First, we tested the kinetics of the nucleotide itself, i.e., without incorporating it in a DNA hairpin (modified thymine, Fig. ). The addition of 4 mM EDC to 8 mM of the modified thymine in 200 mM MES at pH 6 yielded the formation of the corresponding anhydride product, as evidenced by HPLC. As the reaction progressed, both the concentrations of fuel and anhydride decayed (Fig. ). The kinetics of the reaction cycle could be accurately captured with a kinetic model (See Supporting Note 1), which allowed us to calculate the half-life of the anhydride product (t1/2) to be 23 sec.