Fig. 1

Principle and optimization of the antibody-controlled DNA circuit. a To engineer a strand displacement reaction controlled by antibody we have split the input strand responsible for the toehold displacement reaction into two portions (red and green) and flanked them with two complementary portions (orange) and with a 12-nt poly-T tail (black). At the two ends of such tails we have conjugated a molecule (antigen) responsible for antibody recognition. The binding of the antibody to the two antigen-conjugated split-inputs co-localizes them and induces stem formation and reconstitution of the functional input strand. b As a first test-bed we used Digoxigenin (Dig) as the antigen. We tested different stem lengths with various predicted ΔG values. c Fluorescent kinetic traces of strand displacement reactions observed by adding one of the Dig-conjugated split-inputs (60 nM) into a solution containing the other Dig-conjugated split-input (60 nM) and the optically-labeled target duplex (30 nM). d The same experiment described in c but in the presence of the specific anti-Dig antibody (300 nM). e Ratio between the end-point fluorescent values obtained in the presence and absence of the anti-Dig antibody vs. the predicted ΔG values of the different split-inputs employed. f Stem formation experiment performed by adding increasing concentrations of the Dig-conjugated split-input strand modified with a quencher (BHQ) to a solution containing the other split-input strand modified with a fluorophore (FAM) (60 nM) in the absence (gray curve) and presence (red curve) of the anti-Dig antibody (300 nM). The strand displacement experiments in this figure were performed using a target duplex labeled with a FRET couple (Cy3-Cy5) so that the displacement reaction can be easily followed through increase of the fluorescence signal. All experiments were performed in 50 mM Na₂HPO₄, 150 mM NaCl at pH 7.0, 25 °C. In all sketches, the 3′ ends are marked with an arrow. The experimental values represent averages of three separate measurements and the error bars reflect the standard deviations.