Linker Molecules Convert Commercial Fluorophores into Tailored Functional Probes during Biolabelling.

Many life-science techniques and assays rely on selective labeling of biological target structures with commercial fluorophores that have specific yet invariant properties. Consequently, a fluorophore (or dye) is only useful for a limited range of applications, e.g., as a label for cellular compartments, super-resolution imaging, DNA sequencing or for a specific biomedical assay. Modifications of fluorophores with the goal to alter their bioconjugation chemistry, photophysical or functional properties typically require complex synthesis schemes. We here introduce a general strategy that allows to customize these properties during biolabelling with the goal to introduce the fluorophore in the last step of biolabelling. For this, we present the design and synthesis of 'linker' compounds, that bridge biotarget, fluorophore and a functional moiety via well-established labeling protocols. Linker molecules were synthesized via the Ugi four-component reaction (Ugi-4CR) which facilitates a modular design of linkers with diverse functional properties and bioconjugation- and fluorophore attachment moieties. To demonstrate the possibilities of different linkers experimentally, we characterized the ability of commercial fluorophores from the classes of cyanines, rhodamines, carbopyronines and silicon-rhodamines to become functional labels on different biological targets in vitro and in vivo via thiol-maleimide chemistry. With our strategy, we showed that the same commercial dye can become a photostable self-healing dye or a sensor for bivalent ions subject to the linker used. Finally, we quantified the photophysical performance of different self-healing linker-fluorophore conjugates and demonstrated their applications in super-resolution imaging and single-molecule spectroscopy.

Characterization of Fluorescent Proteins with Intramolecular Photostabilization*.

Genetically encodable fluorescent proteins have revolutionized biological imaging in vivo and in vitro. Despite their importance, their photophysical properties, i. e., brightness, count-rate and photostability, are relatively poor compared to synthetic organic fluorophores or quantum dots. Intramolecular photostabilizers were recently rediscovered as an effective approach to improve photophysical properties of organic fluorophores. Here, direct conjugation of triplet-state quenchers or redox-active substances creates high local concentrations of photostabilizer around the fluorophore. In this paper, we screen for effects of covalently linked photostabilizers on fluorescent proteins. We produced a double cysteine mutant (A206C/L221C) of α-GFP for attachment of photostabilizer-maleimides on the ß-barrel near the chromophore. Whereas labelling with photostabilizers such as trolox, a nitrophenyl group, and cyclooctatetraene, which are often used for organic fluorophores, had no effect on α-GFP-photostability, a substantial increase of photostability was found upon conjugation to azobenzene. Although the mechanism of the photostabilizing effects remains to be elucidated, we speculate that the higher triplet-energy of azobenzene might be crucial for triplet-quenching of fluorophores in the blue spectral range. Our study paves the way for the development of fluorescent proteins with photostabilizers in the protein barrel by methods such as unnatural amino acid incorporation.