One of the key innovations in biomolecular science over the past 50 years has been the ability to directly observe and measure single molecules. Single molecule experiments have revealed a wealth of information that has not been accessible to ensemble measurements, and have helped shed light on biological processes as diverse as RNA transcription, DNA replication, protein synthesis, membrane pore channel modulation, motor protein translocation, and protein folding. Among the tools available, atomic force microscopy (AFM) is uniquely powerful in its ability to directly image and manipulate single biomolecules and biomolecular complexes with nanometer precision. A large research effort has been focused on improved instrumentation to expand the capabilities of AFM, such as high-resolution non-contact mode, force mapping, and high-speed imaging. In contrast, there has been a lack of similar progress in the development of improved surface chemistry for immobilizing single biomolecules, a key step for in situ imaging. Indeed the standard approach for AFM imaging of nucleic acids, adsorption on mica, has been in use for over twenty years and has seen relatively few changes. There is a potential for a more dynamic surface to provide active control over the molecule-surface interactions, in turn enabling more complex measurements of biomolecules in their native states. This would help to address a longstanding challenge in AFM imaging: molecules adsorbed on a static surface for imaging are unlikely to retain their native properties.

In this dissertation I describe a number of novel surface chemistry tools that I have developed toward this end, and I demonstrate their use in AFM studies of single DNA molecules that would be impossible with previously existing substrates. Chapter 1 provides the background and historical context of the imaging and manipulation of DNA with scanning probe microscopy, with a special focus on the role of the surfaces being used. In Chapter 2 I describe dynamic, switchable surface chemistry that overcomes the challenge inherent in using a static surface for AFM imaging, and I demonstrate its use in studying the hybridization kinetics of single DNA molecules at the solid/liquid interface. In Chapter 3 I present a method for covalently and sequence-specifically tethering long DNA molecules to the switchable surface for AFM imaging or force spectroscopy measurements. Then, in Chapter 4, I describe a strategy for measuring and suppressing the oxidative damage that occurs during copper-catalyzed bioconjugation reactions such as the surface-tethering reaction that was described in the previous chapter. In Chapter 5 I demonstrate how the switchable surface chemistry can be used to study the self-assembly kinetics of a synthetic DNA tile. And finally, in Chapter 6 I summarize the work presented in this dissertation, and I also discuss its potential utility in studying other biomolecular processes, including transcription, replication, and DNA packaging and repair.