The Uncoiling of Supercoiled Plasmid DNA Over Time Observed by Atomic Force Microscopy

Long term stability of DNA structures in a cell is critical to sustaining life. The DNA structures could be degraded biologically (e.g. enzymes), chemically (e.g. drugs), and physically (e.g. thermal agitation process) with time. The DNA structures are maintained by being regenerated and/or being recovered by proteins within a cell. However, even though it is important, it is difficult to observe the time-evolution of DNA structures for extended periods at a molecular resolution. Here, we observed the time evolution of DNA structures for two months, in order to understand the long term stability of DNA structures. For this study, we used purified plasmid DNA molecules extracted from Escherichia coli (E-coli) as a sample. We also employed atomic force microscopy (AFM) to observe the plasmid DNA structures at a molecular resolution. The purified plasmid DNA molecules were diluted with pure water, deposited on a mica surface, and observed by an AFM on a regular basis in an ambient environment for two months. The sequential AFM images show the plasmid DNA formed globular structures at the beginning and transformed into uncoiled plasmid DNA network structures after two months. The globular structures appeared to be the supercoiled state of plasmid DNA, a well-known strategy to store genetic information in a confined space for bacterial systems. The observed DNA network structures are believed to be results of long periods of unwinding and rejoining processes of the supercoiled plasmid DNA. The unwinding and rejoining processes would have been caused by small residual proteins (or enzymes) possibly present in the plasmid DNA solution. This study reveals DNA stability is dramatically influenced by prolonged (~ a few months) exposure to small amounts of residual proteins (or enzymes). The result also suggests the AFM is a powerful tool in observing the biological process at the molecular level over extended periods of time