- While numerous DNA-based molecular machines have been developed in recent years, high operational yield and speed remain a major challenge. To understand the reasons for the limited performance, and to find rational solutions, we applied single-molecule fluorescence techniques and conducted a detailed study of the reactions involved in the operation of a model system, comprised of a bipedal DNA walker that strides on a DNA origami track powered by interactions with fuel and anti-fuel strands. Analysis of the kinetic profiles of the leg-lifting reactions indicates a pseudo first-order anti-fuel binding mechanism leading to a rapid and complete leg-lifting, indicating that the fuel-removal reaction is not responsible for the 1% operational yield observed after six steps. Analysis of the leg-placing reactions showed that although increased concentrations of fuel increase the reaction rate, they decrease the yield by consecutively binding the motor and leading to an undesirable trapped state. Recognizing this, we designed asymmetrical hairpin fuels that by regulating the reaction hierarchy avoid consecutive binding. Motors operating with the improved fuels show 74% yield after twelve consecutive reactions, a dramatic increase over the 1% observed for motors operating with non-hairpin fuels. This work demonstrate that studying the mechanisms of the reactions involved in the operation of DNA-based molecular machines using single-molecule fluorescence can facilitate rationally designed improvements that increase yield and speed, and promote the applicability of DNA-based machines.