New Methods of Deposition for Atomic Force Microscopy Studies of Biomolecules

Atomic force microscopy (AFM) can directly visualize single protein and DNA molecules and protein-DNA complexes. To image single molecules with AFM, the sample must first be deposited on an atomically flat mica surface. Proteins generally adhere well to mica, but DNA has poor adhesion due to electrostatic repulsion between the phosphate backbone and the negatively charged mica surface. There are two methods currently used to deposit DNA on mica. The first method utilizes divalent cations like magnesium to act as a salt bridge to bind DNA to the surface. The second method chemically modifies the mica surface to be positively charged so that DNA can bind electrostatically. Each method has its own limitations. Depositions using divalent cation are limited to buffers with a low concentration of monovalent cations and therefore cannot be used with physiological salt conditions. Chemical modification is not limited by salt concentration but can introduce surface effects that can affect the conformation of the molecules on the surface. Currently there is no method to deposit DNA on mica in higher salt conditions without modifying the surface. In this work, I developed a new deposition method for AFM imaging of single protein and DNA molecules in air. In chapter 2, I describe the fabrication of a silicon-based device to transfer DNA samples in physiological salt conditions from shallow microwells to untreated mica for AFM imaging. I fabricated and characterized several different silicon-based devices and tested their ability to transfer DNA samples in high salt conditions to mica. Using polymer chain statistics, I determined that DNA deposited onto mica with this method adopted a 2D conformation. Additionally, I applied this deposition method to protein-DNA complexes in physiological salt conditions. In chapter 3, I used the transfer deposition method for Thermus aquaticus (Taq) MutS-mismatched DNA and RNA polymerase-promoter DNA experiments. Using volume, position, and bend angle analysis, I found that AFM results obtained from samples using the transfer method of deposition agreed with previous results from samples deposited with conventional deposition methods. I also demonstrated the potential application of the deposition method for high-throughput AFM imaging.Doctor of Philosoph