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Open AccessArticle

Ratiometric BRET Measurements of ATP with a Genetically-Encoded Luminescent Sensor

Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA
Hall for Discovery Learning 399, Purdue Institute for Integrative Neuroscience, 207 South Martin Jischke Drive, West Lafayette, IN 47907, USA
Department of Chemistry, Wellesley College, 106 Central Street, Wellesley, MA 02481, USA
Author to whom correspondence should be addressed.
Sensors 2019, 19(16), 3502;
Received: 2 July 2019 / Revised: 29 July 2019 / Accepted: 31 July 2019 / Published: 10 August 2019
(This article belongs to the Section Biosensors)
PDF [1799 KB, uploaded 10 August 2019]


Luciferase-based reporters provide a key measurement approach in a broad range of applications, from in vitro high-throughput screening to whole animal imaging. For example, luminescence intensity is widely used to measure promoter activity, protein expression levels, and cell growth. However, luminescence intensity measurements are subject to quantitative irregularities caused by luminescence decay and variation in reporter expression level. In contrast, bioluminescence resonance energy transfer (BRET) sensors provide the advantages of luciferase-based reporters but overcome the aforementioned irregularities because of the inherently ratiometric readout. Here, we generated a new ratiometric BRET sensor of ATP (ARSeNL—ATP detection with a Ratiometric mScarlet-NanoLuc sensor), and we demonstrated that it provides a stable and robust readout across protein, cell, and whole animal tissue contexts. The ARSeNL sensor was engineered by screening a color palette of sensors utilizing variants of the high photon flux NanoLuc luciferase as donors and a panel of red fluorescent proteins as acceptors. We found that the novel combination of NanoLuc and mScarlet exhibited the largest dynamic range, with a 5-fold change in the BRET ratio upon saturation with ATP. Importantly, the NanoLuc-mScarlet BRET pair provided a large spectral separation between luminescence emission channels that is compatible with green and red filter sets extensively used in typical biological microscopes and animal imaging systems. Using this new sensor, we showed that the BRET ratio was independent of luminescence intensity decay and sensor expression level, and the BRET ratio faithfully reported differences in live-cell energy metabolism whether in culture or within mouse tissue. In particular, BRET analyte sensors have not been used broadly in tissue contexts, and thus, in principle, our sensor could provide a new tool for in vivo imaging of metabolic status. View Full-Text
Keywords: bioluminescence; BRET; ATP; genetically-encoded biosensor; NanoLuc; mScarlet bioluminescence; BRET; ATP; genetically-encoded biosensor; NanoLuc; mScarlet

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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Min, S.-H.; French, A.R.; Trull, K.J.; Tat, K.; Varney, S.A.; Tantama, M. Ratiometric BRET Measurements of ATP with a Genetically-Encoded Luminescent Sensor. Sensors 2019, 19, 3502.

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