Abstract
Synthetic dyes with photoactivatable fluorescence allow the monitoring of dynamic events with time-lapse measurements and the reconstruction of images with subdiffraction spatial resolution. Their application to the investigation of cellular processes, however, requires first the identification of appropriate protocols to label selectively subcellular structures without compromising their photochemical and photophysical properties. To this end, the ability of self-labeling protein tags to connect synthetic dyes selectively to intracellular targets with structural control rivaling that possible with fluorescent proteins is extremely attractive. Indeed, we designed multistep synthetic strategies to install ligands for the selective labeling of HaloTag or SNAP-tag on the
-position of borondipyrromethene (BODIPY) chromophores with photoactivatable fluorescence. We demonstrated that the targeting ability of the ligand and the photochemical and photophysical properties of the chromophoric assembly are preserved in the resulting molecular constructs. We also assessed the influence of the protein on the connected fluorescent chromophore with ensemble and single-molecule spectroscopic measurements as well as with molecular dynamics simulations. We additionally showed that our photoresponsive dyes label fusion proteins in the interior of model cells to enable intracellular fluorescence photoactivation with optimal contrast. Thus, our synthetic dyes may evolve into invaluable molecular probes for the investigation of the structures and dynamics of intracellular proteins with the level of spatiotemporal control that is only possible with fluorescence photoactivation.