Extended Data Fig. 7: Calculated pulling and re-opening rates.

Box-plot parameters in (a,b) Edges: 25^th and 75^th percentiles. Lines: 50^th percentile. Whisker size: 1.5× the IQR or the largest/ smallest point, whichever is closer to the centre. Black dots: outliers. (a) Calculated pulling rates relative to the mean of the nicked-nanoengine at 37 °C under 16 pN applied force between the polymerase attachment point and the terminator sequence on the dsDNA-t for each design at 23 °C and 37 °C. The only notable difference between the two temperatures was observed for the nicked-nanoengine and the nanoengine, where decreasing the number of base pairs in the flexure substantially increased the rate at which the leaf-spring was able to close (n = 10 for all boxes; nNE_23: min = −1.12, max = −0.53, Q2 = −0.74; nNE_37: mi= -1.54 max = −0.62, median = −0.97; nNE_NS_23: min = −1.72, max = −0.35, median = −1.09; nNE_NS_37: min = −1.79, max = −0.47, median = −1.10; NE_23: min = −0.96, max = −0.40, median = −0.57; NE_37: min = −1.52, max = −0.13, median = −0.99; NE_NS_23: min = −1.60, max = −0.63, median = −1.13; NE_NS_37: min = −1.67, max = −0.60, median = −0.91). (b) Calculated re-opening rates after release of the 16 pN force. At elevated temperature, both the average rate and the variance for the structures where secondary structures can form in the flexure are increased. If no structure was permitted in the flexure the average rate decreased and no trend in variance for structures was observed (nNE_23: min=0.02, max=1.41, median = 0.81; nNE_37: min=0.26, max=1.65, median = 0.93; nNE_NS_23: min=1.39, max=2.45, median = 1.90; nNE_NS_37: min=0.80, max=2.63, median = 1.52; NE_23: min=0.27, max=1.11, median = 0.67; NE_37: min=0.09, max=1.61, median = 0.87; NE_NS_23: min=1.29, max=2.47, median = 1.53; NE_NS_37: min=1.27, max=2.36, median = 1.58; NTS_23: min = −0.29, max=1.24, median = 0.39; NTS_37: min = −0.17, max=1.52, median = 0.66; NTS_NS_23: min=0.52, max=2.30, median = 1.21; NTS_NS_37: min=0.45, max=1.89, median = 1.15). (c) Histogram of the distance between the polymerase attachment site and the start of the T7 promoter on the dsDNA-t. The diameter of T7RNAP is ~8.6 nm. The frequency with which the nicked-nanoengine construct (orange, nNE) dwells within this ideal spacing bubble is higher than when the dsDNA-t is only attached next to T7RNAP (blue, nNE_LB). (d) oxView image showing the relative size of T7RNAP (orange; without HT, PDB ID: 3E2E)⁴⁵ compared with the mean structure of the nicked-nanoengine (red: chloroalkane-modified overhang, blue: dsDNA-t). End-to-end length of dsDNA-t: 37.5 nm. Largest dimension of T7RNAP: ~8.6 nm. Glossary - nNE_23: nicked-nanoengine at 23 °C, nNE_37: nicked-nanoengine at 37 °C, nNE_NS_23: non-structured nicked-nanoengine at 23 °C, nNE_NS_37: non-structured nicked-nanoengine at 37 °C, NE_23: nanoengine at 23 °C, NE_37: nanoengine at 37 °C, NE_NS_23: non-structured nanoengine at 23 °C, NE_NS_37: non-structured nanoengine at 37 °C, NTS_23: origami lacking the dsDNA-t at 23 °C, NTS_37: origami lacking the dsDNA-t 37 °C, NTS_NS_23: non-structured origami lacking the dsDNA-t at 23 °C, NTS_NS_37: non-structured origami lacking the dsDNA-t at 37 °C.