Fig. 4: Remotely activated double-stranded DNA Nano-winch characterization and activity.

A Addition of extension oligonucleotides complementary to the connector strands between the backstop and cylinder ratchets the piston from the membrane surface. Annealing of the oligonucleotides to these connectors transitions them from single-stranded, with a persistence length of ~1 nm, to double-stranded, which is fifty times stiffer, at 50 nm. B Nano-winches tuned with 30-bp, 60-bp, and 97-bp connectors between the backstop and cylinder with corresponding distance d measurements and reference-free class averages (particle sets available in Supplementary Figs. 33, 35, and 37). C The mean distance extension of 30-bp, 60-bp, and 97-bp connectors (red dots) overlaid on the WLC model of Piston-cylinder extension (black line) enables the user to rationally define the connectors length for a required distance d. Error bars represent standard deviation. D Nano-winch was folded with a DNA hairpin tethering the backstop to the cylinder (red strands) requiring F1/2 of approximately 20pN to unzip. This hairpin serves as a benchmark to cross-validate the force applied by annealing of the extension oligonucleotides estimated by coarse-grained molecular dynamics simulation. E The distance d of tethered Piston-cylinders with the hairpin were measured before (blue) and after (red) incubation with extension oligonucleotides (particle sets available in Supplementary Figs. 39–40). F BtuB is a mechanically gated β-barrel channel occluded by a globular plug domain. The N-terminal 49 residues (red) are dislocated to form a channel and a N- terminal linker was engineered with a cysteine residue into the protein to serve as a linker for conjugation with a thiolated oligonucleotide. Addition of extension oligonucleotides retracts the piston and unfolds part of the plug domain to open a channel. G Selected traces and corresponding trace count histogram of BtuB-oligo reconstituted into planar lipid bilayers with Nano-winches after addition of 400 nM extension oligonucleotides. Closed channels (blue) transitioned to an open state (red) over two seconds after addition of 400 nM extension oligonucleotides. H Reducing the connection between BtuB and the Nano-winch allowed the plug domain to refold and transition from an open channel back to a closed state. Breaks in the traces represent preparations for thoroughly mixing the cis compartment. Arrows on the graphs indicate where the extension oligonucleotides and the DTT reducing agent solutions are added. Source data are provided as a Source Data file.