Detection of Stochastic and Heterogeneous Behaviors in DNA Nanodevices by Super-Resolution Fluorescence Microscopy.

Decades of advances in structural biology have inspired efforts to emulate and expand upon the functional capabilities of natural nanomachines. Recently, DNA nanotechnology has emerged as a promising route to realizing this ambition. With the help of advanced approaches like DNA origami, the structural and functional repertoire of this biopolymer has expanded far beyond the humble double helix, permitting designs of nearly arbitrary shape and growing sophistication. The increased complexity and multifunctionality of synthetic devices constructed of DNA introduces more opportunities for erroneous assembly and otherwise heterogeneous performance. However, in most cases, functional characterization of DNA nanodevices is carried out in bulk or by techniques like atomic force microscopy (AFM) and transmission electron microscopy (TEM). Such approaches, while undeniably powerful, do not provide full access to the coupling between spatial, temporal, and chemical properties of individual nanodevices, rendering it difficult to understand how reproducibly they perform. Recent progress in single-molecule fluorescence detection and super-resolution microscopy make it possible to address this gap in understanding. In this dissertation, I report the development and application of several such techniques to elucidate spatiotemporal properties of single DNA nanodevices. First, a single-particle tracking assay is developed to characterize the complex movement of a synthetic DNA-based walker on prescriptive landscapes. The assay, combined with numerical modeling and population-level AFM results, confirms the designed path-following behavior of individual spiders and provides insights into their stochastic and heterogeneous behavior. Second, I develop a two-color version of the super-resolution technique PAINT (points accumulation for imaging in nanoscale topography) to interrogate the spatiotemporal dependence of oligonucleotide hybridization to arrays of dense targets on individual DNA origami, revealing surprisingly variable and persistent spatial patterns of binding kinetics. Finally, I examine the kinetics of oligonucleotide hybridization reactions on single DNA origami arrays bearing different densities of targets, showing evidence of at least two mechanisms by which hybridization kinetics differ from those in solution. Together, these results provide new insights into the degree of stochasticity and heterogeneity in the performance of DNA nanodevices and furnish new tools with which to design more sophisticated devices in the future.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/97807/1/alebuck_1.pd