Abstract
Intramolecular photostabilization via triple-state quenching was recently revived as a tool to impart synthetic organic fluorophores with ‘self-healing’ properties. To date, utilization of such fluorophore derivatives is rare due to their elaborate multi-step synthesis. Here we present a general strategy to covalently link a synthetic organic fluorophore simultaneously to a photostabilizer and biomolecular target via unnatural amino acids. The modular approach uses commercially available starting materials and simple chemical transformations. The resulting photostabilizer–dye conjugates are based on rhodamines, carbopyronines and cyanines with excellent photophysical properties, that is, high photostability and minimal signal fluctuations. Their versatile use is demonstrated by single-step labelling of DNA, antibodies and proteins, as well as applications in single-molecule and super-resolution fluorescence microscopy. We are convinced that the presented scaffolding strategy and the improved characteristics of the conjugates in applications will trigger the broader use of intramolecular photostabilization and help to emerge this approach as a new gold standard.
Highlights
Intramolecular photostabilization via triple-state quenching was recently revived as a tool to impart synthetic organic fluorophores with ‘self-healing’ properties
As a proof-of-concept, we synthesized photostabilizer–dye conjugates of fluorophores that could far not be tested for intramolecular photostabilization due to limited scaffolding options
We focused on rhodamines and carbopyronines, that is, fluorophores from the ATTO and Alexa series, which are extremely popular for applications. (S)-Nitrophenylalanine, NPA (Fig. 2), was used as a scaffold for the first generation of compounds, which consists of single-stranded DNA as the biomolecular target, a commercially available organic fluorophore (Alexa[555], ATTO647N and Cy5) and the p-nitrophenyl group of the known photostabilizer NPA16,17,19 (Fig. 2, compounds 5, 6 and 7)
Summary
Intramolecular photostabilization via triple-state quenching was recently revived as a tool to impart synthetic organic fluorophores with ‘self-healing’ properties. The resulting photostabilizer–dye conjugates are based on rhodamines, carbopyronines and cyanines with excellent photophysical properties, that is, high photostability and minimal signal fluctuations Their versatile use is demonstrated by single-step labelling of DNA, antibodies and proteins, as well as applications in single-molecule and super-resolution fluorescence microscopy. The synthesized fluorophore derivatives comprise of either a reducing or oxidizing photostabilizer in the form of the antioxidant Trolox (TX) or a nitrophenyl group[10,12] We characterized their photophysical properties with singlemolecule fluorescence microscopy on the biomolecular target DNA and observed significant increases in photostability for all compounds including suppression of triplet-based blinking. We demonstrate state-of-the-art applications of photostabilizer–dye conjugates in singlemolecule Forster resonance energy transfer (smFRET) and super-resolution stimulated emission depletion (STED) microscopy, with significantly increased sensitivity and photostability of the conjugates compared to their nonstabilized parent fluorophores
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