Cargando…

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

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, lumin...

Descripción completa

Detalles Bibliográficos
Autores principales: Min, Se-Hong, French, Alexander R., Trull, Keelan J., Tat, Kiet, Varney, S. Ashley, Tantama, Mathew
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721044/
https://www.ncbi.nlm.nih.gov/pubmed/31405152
http://dx.doi.org/10.3390/s19163502
_version_ 1783448259050602496
author Min, Se-Hong
French, Alexander R.
Trull, Keelan J.
Tat, Kiet
Varney, S. Ashley
Tantama, Mathew
author_facet Min, Se-Hong
French, Alexander R.
Trull, Keelan J.
Tat, Kiet
Varney, S. Ashley
Tantama, Mathew
author_sort Min, Se-Hong
collection PubMed
description 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.
format Online
Article
Text
id pubmed-6721044
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-67210442019-09-10 Ratiometric BRET Measurements of ATP with a Genetically-Encoded Luminescent Sensor Min, Se-Hong French, Alexander R. Trull, Keelan J. Tat, Kiet Varney, S. Ashley Tantama, Mathew Sensors (Basel) Article 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. MDPI 2019-08-10 /pmc/articles/PMC6721044/ /pubmed/31405152 http://dx.doi.org/10.3390/s19163502 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Min, Se-Hong
French, Alexander R.
Trull, Keelan J.
Tat, Kiet
Varney, S. Ashley
Tantama, Mathew
Ratiometric BRET Measurements of ATP with a Genetically-Encoded Luminescent Sensor
title Ratiometric BRET Measurements of ATP with a Genetically-Encoded Luminescent Sensor
title_full Ratiometric BRET Measurements of ATP with a Genetically-Encoded Luminescent Sensor
title_fullStr Ratiometric BRET Measurements of ATP with a Genetically-Encoded Luminescent Sensor
title_full_unstemmed Ratiometric BRET Measurements of ATP with a Genetically-Encoded Luminescent Sensor
title_short Ratiometric BRET Measurements of ATP with a Genetically-Encoded Luminescent Sensor
title_sort ratiometric bret measurements of atp with a genetically-encoded luminescent sensor
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721044/
https://www.ncbi.nlm.nih.gov/pubmed/31405152
http://dx.doi.org/10.3390/s19163502
work_keys_str_mv AT minsehong ratiometricbretmeasurementsofatpwithageneticallyencodedluminescentsensor
AT frenchalexanderr ratiometricbretmeasurementsofatpwithageneticallyencodedluminescentsensor
AT trullkeelanj ratiometricbretmeasurementsofatpwithageneticallyencodedluminescentsensor
AT tatkiet ratiometricbretmeasurementsofatpwithageneticallyencodedluminescentsensor
AT varneysashley ratiometricbretmeasurementsofatpwithageneticallyencodedluminescentsensor
AT tantamamathew ratiometricbretmeasurementsofatpwithageneticallyencodedluminescentsensor