We measured the increase in α upon the addition of fuel for all the hairpins in this work (Fig. ). We compared those values to the increase in α caused by the addition of EDU. As expected, for nonmodified H-0 the increase in α was minimal. Moreover, it was similar to the increase upon the addition of EDU. The same trend is observed for H-3, i.e., the molecular beacon that has the modified thymine in the loop region. For the final control compound H-5, where the carboxylic acids are protected as methyl esters, the change induced by the fuel and the waste was modest, and we attribute it to the change in the ionic strength of the solution. In contrast, H-1, H-2, and H-4 displayed a striking difference between the addition of EDC or EDU. In the case of H-4, we started with an almost open state before applying fuel (α = 0.21). The addition of fuel pushed the system towards a closed state (α = 0.66). In other words, the fuel switched the system from a condition where the open state is dominant to a new, temporary distribution where the closed state is in excess. We then explored the effect of different amounts of fuel on the folding of H-1. We chose to focus on H-1 because it has only one modified thymine, thus reducing the number of transient species. When increasing amounts of fuel are added, a greater degree of folding is achieved (Fig. ), which is mainly attributed to the higher yield of anhydride in a steady state. In contrast, when a similar amount of EDU was added, the change in α was much lower (Fig ). These combined experiments conclude that the addition of fuel increases the population of folded hairpins by activating the modified thymine. The fluorescence data provided information about the overall fraction of the folded hairpin. However, both precursor and activated states are simultaneously present, and each can form hairpins (See Scheme 2). Our system was designed such that the activated state is more likely to form a hairpin, and we were interested in understanding the contributions of each of the two possible hairpin species on the overall fraction of folded hairpins. We can rewrite eq. 1 as the sum of two terms like in the following: