DNA Assembly of Modular Components into a Rotary Nanodevice

The bacterial flagellar motor is a rotary machine composed of functional modular components, which can perform bidirectional rotations to control the migration behavior of the bacterial cell. It resembles a two-cogwheel gear system, which consists of small and large cogwheels with cogs at the edges to regulate rotations. Such gearset models provide elegant blueprints to design and build artificial nanomachinery with desired functionalities. In this work, we demonstrate DNA assembly of a structurally well-defined nanodevice, which can carry out programmable rotations powered by DNA fuels. Our rotary nanodevice consists of three modular components, small origami ring, large origami ring, and gold nanoparticles (AuNPs). They mimic the sun gear, ring gear, and planet gears in a planetary gearset accordingly. These modular components are self-assembled in a compact manner, such that they can work cooperatively to impart bidirectional rotations. The rotary dynamics is optically recorded using fluorescence spectroscopy in real time, given the sensitive distance-dependent interactions between the tethered fluorophores and AuNPs on the rings. The experimental results are well supported by the theoretical calculations.


I. DNA origami design
Small origami ring. The small ring structure was designed using the caDNAno software. 1 It consists of a 10-helix bundle arranged in a 'honeycomb' lattice. Constant bending of the DNA bundle was achieved by the deletion and addition of base pairs along the helices. The DNA bundle was split into four parts. Helices of the innermost part (helices 3 and 9, 1 st layer) have 44 deletions, helices of the 2 nd layer (helices 2, 4 and 8) have 22 deletions, helices of the 3 rd layer (helices 1, 5 and 7) have 22 insertions and helices of the 4 th layer (helices 0 and 6) have 44 insertions. To fold the structure, the p7249 scaffold was used. Due to the small size of the origami, only 2309 bases were used. To prevent the remaining scaffold to disturb the rotation process, the unused part of the scaffold strand points towards the center of the ring. At the end of the DNA bundle, scaffold and staple strand overhangs connect each head and tail region to ensure a closed ring geometry (for more details see Supplementary Figure S1).
Large origami ring. The large ring structure was designed with caDNAno 1 software using the p8064 scaffold. The large ring consists of a 12-helix bundle arranged in a 'honeycomb' lattice.
Constant bending of the DNA bundles was achieved by the deletion and insertion of base pairs along the helices. The DNA bundle was split into six parts. Helices of the innermost part (helices 0 and 11, 1 st layer) have 90 deletions, helices of the 2 nd (helices 1 and 10) and 3 rd part (helices 2 and 9) have 54 and 18 deletions, respectively, helices of the 4 th (helices 3 and 8), 5 th (helices 4 and 7) and 6 th layer (helices 5 and 6) have 18, 54 and 90 insertions, accordingly. At the end of the DNA bundle, staple strand overhangs connect each head and tail region to ensure a closed ring geometry (for more details see Supplementary Figure S2).

II. Supplementary Figures
Supplementary Figure S1. (a) caDNAno design and strand routing for the small DNA origami ring. Blue: p7249 scaffold; light gray: core staples; red: footholds; green: ATTO550 staples; black: ATTO647N staples; purple: locking strands; pink: foothold strands with locking region at the 3' end. According to whether only the small DNA origami ring or the rotary device was assembled, addition of ATTO647 and locks (black and pink staples) had to be adjusted. (b) Cross section of the DNA bundle layout. Figure S2. (a) caDNAno design and strand routing for the large DNA origami ring. Blue: p8064 scaffold; light gray: core staples; red: footholds; green: ATTO647N staples; black: ATTO550 position and locking strand, respectively; purple: locking strands; pink: foothold strands with locking region or ATTO550 hybridization region at the 3' end. According to whether only the large DNA origami ring or the rotary device was assembled, addition of ATTO550 staples and locks (black and pink staples) had to be adjusted. (b) Cross section of the DNA bundle layout. Foothold design and blocking/releasing scheme. Each foothold comprises a unique stem region (black) connected to the DNA origami structure and a AuNP-capture sequence (green) to bind the AuNPs. Blocking strand deactivates the foothold by hybridizing with the stem and AuNP-capturing regions. Each blocking strand further comprises a toehold region (blue). Activation of the foothold is achieved by the addition of the complementary releasing strand, which completely hybridizes with the blocking strand to release it from the foothold. to calculate the averaged TEM image (see Supplementary Figure 29). Scale bar, 50 nm.

Supplementary Tables
Supplementary Table S1. Samples added to drive the rotation for real time fluorescence detection for the small rings in Figure 2. The blocking and releasing strands were prepared at a concentration of 400 μM in H2O. Generally, small volumes but high concentrations of the blocking and releasing DNA strands were used to power the AuNPs to reduce the dilution effect. The total volume increase was 4.5 μL (4.3%) after the rotation process.
Step  Fig. 3. The blocking and releasing strands were prepared at a concentration of 400 μM in H2O. To reduce the dilution effect, small volumes and high concentrations of the blocking and releasing DNA strands were used. After the rotation process, the total volume increase was 4.5 μL (4.3%).
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