Modular Measurements and Manipulations in Living Organisms Using Dye-targeting Proteins

Our group has developed a chemo-genetic hybrid-tagging approach that allows us to target and activate various fluorescent dyes at specific subcellular locations in cultured cells and living zebrafish embryos.  Specific dye-binding single chain variable fragment antibodies are selected for dye activation properties using high-speed fluorescence activated cell sorting, resulting in a diverse collection of fluorogen activating proteins.  Subsequent engineering of the dyes allows us to dramatically alter the biological and spectroscopic properties of the fluorescent complexes formed with the proteins.  By linking these fluorogenic dyes to other environmentally sensitive donor or acceptor dyes, we produce unique genetically targeted and ratiometric physiological indicators that are only active when targeted to these subcellular locations.  We have produced sensors that respond to changes in local pH, reactive oxygen species, Ca(II) and other physiological analytes.  Genetic fusion of specific affinity molecules to the FAP protein can be exploited to make compact tagging proteins that allow use of the wide diversity of modified fluorogenic dyes to visualize and analyze endogenous receptors on living cells and in small animal studies. Dyes with unique photochemical properties enable chemo-optogenetic manipulation of living systems from manipulation of specific proteins to ablation of distinct cellular lineages.