Photostable probes for imaging proteins with high temporal resolution – Tag-light
Next-generation chemical-genetic fluorescent markers for advanced biological imaging
Next-generation chemical-genetic fluorescent markers for advanced biological imaging
Pushing the limit of biological imaging
Deciphering the complex mechanisms controlling cells and organisms requires effective imaging systems and fluorescent probes to observe and quantify biomolecules in real time with high spatiotemporal resolution. A common strategy for imaging proteins is to fuse them to peptide or protein sequences that provide fluorescence, such as autofluorescent proteins. Although the last decade's advances in imaging have led (and will lead) to new discoveries in biology, there are still extraordinary opportunities for basic and clinical research in developing new fluorescent markers for further advancing imaging capabilities. The most useful techniques will enable to interrogate quantitatively and comprehensively living systems at the molecular, cellular and network levels. In this project, we developed next generation fluorescent markers for pushing the limits of biological imaging.
Our approach uses exogenously applied small synthetic fluorescent probes to label proteins. Selectivity is ensured through fusion to a genetic tag that binds selectively the tailored fluorescent molecules. The modular nature of such an approach enables one to tune the synthetic part by molecular engineering, in order to address biological questions with the molecular diversity offered by modern chemistry. To avoid unspecific background in cells and achieve high imaging contrast, we used fluorescent probes that display no fluorescence until labeling occurs. Such probes are often called fluorogenic probes to highlight their ability to generate fluorescence upon interaction with their target. Our chemical-genetic fluorescent markers are thus composed of a genetically encoded protein tag that forms fluorescent complexes with small organic fluorogenic chromophores (also called fluorogens). Such fluorogenic labeling allows selective background-free imaging even in presence of an excess of free fluorogen, opening great prospects for imaging in complex samples such as tissues and whole organisms.
We developed an inducible chemical-genetic fluorescent marker named FAST. FAST is a small protein tag that binds and switches on the fluorescence of hydroxybenzylidene rhodanine (HBR) analogs. FAST was evolved from the photoactive yellow protein (PYP). FAST proved to be highly effective to fluorescently label proteins in living cells and multicellular organisms.
This unique marker has enabled the development of innovative labeling protocols, and opens unprecedented perspectives for addressing current challenges in imaging, in particular in multiplexing, super-resolution microscopy and biosensing. The development of FAST led to the creation of the start-up Twinkle Bioscience in charge of its commercial development.
This work led to 8 publications (5 research articles and 3 review articles), 1 book chapter and 2 patents.
Publications
Gautier in Optogenetics, Photochemical and Photobiological Sciences Series (S. Vriz & T. Ozawa Eds), The Royal Society of Chemistry (just accepted).
Li, […], Gautier submitted
Pimenta, […], Jullien, Gautier . Sci. Rep. 7, 12316 (2017).
Li, Tebo, Gautier. Int. J. Mol. Sci. 18, 1473 (2017).
Jullien & Gautier Médecine/Sciences 33 (6-7), 576-578 (2017).
Li, […], Jullien, Gautier Chem. Sci. 8, 5598-5605 (2017).
Li, […], Jullien, Gautier Org. Biomol. Chem. 14, 9253-9261 (2016).
Pamont, […], Jullien, Gautier Proc. Natl. Acad. Sci. USA 113 (3), 497-502 (2016).
Jullien & Gautier Methods Appl. Fluoresc. 3, 042007 (2015).
Patents
Patent 1: Pub. No : WO/2016/001437, International Application No.: PCT/14175837.
Patent 2: Application number : EP17305591.4.
Imaging has become an essential tool in Biology thanks to major advances in instrumentation and probes. Fluorescence microscopy enables now submicrometric observation of proteins labeled with fluorescent probes in live cells. However approaches relying on genetic or chemical covalent labeling are impeded by non-optimal temporal resolution, rendering delicate the study of fast and transient processes, and poor photostability, preventing long-term imaging of single molecules. We propose to develop a collection of probes enabling the study of fast processes and long-term imaging with high contrast. Our design will rely on protein tags binding non-covalently fluorogenic molecules. Binding will both enhance the fluorogen’s brightness and alter its chromatic properties, enabling fast wash-free labeling and high contrast imaging. High photostability will be engineered by tuning the binding affinity to obtain fast fluorogen-exchange dynamics, allowing for overcoming bleaching by fluorogen replacement.
Project coordination
Arnaud Gautier (UMR CNRS ENS UPMC 8640 PASTEUR)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Partnership
UMR 8640 PASTEUR UMR CNRS ENS UPMC 8640 PASTEUR
Help of the ANR 198,640 euros
Beginning and duration of the scientific project:
September 2014
- 36 Months
