Modular Design of a Tandem Dye Tunes the Photophysical Properties of a Biosensor

Abstract

Fluorogen Activating Peptides (FAPs) are protein reporters which activate the fluorescence of a non-covalently bound dark molecule (fluorogen). Light-harvesting dendrimers consisting of multiple chromophores have been built to improve the brightness of a biosensor. The multiple donor dyes transfer energy to the lone acceptor, thus increasing the brightness of the biosensor. Although bright, these light harvesting structures (dyedrons) displayed reduced association constants with the cognate protein, and reduced photostability of the fluorogen-FAP complex. To understand and potentially overcome these limitations, we have synthesized Cy3-Malachite Green based donor-acceptor structures with linker lengths from 10 to 30 atoms and found that longer linker lengths correspond to reduced quantum yield, but not reduced energy transfer efficiency, and that this arises from protein-dye changes that are slow relative to protein-dye association. Ensemble and single molecule level investigation of triplet state quenchers (TSQs) covalently linked to the fluorogen and the tandem dyes provide deeper insights into the development of bright, photostable probes for imaging applications.