An expanded palette of fluorogenic HaloTag probes with enhanced contrast for targeted cellular imaging

We report the development of HaloTag fluorogens based on dipolar flexible molecular rotor structures. By modulating the electron donating and withdrawing groups, we have tuned the absorption and emission wavelengths to design a palette of fluorogens with emissions spanning the green to red range, opening new possibilities for multicolor imaging. The probes were studied in glycerol and in the presence of HaloTag and exhibited good fluorogenic properties thanks to a viscosity-sensitive emission. In live-cell confocal imaging, the fluorogens yielded only a very low non-specific signal that enabled wash-free targeted imaging of intracellular organelles and proteins with good contrast. Combining experimental studies and theoretical investigation of the protein/fluorogen complexes by molecular dynamics, these results offer new insight into the design of molecular rotor-based fluorogenic HaloTag probes in order to improve reaction rates and the imaging contrast.

Automated summary

This study reports the development of HaloTag fluorogens based on dipolar flexible molecular rotor structures. By modulating the electron donating and withdrawing groups, the absorption and emission wavelengths were tuned to design a palette of fluorogens with emissions spanning the green to red range, enabling multicolor imaging. Experimental and theoretical investigations were conducted, offering new insights into the design of molecular rotor-based fluorogenic HaloTag probes to improve reaction rates and imaging contrast.