Harnessing Cyanine-like Properties to Develop Bright Fluorogenic Probes Based on Viscosity-Sensitive Molecular Rotors.

Dipolar fluorescent molecular rotors (FMRs) are environmentally-sensitive fluorophores that can be used in bioimaging applications to sense local viscosity and polarity. Their sensitivity to viscosity can also be used for the fluorogenic labeling of biomolecules such as DNA or proteins. In particular, we have previously used FMRs to develop a series of tunable fluorogens targeting the self-labeling protein tag Halotag for wash-free protein imaging in live cells.

A Fluorogenic Chemogenetic pH Sensor for Imaging Protein Exocytosis.

Fluorescent protein-based pH biosensors enable the tracking of pH changes during protein trafficking and, in particular, exocytosis. The recent development of chemogenetic reporters combining synthetic fluorophores with self-labeling protein tags offers a versatile alternative to fluorescent proteins that combines the diversity of chemical probes and indicators with the selectivity of the genetic encoding. However, this hybrid protein labeling strategy does not avoid common drawbacks of organic fluorophores such as the risk of off-target signal due to unbound molecules.

Design of Bright Chemogenetic Reporters Based on the Combined Engineering of Fluorogenic Molecular Rotors and of the HaloTag Protein.

The combination of fluorogenic probes (fluorogens) and self-labeling protein tags represent a promising tool for imaging biological processes with high specificity but it requires the adequation between the fluorogen and its target to ensure a good activation of its fluorescence. In this work, we report a strategy to develop molecular rotors that specifically target HaloTag with a strong enhancement of their fluorescence. The divergent design facilitates the diversification of the structures to tune the photophysical and cellular properties.

FRET-Sensing of Multivalent Protein Binding at the Interface of Biomimetic Microparticles Functionalized with Fluorescent Glycolipids.

Cell adhesion is a central process in cellular communication and regulation. Adhesion sites are triggered by specific ligand-receptor interactions inducing the clustering of both partners at the contact point. Investigating cell adhesion using microscopy techniques requires targeted fluorescent particles with a signal sensitive to the clustering of receptors and ligands at the interface.

Fluorogenic and genetic targeting of a red-emitting molecular calcium indicator.

We introduce a strategy for the fluorogenic and genetic targeting of a calcium indicator by combining a protein fluorogen with the BAPTA sensing group. The resulting dual-input probe acts like a fluorescent AND logic gate with a Ca-sensitive red emission that is activated only upon reaction with HaloTag with a 25-fold intensity enhancement and can be used for wash-free calcium imaging in HeLa cells. The modular all-molecular design relying on a well-established self-labeling protein tag opens future possibilities for tuning the photophysical properties or targeting different analytes.