Nanolithographic Control of Double-Stranded DNA at the Single-Molecule Level

• Main Author: Fazio, Teresa
• Language: English
• Published: 2012
• Subjects: Materials science; Nanolithography; Nanobiotechnology; DNA > Analysis; Nanostructured materials
• Online Access: https://doi.org/10.7916/D8SQ9C1T

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.