Dynamically elongated associative toehold for tuning DNA circuit kinetics and thermodynamics

Associative toehold is a powerful concept enabling efficient combinatorial computation in DNA circuit. A longer association length boosts circuit kinetics and equilibrium signal but results in higher leak rate. We reconcile this trade-off by using a hairpin lock design to dynamically elongate the effective associative toehold length in response to the input target. Design guidelines were established to achieve robust elongation without incurring additional leakages. Three hairpin initiators with different combinations of elongated associative toehold (4 → 6 nt, 5 → 8 nt and 6 → 9 nt) were shortlisted from the design framework for further discussion. The circuit performance improved in terms of reaction kinetics, equilibrium signal generated and limit of detection. Overall, the elongated associative toehold served as a built-in function to stabilize and favour the forward, desired reaction when triggered.