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Engineering Approaches to Biomolecular Motors: From in vitro to in vivo Wednesday Speaker Abstracts
12
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.