Genetically Targetable Cell Surface Selected pH Sensor

Abstract

The specific noncovalent interactions between a monomethine dye and a single chain variable fragment antibody can be used to target and activate dye molecules that are otherwise nonfluorescent in solution at a specific protein partner in living cells. This interaction/activation process allows detection of bound dye in the presence of a large excess of unbound dye, a useful property for dynamic imaging. To apply this approach to environmental sensing, a series of tandem dye molecules were prepared consisting of a fluorogen and a pH sensitive cyanine dye linked at distances where intramolecular energy transfer was dominant. The binding of these molecules to their respective fluorogen activating peptides provided a series of genetically targeted and activated probes that function as single excitation ratiometric emitter probes, or as ratiometric excitation probes, depending on the arrangement of the energy transfer pair, and displayed pH responses with pKa values from 6.0 to 8.0. These probes allowed tracking and repeated pH measurements of b2 adrenergic receptor internalization and sorting at a single vesicle level, in 4-d confocal microscopy.