Engineering Approaches to Biomolecular Motors

Engineering Approaches to Biomolecular Motors: From in vitro to in vivo Wednesday Speaker Abstracts

Rapid Unbiased Transport by a DNA Walker Jieming Li 1 , Alexander Johnson-Buck 4 , Yuhe Renee Yang 2,3 , Hao Yan 2,3 , Nils Walter 1 . 1 University of Michigan, Ann Arbor, MI, USA, 2 Arizona State University, Tempe, AZ, USA, 3 Arizona State University, Tempe, AZ, USA, 4 Dana-Farber Cancer Institute, Boston, MA, USA. Ever since the step-by-step movement of biomolecular motors such as myosin and kinesin super families was mechanistically characterized, attempts have been made to mimic their dynamic behavior in the form of synthetic molecular walkers. Several DNA-based molecular walkers have been synthesized, motivated by the long-term goal of controlling molecular transport processes with the programmability and structural robustness. Previous studies show that DNA walkers can walk directionally along a track upon sequential addition of a DNA strand as chemical “fuel”. Despite this progress, the DNA walkers reported so far have been constrained by slow translocation rates, typically on the order of a few nm/min. By comparison, natural protein motors have translocation rates of ~1μm/s under saturating ATP conditions. It is desirable to reduce this gap if synthetic DNA walkers can serve as useful agents of molecular transport. Slow catalytic steps or slow release of cleavage products limits the translocation rate of many DNA walkers. In contrast, the displacement of one strand in a DNA duplex by another can be catalyzed by the nucleation of short single-stranded overhangs, or “toeholds”, a process that can be very rapid when the reagents are present at high concentration. Here we report the design and single-molecule fluorescence resonance energy transfer characterization of a novel class of reversible DNA transporters that utilizes strand displacement mediated by toehold exchange. The fastest rate constant of stepping approaches 1 s -1 , which is ~100-fold higher than typical DNA-based transporters. We present evidence that the walking occurs by a rapid branch migration step followed by slower dissociation and rebinding of toehold sequences. While branch migration is rapid and may be treated as a rapid equilibrium process, the rate constant of stepping between adjacent track sites is dependent on the length of the associated toeholds.

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