Fig. 1: Setup and measurement principle.

A A dual-beam optical trap setup is used to measure force-dependent hairpin opening promoted by a trigger strand in a toehold-mediated strand displacement (TMSD) process. DNA (or RNA) toehold hairpin molecules are tethered between two beads using 545 base pair (bp) long DNA handles as indicated. B Microfluidics setup used for TMSD measurements. The hybridized constructs with attached beads are incubated and then pumped into the microfluidics device. The bead mix is introduced into the beads channel together with the pure buffer channel (20 mM MgCl₂, 300 mM KCl, 50 mM HEPES) and trigger channel with the same buffer containing the 100 nM trigger strand. The pure buffer and the buffer containing the trigger strand are separated by laminar flow. The bead pairs are trapped within the beads channel and then transferred to the buffer channel, where two beads are brought into close proximity to form a tether between the DNA handle and anti-digoxigenin beads. Once the tether is formed, pulling and passive mode experiments are performed in the buffer channel, and subsequently, TMSD measurements are executed in the trigger channel.