Conspectus
DNA nanotechnology relies on the molecular recognition properties
of DNA to produce complex architectures through self-assembly. The
resulting DNA nanostructures allow scientists to organize functional
materials at the nanoscale and have therefore found applications in
many domains of materials science over the past several years. These
scaffolds have been used to position proteins, nanoparticles, carbon
nanotubes, and other nanomaterials with high spatial resolution. In
addition to their remarkable performance as frameworks for other species,
DNA constructs also possess interesting dynamic properties, which
have led to their use in logic circuits, drug delivery vehicles, and
molecular walkers.
Although DNA nanostructures have become increasingly
complex, the
development of tools to study them has lagged. Currently, gel electrophoresis,
dynamic light scattering, and ensemble fluorescence measurements are
widely used to characterize DNA-based assemblies. Unfortunately, ensemble
averaging in these methods obscures malformed structures and may mask
properties associated with structure, length, and shape in polydisperse
samples. While atomic force microscopy allows for the determination
of morphology at the single-molecule level, this technique cannot
typically be used to assess the dynamic properties of these constructs.
To analyze the function of DNA-based devices such as molecular motors
and reconfigurable nanostructures in real time, new single-molecule
techniques are required.
This Account details the work from
our laboratories toward developing
single-molecule fluorescence (SMF) methodologies for the structural
and dynamic characterization of wireframe DNA nanostructures, one
at a time. The methods described herein provide us with two separate
yet related sets of information: First, we can statically examine
the nanostructures one by one to assess their robustness, structural
fidelity, and morphology. This is primarily done using two-color stepwise
photobleaching, wherein we can examine the subunit stoichiometry of
our assemblies before and after various perturbations to the structures.
For example, we can introduce length mismatches to cause the nanotube
to bend or perform strand displacement reactions to generate single-stranded,
flexible analogues of our materials. Second, due to the unmatched
spatiotemporal resolution of SMF techniques, we can study the dynamic
character of these assemblies by implementing structural changes to
the nanotube and monitoring them in real time.
With this structural
and dynamic information in hand, our groups
have additionally developed new tools for the improved construction
of DNA nanotubes, inspired by solid-phase DNA synthesis. By assembling
the nanotubes in a stepwise manner, highly monodisperse nanostructures
of any desired length can be made without a template strand. In this
way, unique building blocks can also be added sequence-specifically,
allowing for the production of user-defined scaffolds to organize
nanoscale materials in three dimensions. This method,...
