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Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

ABSTRACT: This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

SUBMITTER: Plamont MA

PROVIDER: S-EPMC4725535 | biostudies-other | 2016 Jan

REPOSITORIES: biostudies-other

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Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

Plamont Marie-Aude MA Billon-Denis Emmanuelle E Maurin Sylvie S Gauron Carole C Pimenta Frederico M FM Specht Christian G CG Shi Jian J Quérard Jérôme J Pan Buyan B Rossignol Julien J Moncoq Karine K Morellet Nelly N Volovitch Michel M Volovitch Michel M Lescop Ewen E Chen Yong Y Triller Antoine A Vriz Sophie S Le Saux Thomas T Jullien Ludovic L Gautier Arnaud A

Proceedings of the National Academy of Sciences of the United States of America 20151228 3

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption re ...[more]

PMID: 26711992

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Project description:A big challenge in the clinical use of Cerenkov luminescence (CL) imaging is its low signal intensity, which is several orders of magnitude below ambient light. Consequently, highly sensitive cameras, sufficient shielding from background light, and long acquisition times are required. To alleviate this problem, we hypothesized a strategy to convert the weak CL signal into a stronger fluorescence signal by using CL-activated formation of nitrenes from azides to locally fix a fluorescent probe in tissue by the formation of a covalent bond. CL-activated drug delivery was also evaluated using the same azide chemistry. The specific delivery of the CL-activated drug to cancer cells could reduce systemic toxicity, which is a limitation in chemotherapy. Methods: A cyanine-class near-infrared fluorescent dye, Cy7, and doxorubicin were synthetically attached to polyfluorinated aryl azide to form Cy7 azide and DOX azide, respectively. Fibrosarcoma cells were incubated with 18F-FDG and exposed to Cy7 azide with subsequent fluorescence imaging. For CL-activated tagging in vivo, tumor-bearing mice were injected first with 90Y-DOTA-RGD, targeting ?v?3 integrins, and then with the Cy7 azide. Fluorescence signal was imaged over time. Breast cancer cells were incubated with DOX azide and 68Ga, after which cell viability was quantified using an assay. Results: CL photoactivation of Cy7 azide in vitro showed significantly higher fluorescence signal from 18F-FDG-treated than untreated cells. In vivo, CL photoactivation could be shown by using the tumor-specific, integrin-targeting 90Y-DOTA-RGD and the localized activation of Cy7 azide. Here, localized CL-induced fluorescence was detected in the tumors and remained significantly higher over several days than in tumors without CL. We also established as a next step CL-activated drug delivery of DOX azide by showing significantly decreasing cell viability of breast cancer cells in a CL dose-dependent manner in vitro using CL photoactivation of DOX azide. Conclusion: We were able to develop a CL-activated "sticky tag" that converts the low CL signal into a stable and long-lasting, highly intense fluorescence signal. This fluorescent footprint of the radioactive signal might be clinically used for intraoperative surgery. The CL-targeted drug delivery strategy may potentially be used for dual-step targeted therapy.

Dataset Information

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

Publications

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

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