| Summary: | This thesis describes methods for constructing nanopatterned surfaces to array DNA. These surfaces enable direct observation of heretofore-unseen single-molecule reactions, eliminating bulk effects and enabling scientists to examine DNA mismatch repair and replication, including the first direct visualization of proteins binding to a target mismatch. This also facilitates directed self-organization of nanoscale features on a patterned substrate using DNA as an assembly tool.
To make techniques for single-molecule visualization of biological processes more accessible, we have developed a novel technology called "DNA curtains," in which a combination of fluid lipid bilayers, nanofabricated barriers to lipid diffusion, and hydrodynamic flow can organize lipid-tethered DNA molecules into dened patterns on the surface of a microfluidic sample chamber.
Using DNA curtains, aligned DNA molecules can be visualized by total internal reflection fluorescence microscopy, allowing simultaneous observation of hundreds of individual molecules within a field-of-view. Ultimately, this results in a 100X improvement in experimental throughput, and a corresponding increase in statistically signicant amounts of data.
We also demonstrate site-specific labeling of DNA using DNA analogues, such as peptide nucleic acid (PNA), locked nucleic acid (LNA), and techniques such as nick-translation. Through PNA invasion, labeled DNA was self-assembled in arrays on surfaces and tagged with gold nanoparticles. In this work, DNA formed a template to self-assemble a nanoparticle in between nanoimprinted AuPd dots. Surface-based self-assembly methods offer potential for DNA employment in bottom-up construction of nanoscale arrays. This offers further proof that DNA can be useful in directed self-assembly of nanoscale architectures.
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