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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, in combination with our imaging and analysis protocols, may be extended to assemble and inspect other supramolecular constructs in a controlled manner. Overall, by combining synthesis, characterization, and analysis, these single-molecule techniques hold the potential to advance the study of DNA nanostructures and dynamic DNA-based devices.

Dynamic wireframe DNA structures have gained significant attention in recent years, with research aimed toward using these architectures for sensing and encapsulation applications. For these assemblies to reach their full potential, however, knowledge of the rates of strand displacement and hybridization on these constructs is required. Herein, we report the use of single-molecule fluorescence methodologies to observe the reversible switching between double- and single-stranded forms of triangular wireframe DNA nanotubes. Specifically, by using fluorescently labeled DNA strands, we were able to monitor changes in intensity over time as we introduced different sequences. This allowed us to extract detailed kinetic information on the strand displacement and hybridization processes. Due to the polymeric nanotube structure, the ability to individually address each of the three sides, and the inherent polydispersity of our samples as a result of the step polymerization by which they are formed, a library of compounds could be studied independently yet simultaneously. Kinetic models relying on mono-exponential decays, multi-exponential decays, or sigmoidal behavior were adjusted to the different constructs to retrieve erasing and refilling kinetics. Correlations were made between the kinetic behavior observed, the site accessibility, the nanotube length, and the structural robustness of wireframe DNA nanostructures, including fully single-stranded analogs. Overall, our results reveal how the length, morphology, and rigidity of the DNA framework modulate the kinetics of strand displacement and hybridization as well as the overall addressability and structural stability of the structures under study.

A wide variety of approaches have become available for the fabrication of nanomaterials with increasing degrees of complexity, precision, and speed while minimizing cost. Their quantitative characterization, however, remains a challenge. Analytical methods to better inspect and validate the structure and composition of large nanoscale objects are required to optimize their applications in diverse technologies. Here, we describe single-molecule fluorescence-based strategies relying on photobleaching and multiple-color co-localization features toward the characterization of supramolecular structures. By optimizing imaging conditions, including surface passivation, excitation power, frame capture rate, fluorophore choice, buffer media, and antifading agents, we have built a robust method by which to dissect the structure of synthetic nanoscale systems. We showcase the use of our methods by retrieving key structural parameters of four DNA nanotube systems differing in their preparation strategy. Our method rapidly and accurately assesses the outcome of synthetic work building nano- and mesoscale architectures, providing a key tool for product studies in nanomaterial synthesis.

DNA nanotubes hold great potential as drug delivery vehicles and as programmable templates for the organization of materials and biomolecules. Existing methods for their construction produce assemblies that are entirely double-stranded and rigid, and thus have limited intrinsic dynamic character, or they rely on chemically modified and ligated DNA structures. Here, we report a simple and efficient synthesis of DNA nanotubes from 11 short unmodified strands, and the study of their dynamic behavior by atomic force microscopy and in situ single molecule fluorescence microscopy. This method allows the programmable introduction of DNA structural changes within the repeat units of the tubes. We generate and study fully double-stranded nanotubes, and convert them to nanotubes with one, two and three single-stranded sides, using strand displacement strategies. The nanotubes can be reversibly switched between these forms without compromising their stability and micron-scale lengths. We then site-specifically introduce DNA strands that shorten two sides of the nanotubes, while keeping the length of the third side. The nanotubes undergo bending with increased length mismatch between their sides, until the distortion is significant enough to shorten them, as measured by AFM and single-molecule fluorescence photobleaching experiments. The method presented here produces dynamic and robust nanotubes that can potentially behave as actuators, and allows their site-specific addressability while using a minimal number of component strands.

DNA nanotubes offer a high aspect ratio and rigidity, attractive attributes for the controlled assembly of hierarchically complex linear arrays. It is highly desirable to control the positioning of rungs along the backbone of the nanotubes, minimize the polydispersity in their manufacture and reduce the building costs. We report here a solid-phase synthesis methodology in which, through a cyclic scheme starting from a 'foundation rung' specifically bound to the surface, distinct rungs can be incorporated in a predetermined manner. Each rung is orthogonally addressable. Using fluorescently tagged rungs, single-molecule fluorescence studies demonstrated the robustness and structural fidelity of the constructs and confirmed the incorporation of the rungs in quantitative yield (>95%) at each step of the cycle. Prototype structures that consisted of up to 20 repeat units, about 450 nm in contour length, were constructed. Combined, the solid-phase synthesis strategy described and its visualization through single-molecule spectroscopy show good promise for the production of custom-made DNA nanotubes.

DNA nanotubes have great potential as nanoscale scaffolds for the organization of materials and the templation of nanowires and as drug delivery vehicles. Current methods for making DNA nanotubes either rely on a tile-based step-growth polymerization mechanism or use a large number of component strands and long annealing times. Step-growth polymerization gives little control over length, is sensitive to stoichiometry, and is slow to generate long products. Here, we present a design strategy for DNA nanotubes that uses an alternative, more controlled growth mechanism, while using just five unmodified component strands and a long enzymatically produced backbone. These tubes form rapidly at room temperature and have numerous, orthogonal sites available for the programmable incorporation of arrays of cargo along their length. As a proof-of-concept, cyanine dyes were organized into two distinct patterns by inclusion into these DNA nanotubes.