Cellular delivery and site-specific targeting of organic fluorophores for super-resolution imaging in living cells

Abstract

Recent advances in super-resolution fluorescence microscopy have pushed the spatial resolution of biological imaging down to a few nanometers. The key element to the development of such imaging modality is synthetic organic fluorophores with suitable brightness and photostability. However, organic fluorophores are very difficult to use in live cells because of their chemical compositions. Many excellent fluorophores, such as cyanine and Alexa Fluor dyes, are highly charged with sulfonate groups and do not cross the plasma membrane. Even if the fluorophores get inside cells, there exist few methods that can be used to target these nongenetically encoded probes to specific cellular proteins with high specificity and minimal interference. We describe herein the development of new methods for cellular delivery and sitespecific targeting of organic fluorophores to proteins in living cells. Building on our lab's previous work on engineering new substrate specificity for E. coli lipoic acid ligase (LplA), we created a mutant ligase that catalyzes covalent conjugation of a 7-hydroxycoumarin fluorophore onto a 13-amino acid peptide substrate, called LAP. We showed that enzymatic fluorophore ligation is compatible with the living cell interior and is highly specific for LAP fusion proteins. To extend the repertoire of fluorophores targetable by LplA inside cells, we devised a two-step labeling approach based on enzymatic azide ligation, followed by chemoselective derivatization with any membrane-permeable fluorophore via strain-promoted cycloaddition. As an auxiliary tool for enzymatic probe ligation, we also developed a very efficient and biocompatible variant of copper-catalyzed azide-alkyne cycloaddition that can be used for modification of cell-surface proteins. To overcome the lack of membrane permeability of sulfonated fluorophores, we identified a chemical reaction that efficiently masks charged sulfonate groups as esterase-labile sulfonate esters. Such masked sulfonated fluorophores enter cells readily and can be sitespecifically targeted to intracellular proteins. Our efforts in developing protein labeling and fluorophore delivery methods culminated in their application to super-resolution imaging of cellular proteins in living cells.