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

Abstract

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 in situ structural dynamics information, inaccessible for traditional methods, constitutes an excellent tool for chaperoning the development of DNA-based technology.

Nucleosome Core Particle Disassembly and Assembly Kinetics Studied Using Single-Molecule Fluorescence

“…For a detailed description of the technique, see Fig. S5 and previous publications (34)(35)(36)(37). The technique provides observation snapshots (also known as bursts) that capture the state of an NCP complex during the time it transits the confocal spot (1-5 ms duration, depending on the trajectory of the molecule and the focus size).…”

“…Single-pair Förster resonance energy transfer (FRET) provides high-resolution information about conformations and distribution of conformations. Alternating laser excitation (ALEX) techniques will, in addition, allow the removal of signal from an incompletely labeled sample, significantly improving the reliability of the data (34)(35)(36)(37). These methods can provide equilibrium data and nonequilibrium kinetic data at picomolar concentrations, which is essential due to the strong interactions (high affinity) between histones and DNA and between histone proteins.…”

The Effect of Basepair Mismatch on DNA Strand Displacement

“…The invader can then be viewed as an input signal while the incumbent can be seen as an output signal. At this level of abstraction, DNA strand displacement can be idealized into "tinker toys" that fit together to form complex, interactive networks in the field of nanotechnology (4)(5)(6)(7) with applications in diverse areas such as biosensing (8,9), DNA construction (10)(11)(12), DNA motors (13)(14)(15)(16)(17), and DNA computation (18)(19)(20)(21)(22)(23).…”

Engineered, harnessed, and hijacked: synthetic uses for cytoskeletal systems

“…Furthermore, a DNA motor was visualized in real time taking continuous, discrete steps [84], as was recently observed for myosin-V by atomic force microscopy [93]. The visualization of single synthetic motor steps has also been achieved using single-molecule motility assays (Box 2, Figure I) [81,94]. Future designs will likely incorporate hybrid approaches that make use of both de novo and natural motor elements.…”