figure 5. Pseudorotaxane filaments with multiple rings.

(a) 3D model of filaments composed of polymerized axis modules with R1 rings attached. (b) TEM image of a pseudorotaxane filament. Rings are still attached at their starting position. Scale bar, 500 nm. (c) Fast scan AFM (scan rate 19.5 Hz, 0.076 frames s⁻¹) snapshots of a pseudorotaxane electrostatically immobilized on the mica substrate (the full image sequence is available as Supplementary Movie 1). The macrocycle is pushed along the filament by the AFM tip in the slow scanning direction. An overview AFM image of a pseudorotaxane sample is shown in Supplementary Fig. 19B. Scale bar, 100 nm. (d) Y-position or the ring for all images shown in Supplementary Movie 1. The slow scan direction is indicated by the triangles. 3D, three dimensional.