Figure 2.

(a) Upstream pH-dependent DNA circuit. (b) Triplex formation in the pH-dependent substrate complex, studied by incorporating a pH-insensitive FRET pair at the ends of the clamp-like triplex-forming strand (left) and measuring the fluorescence signal at different pHs (right). Stable triplex formation is observed only at pHs below 7.0. (c) The pH-dependent triplex complex in the substrate inhibits strand displacement reaction upon C addition (pH 5.0, left). At basic pHs the destabilization of Hoogsteen interactions leads to substrate activation, which allows strand displacement reaction in the presence of catalyst strand (C) (pH 8.0, right). (d) The pH dependence of the catalyst/substrate reaction can be finely controlled at different pHs. Here, strand displacement reaction is followed by fluorescence measurements in a solution containing the pH-dependent substrate (10 nM), the fuel strand (F) (20 nM), and an external, optically labeled reporter (30 nM) (see experimental details in the SI) that stoichiometrically reacts with the released D to give a fluorescence signal. The catalyst was added at a high concentration (30 nM) to better highlight the pH dependence of the circuit. All experiments were performed in a TAE 1x buffer + 15 mM MgCl₂ at 25 °C with the pH adjusted using small aliquots of HCl (1 M) and NaOH (1 M). Error bars here and in the following figures represent the average and standard deviations (average RSD = 6%) of three independent measurements.