Chemogenetic Approaches for Interrogation and Influence of Biological Systems

In current biology, there is a noticeable trend in developing new tools and methods for targeting proteins of interest as well as various cell compartments for interrogating their position and function. Fluorogen activating proteins (FAPs) offer an approach for specific protein targeting with a high activation ratio which allows their vast applications. Their inherent modularity allows researchers to modify only fluorogen to alter the properties or function of the resulting fluorogen-FAP complex. This work focuses on a rational design and validation of several new fluorogens for already existing FAP systems.We developed a novel fluorogenic dye, an azetidine modified variant of malachite green esters previously used in our lab, that preferentially labels FAPs located in acidic compartments of the cell due to a shifted carbinol-chromophore equilibrium. This fluorogen-FAP pair shows high colocalization with LysoTracker without noticeable background from protein located in neutral cellular locations. This fluorogen provides a novel chemoselective labeling platform for studies of protein trafficking in acidic compartments of the cell which might be applicable for lysosomal storage disorders.Another fluorogenic dye that we developed is an iodinated analog of the synthetic GFP chromophore that preserves tight binding to a previously published de novo designed protein mFAP2b. In addition to having high activation upon binding to the protein, this fluorogen also demonstrates singlet oxygen production capabilities. Addition of this new fluorogen-fluorophore pair to our toolbox of cellular probes would allow researchers to induce targeted ROS-mediated damage in live cells in two separate genetically targeted locations.In order to make population-scale experiments that include photoablation and targeted protein damage possible, we have worked on the development and validation of several different versions of LED-based illumination setups (Lightbox 1.1, Lightbox 2.0, LightCoffin). With these setups researchers can select what wavelength they want to use for their illumination experiment, if they want to have a flat illumination field or a spread of different light doses received by samples and perform the experiments in a reproducible fashion.