Conformational Dynamics of DNA Hairpins at Millisecond Resolution Obtained from Analysis of Single-Molecule FRET Histograms

Here we provide high resolution study of DNA hairpin dynamics achieved by probability distribution analysis (PDA) of diffusion-based single-molecule Förster resonance energy transfer (sm-FRET) histograms. The opening and closing rates of three hairpins both free and attached to DNA origami were determined. The agreement with rates previously obtained using the total internal reflection (TIRF) technique and between free hairpins and hairpins attached to origami validated the PDA and demonstrated that the origami had no influence on the hairpin dynamics. From comparison of rates of four DNA hairpins, differing only in stem sequence, and from comparison with rates calculated using nearest-neighbor method and standard transition state theory, we conclude that the unfolding reaction resembles that of melting of DNA duplex with a corresponding sequence and that the folding reaction depends on counterion concentration and not on stem sequence. Our validation and demonstration of the PDA method will encourage its implementation in future high-resolution dynamic studies of freely diffusing biomolecules

Rational Design of DNA Motors: Fuel Optimization through Single-Molecule Fluorescence

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 antifuel strands. Analysis of the kinetic profiles of the leg-lifting reactions indicates a pseudo-first-order antifuel 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 12 consecutive reactions, a dramatic increase over the 1% observed for motors operating with nonhairpin fuels. This work demonstrates 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

Studying the Structural Dynamics of Bipedal DNA Motors with Single-Molecule Fluorescence Spectroscopy

We present a test case example of a detailed single-molecule fluorescence study of one of the most sophisticated and complex DNA devices introduced to date, a recently published autonomous bipedal DNA motor. We used the diffusion-based single-molecule Förster resonance energy transfer technique, coupled to alternating laser excitation (sm-FRET–ALEX), to monitor the motor assembly and operation. The study included verification of the formation of the correct structures, and of the correct motor operation, determination of the formation and stepping reaction yields, and identification of side products. Finally, the mechanisms of the motor assembly and operation were elucidated by measuring the reaction kinetics profile of track-walker binding and of lifting of the walker’s leg upon fuel addition. The profiles revealed a fast phase, in which about half of the reaction was completed, followed by a slow phase which adds somewhat to the yield, reflecting the incomplete motor assembly and operation identified in the equilibrium experiments. Although further study is needed to fully understand the reasons for the incomplete assembly and operation, this work demonstrates that single-molecule fluorescence, based on its ability to provide detailed <i>in situ</i> structural dynamics information, inaccessible for traditional methods, constitutes an excellent tool for chaperoning the development of DNA-based technology