Fig. 3. Antibody-regulated DNA-nanoswitches. (A) Doxorubicin is released from a “Load” state upon the binding of a specific antibody (here the anti-Dig antibody) to two recognition elements (here Dig) appended at the two ends of a DNA-nanoswitch. (B and C) We designed three variants of DNA-nanoswitches with different stabilities of the “Load” states (indicated are the free energies predicted using mfold) showing different doxorubicin intercalation efficiencies. (D) Doxorubicin release at increasing concentrations of anti-Dig antibody followed by steady-state fluorescence experiments. (E) Using variant #2, doxorubicin release occurs only in the presence of the specific antibody and no significant release is observed in the presence of other non-specific antibodies (at 300 nM). Here doxorubicin loading experiments have been performed by adding the relevant antibody-regulated DNA-nanoswitch to a solution of 100 nM of doxorubicin and measuring the fluorescence anisotropy signal of doxorubicin. For releasing experiments, increasing concentrations of anti-Dig antibody were added to an equimolar solution of doxorubicin and the DNA-nanoswitch (100 nM) and the doxorubicin signal was measured via steady-state fluorescence. In both cases the excitation and emission wavelengths were fixed at 480 nm (±7 nm) and 592 (±10) nm, respectively. All experiments were conducted at pH 7.0 in 50 mM sodium phosphate, 150 mM NaCl and 10 mM MgCl₂ in a 100 μL cuvette at 25 °C. For further information about the experimental set up see the Experimental section.