Development of single-molecule tracking using fluorescence microscopy

In this project we want to use single-molecule tracking (SMT) to gain insight into the mechanisms of DNA repair by the real-time tracking and analysis of single DNA repair proteins in action. The main challenges in SMT of DNA-protein complexes are (1) having the DNA locked in a linear form in order to be able to track the protein that is moving along the DNA contour, (2) being able to detect the rather weak signal from one single protein labeled with a fluorescent reporter probe, and (3) perform a robust data acquisition and analysis in order to reliably obtain quantitative biological and biophysical results. Studies of single-molecule imaging of DNA repair proteins in action have so far been performed either using a constant flow of buffer to stretch the DNA substrate, or labeling of proteins using quantum dots and antibodies, to obtain high quantum efficiency and long-lived signals. Both these methods have severe fundamental drawbacks with respect to extracting relevant biophysical parameters. Therefore, we have developed a new protocol combining DNA substrate trapping, protein labeling and data collection strategies which eliminates shortcomings of previous published studies. By trapping the ends of DNA we can optically track the protein while it is moving along the DNA. Overcoming the second challenge is achieved through a series of optimizations to reduce the noise and detect the signal. A unique combination of optical tweezers and high localization precision microscopy meets the core needs of the single molecule experiments very well. There are no such instruments in Norway, and the setup at UBI (Bielefeld, Germany) is ideal for these kinds of experiments by combining the two research groups expertise in biology and optics, respectively.