Imaging and pulling single macromolecules - DNA as case study
A fundamental breakthrough in Nanotechnology was the development of the atomic force microscope (AFM) about 25 years ago. In contrast to a conventional optical microscope the AFM is comprised of a very sharp tip with a few nanometers in radius that is scanned ultra-precisely over a sample. Thereby a topography image of the sample is generated with a very high resolution down to the dimension of single molecules. The AFM enables direct space imaging and stretching or unfolding of individual macromolecules like DNA. Molecules can be attached between AFM tip and sample surface or even be picked or “fished” from the surface by the AFM tip. Upon pulling, molecules are stretched and gradually weak intra-molecular bonds are broken apart. Refolding of the molecules is possible upon approach back to the sample. Repetitive unfolding and refolding experiments give insight into the energy landscapes of individual molecules.
As a very prominent example DNA molecules were studied soon after AFM was introduced, both for imaging and later also for single molecule mechanics studies [1, 2].
In this project the students will prepare DNA samples on ultra-flat mica for imaging in air. This will be achieved by adsorption of DNA molecules from solution on a freshly cleaved mica surface. After rinsing with milli-Q water and drying, the samples will be investigated by tapping mode imaging in air. The contour lengths of individual molecules will be measured and plotted in histograms.
The same DNA solution will be used to modify freshly prepared gold substrates to near saturation coverage. From those samples individual DNA molecules shall be picked by the AFM tip and stretched to full extension. From the obtained force-distance curves the contour lengths will be determined.
Fig.A) AFM tapping mode height image of lambda digest DNA on mica
Fig.B) illustration of a single dsDNA molecule stretching with forced melting to a single strand at f=65 pN .
 Hansma, H. G.; Kasuya, K.; Oroudjev, E. Curr. Opin. Struct. Biol. 2004, 14, 380.
 Clausen-Schaumann, H.; Rief, M.; Tolksdorf, C.; Gaub, H. E. Biophys. J. 2000, 78.