Fig. 2. (A) TATA-binding protein (TBP)-regulated DNA-nanoswitches coexist in two mutually exclusive stem-loop conformations (i.e. “Load” and “Release”). The binding of TBP to the latter conformation pushes this conformational equilibrium towards the “Release” state, thus triggering the release of the drug (i.e. doxorubicin) intercalated in the “Load” state. (B) We engineered three variants of DNA-nanoswitches with increasing stabilities of the “Load” state (indicated are the free energies predicted using mfold). (C) All three variants show a similar affinity for doxorubicin. (D) Drug release efficiency upon TBP addition for the three DNA-nanoswitch variants. (E) Variant #1 shows good loading efficiency while a control DNA-nanoswitch which contains the TBP recognition element but lacks the GC-rich stem portion (control #1) shows a much lower loading efficiency towards doxorubicin intercalation, especially when previously bound to TBP. Variant #1 also shows high specificity against other, non-specific proteins (at 350 nM). Doxorubicin load/release experiments have been performed by measuring doxorubicin fluorescence anisotropy signals. The excitation and emission wavelengths were fixed at 480 nm (±7 nm) and 592 (±10) nm, respectively. For loading experiments, increasing concentrations of the relevant DNA-nanoswitch were added to a solution of 100 nM of doxorubicin. For releasing experiments, increasing concentrations of TBP were added to an equimolar solution of doxorubicin and the DNA-nanoswitch (100 nM). (E) Loading results were also obtained with an equimolar solution of doxorubicin and the DNA-nanoswitch or control (100 nM). 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.
