Fig. 2: Initial TOF is optimized for toeholds and handholds of moderate length.

a, The experimental setup. A small concentration of template (1 nM) is combined with a larger pool of M₁L and N₃ monomers (10 nM) for a range of primary toehold and handhold lengths. The catalytic turnover of M₁L is reported by an increase in fluorescence signal. b, The example trajectories showing the concentration of reacted M₁L over time, for a range of handhold lengths and a primary toehold of 6 nt. Increasing the handhold length above 9 nt results in a decrease of the M₁L catalytic turnover due to increased product inhibition. These results illustrate how the overall reaction rate is a balance between the displacement and M_xN_y detachment from T_xy. The leak reaction in the absence of template could not be detected. Its magnitude for a monomer concentration of 100 nM is shown for M₁N₃ in Fig. 3c, and for all monomer combinations in Supplementary Fig. 12. The concentration of reacted M₁L is inferred from the fluorescence data as outlined in Supplementary Note 5. c, The initial rate of reaction per unit of template (TOF) for each primary toehold and handhold condition. An optimum is obtained for a system with a primary toehold of 6 nt and a handhold of 9 nt (6t/9h) (1.01 ± 0.03 h⁻¹) followed by condition 6t/8h (0.622 ± 0.009 h⁻¹).