C4 - Developing probes for super-resolving bimolecular interactions

The dynamics of biomolecules within a cell span multiple timescales. They range from fast intramolecular dynamics and transient intermolecular interactions in the microsecond-to-millisecond range to structural arrangements that remain stable for hours. Ideally, one would be able to observe the temporal evolution of a single biomolecule successively through its functional states i.e., to observe a single molecule for up to hours with a time resolution of milliseconds or below. Additionally, as the individual molecules are typically not-isolated molecular players but are in constant interaction with other molecules in the cell, one would like to observe these interactions directly. Many biological research questions would benefit from “co-tracking” methods that visualize and quantify interaction partners. To date, only time- and spatially resolved single-molecule fluorescence resonance energy transfer (smFRET) can measure and visualize such interactions but is strongly limited in observation time by the photo-bleaching rate of the fluorescent labels. Co-tracking approaches that follow (transient) molecular interactions using super-resolution methods do not exist.
In this project, we will build and test several variants of probes that decouple the observation time for single molecules from the photobleaching of single fluorescent markers by rapidly exchanging the chromophores. This will allow following individual molecules for minutes to hours while maintaining a high temporal resolution. Furthermore, we will combine smFRET with super-resolution techniques, which allows the co-tracking of interacting molecular partners. To demonstrate the suitability and limits of our new probes, we will follow the dynamics of CRISPR-Cas systems, because their dynamics include processes that occur within milliseconds, such as the scanning of a target, as well as processes that take place in the time range of minutes to hours, such as the residence time of target-bound defense complexes. So far, these different processes can only be studied by consecutive, repetitive experiments with different temporal resolutions. The new probes will allow direct visualization of the CRISPR-Cas dynamics over extended periods, which enables new experimental options for projects A2 and A3 and will also be of great advantage over current probes for several other CRC projects requiring sensitive smFRET and single-molecule measurements.