Where α 3 and α 2 are the folding ratio of the acid and the anhydride state, respectively, and y is the steady-state yield of the reaction cycle. To determine those two α values, we thus have to determine the steady-state concentration of anhydride which we measured by HPLC. As a precursor, we used a short oligonucleotide equipped with our modified thymine and we applied different amounts of fuel to induce different steady-state levels. We quenched these samples after 1 min with a previously established quenching method . In brief, we added EDC to a solution of unlabeled oligo (1 µM) in MES buffer at pH 5.5 and after 1 min, 360 mM benzylamine was added to the sample. We found that two new peaks appeared in the chromatogram, which we attributed to the products of the reaction of the anhydride with benzylamine (see Fig. ). By comparing the areas of the peaks, we calculated the yield of anhydride in a steady-state which we used in our kinetic model to fit the fluorescence data as a function of time (See Supporting Note 1 and Fig. ). From the model, we derived a value for 𝛼 2 , which we estimated to be 0.95. In other words, 95% of the activated oligos are in their folded state, whereas only 50% of the nonactivated oligos are in their folded state. The obtained data creates a clearer picture of the energy landscape of our dynamic hairpins (Scheme 2). In the energy landscapes, four states exist: a DNA can either be folded or unfolded, and it can either be in an activated or deactivated state. Pathways exist between each of these states, however, only the eight dominant ones are shown. These are: an unactivated strand can fold and unfold, and an activated strand can fold and unfold (pathways along the folding coordinate). Along the reaction coordinate, a folded or unfolded strand can become activated, and a folded and unfolded activated strand can become deactivated. While it is technically possible that a folded oligomer simultaneously deactivates and unfolds, it is unrealistic and complicates the landscape. We found that the activated state has a half-life of 23 seconds (vide supra). We also found that its degree of folding is in the range of 95%. Thus, in its lifetime, the activated oligomer will spend roughly 95% of its time in the folded state. However, the half-life for the folding of a DNA strand such as the one used in our experiments is reported to be in the range of milliseconds. That means that even though an activated state will spend roughly 95% of its time in a folded state, it will unfold and refold 100s of times in its lifetime. Interestingly, the anhydride state was significantly more folded compared to the methyl ester control compound (activated H-1 vs H-5, α = 0.95 vs α = 0.71, under the same conditions) even though both species lack the negative charges. A possible explanation is that the formation of the anhydride increases the overall planarity of the functional group thus facilitating intercalation and stacking into the DNA helix. We also observed a modest increase in the activation rate constant (k1 in the kinetic model, see Supporting Table ) for the modified thymine when embedded in the oligo, in agreement with the previous report from our group that a densely charged local environment can affect the reactivity of dicarboxylic acid. In the experiments above, we changed the fraction of folding of hairpins in a steady-state by the addition of fuel. The addition of a fuel-scavenger allows to reversibly control the steady-state by rapidly depleting all fuel. Next, we introduce the idea of a permanently present fuel-scavenger that consumes most fuel and thus controls the lifetime of the folded population. Such an approach opens the door to self-regulating DNA nanodevices with time-dependent behavior. When fuel is added to such a competition experiment, the majority of the fuel is consumed by the scavenger, and the conformation change of the hairpin is only temporary (Fig. ). A simulation by our kinetic model demonstrated the validity of this approach and showed that lifetimes of the folded population could be tuned from few minutes to hours depending on the amount of scavenger that is added (Fig. ). We carried out these experiments using 0.2 µM of H-1 in MES buffer containing variable concentrations of Ac-D-OH as a scavenger at 30°C. When 20 mM EDC is added to the system with 20 mM of scavenger, we observed a marked drop in the fluorescence, followed by a rapid recovery of the signal (Fig. ). The kinetic profile of the fluorescence recovery correlates with the experimentally determined consumption of the EDC (Fig. ). Moreover, the fluorescence signal is accurately modeled by our kinetic model and correlates with the predicted anhydride concentration (Fig. ). The concentration of scavenger could be used to tune the half-life of recovery of the folded states from 4 min to over 13 min in agreement with the predicted half-life of recovery (Fig. ). The ability of the system to autonomously stop allows operating multiple cycles with minimal intervention, which we demonstrate by the addition of five batches of 20 mM of EDC, which resulted in a transient change in the population of folded and unfolded molecules each time (fig. ). In contrast, most of the DNA dynamic system reported so far requires either extensive manipulation, such as buffer exchange, or at least two different stimuli to move between the different states.