BREAKFAST, Bioluminescence Resonance Energy mAKe over with a Fluorescence-Activating absorption-Shifting Tag, they call it. A team of UC Irvine led by Prof. J. Prescher has combined BRET and FAST together into a new customizable platform for in vivo imaging at infra-cell level. Just published in RSC Chem. Biol. 2024.
While bioluminescence is a powerful method for imaging tissues or organisms in vivo, applications are far from routine at the microscale. Indeed, one weak spot of bioluminescence is the visualization of dynamic events. A strategy explored for some time combines bioluminescent reporters together with fluorescent emitters, the so-called BRET (Bioluminescence Resonance Energy Transfer).
BRET and FAST – © 2024 The Authors (after RSC Chem. Biol.)
Pertaining to this strategy, the authors disclose a customizable imaging tool -BREAKFAST- suitable for both bioluminescence and fluorescence microscopy. BREAKFAST comprises pFAST™, the now-established and modular chemogenetic tag of The Twinkle Factory, fused to NanoLuc®, the ultrabright luciferase. Photon emission is triggered by the addition of luciferin and a fluorogen selected out of the wide range of FAST ligands. Different ligand combinations produce distinct spectral outputs that are readily distinguished via phasor analysis. Thus, from a single genetic reporter, multiple colors, and furthermore dynamic color tuning, are possible. Additionally, it is possible to control illumination levels with a “dark fluorogen”, a non-emissive quencher. BREAKFAST can thus serve as an on-demand bioluminescence ‘‘eraser’’.
“The customizability of the platform will enable rapid access to different spectral outputs without the need for additional protein engineering”
Zach Torrey et al.
Coming after the evolution of FAST into a protein dimerizer, dubbed CATCHFIRE, or combination with click chmistry, these findings vividly demonstrate the versatility and the potential of FAST of The Twinkle Factory for imaging the most transient phenomena at molecular level.
Want to give BREAKFAST a try in your research? Nothing easier! The Twinkle Factory distributes pFAST fluorogens, namely TFLime (HMBR), TFAmber (HBR-3,5DM), TFCoral (HBR-3,5DOM), and the quencher, TFDarth (HBIR-3M). And Addgene hosts the luciferase-pFAST plasmid as any other FAST plasmids.
Read more about BRET and FAST
Torrey, Z.R., Halbers, L.P., Scipioni, L., Tedeschi, G., Digman, M.A. and Prescher, J.A., 2024. A versatile bioluminescent probe with tunable color. RSC Chemical Biology. doi:10.1039/d4cb00101j
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