NanoBRET: The Bright Future of Proximity-Based Assays

Natasha C. Dale, Elizabeth K. M. Johnstone, Carl W. White, Kevin D. G. Pfleger

Research output: Contribution to journalReview article

Abstract

Bioluminescence resonance energy transfer (BRET) is a biophysical technique used to monitor proximity within live cells. BRET exploits the naturally occurring phenomenon of dipole-dipole energy transfer from a donor enzyme (luciferase) to an acceptor fluorophore following enzyme-mediated oxidation of a substrate. This results in production of a quantifiable signal that denotes proximity between proteins and/or molecules tagged with complementary luciferase and fluorophore partners. BRET assays have been used to observe an array of biological functions including ligand binding, intracellular signaling, receptor-receptor proximity, and receptor trafficking, however, BRET assays can theoretically be used to monitor the proximity of any protein or molecule for which appropriate fusion constructs and/or fluorophore conjugates can be produced. Over the years, new luciferases and approaches have been developed that have increased the potential applications for BRET assays. In particular, the development of the small, bright and stable Nanoluciferase (NanoLuc; Nluc) and its use in NanoBRET has vastly broadened the potential applications of BRET assays. These advances have exciting potential to produce new experimental methods to monitor protein-protein interactions (PPIs), protein-ligand interactions, and/or molecular proximity. In addition to NanoBRET, Nluc has also been exploited to produce NanoBiT technology, which further broadens the scope of BRET to monitor biological function when NanoBiT is combined with an acceptor. BRET has proved to be a powerful tool for monitoring proximity and interaction, and these recent advances further strengthen its utility for a range of applications.

Original languageEnglish
Article number56
Number of pages13
JournalFrontiers in Bioengineering and Biotechnology
Volume7
Issue numberMAR
DOIs
Publication statusPublished - 26 Mar 2019

Cite this

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title = "NanoBRET: The Bright Future of Proximity-Based Assays",
abstract = "Bioluminescence resonance energy transfer (BRET) is a biophysical technique used to monitor proximity within live cells. BRET exploits the naturally occurring phenomenon of dipole-dipole energy transfer from a donor enzyme (luciferase) to an acceptor fluorophore following enzyme-mediated oxidation of a substrate. This results in production of a quantifiable signal that denotes proximity between proteins and/or molecules tagged with complementary luciferase and fluorophore partners. BRET assays have been used to observe an array of biological functions including ligand binding, intracellular signaling, receptor-receptor proximity, and receptor trafficking, however, BRET assays can theoretically be used to monitor the proximity of any protein or molecule for which appropriate fusion constructs and/or fluorophore conjugates can be produced. Over the years, new luciferases and approaches have been developed that have increased the potential applications for BRET assays. In particular, the development of the small, bright and stable Nanoluciferase (NanoLuc; Nluc) and its use in NanoBRET has vastly broadened the potential applications of BRET assays. These advances have exciting potential to produce new experimental methods to monitor protein-protein interactions (PPIs), protein-ligand interactions, and/or molecular proximity. In addition to NanoBRET, Nluc has also been exploited to produce NanoBiT technology, which further broadens the scope of BRET to monitor biological function when NanoBiT is combined with an acceptor. BRET has proved to be a powerful tool for monitoring proximity and interaction, and these recent advances further strengthen its utility for a range of applications.",
keywords = "BRET, NanoLuc, Nluc, NanoBRET, fluorophore, ligand binding, CRISPR, RESONANCE ENERGY-TRANSFER, PROTEIN-PROTEIN INTERACTIONS, RENILLA-RENIFORMIS LUCIFERASE, BIOLUMINESCENCE-RESONANCE, TRANSFER BRET, COMPLEMENTATION ASSAY, NANOLUC LUCIFERASE, LIGAND-BINDING, DRUG DISCOVERY, SPLIT REPORTER",
author = "Dale, {Natasha C.} and Johnstone, {Elizabeth K. M.} and White, {Carl W.} and Pfleger, {Kevin D. G.}",
year = "2019",
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doi = "10.3389/fbioe.2019.00056",
language = "English",
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journal = "Frontiers in Bioengineering and Biotechnology",
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NanoBRET : The Bright Future of Proximity-Based Assays. / Dale, Natasha C.; Johnstone, Elizabeth K. M.; White, Carl W.; Pfleger, Kevin D. G.

In: Frontiers in Bioengineering and Biotechnology, Vol. 7, No. MAR, 56, 26.03.2019.

Research output: Contribution to journalReview article

TY - JOUR

T1 - NanoBRET

T2 - The Bright Future of Proximity-Based Assays

AU - Dale, Natasha C.

AU - Johnstone, Elizabeth K. M.

AU - White, Carl W.

AU - Pfleger, Kevin D. G.

PY - 2019/3/26

Y1 - 2019/3/26

N2 - Bioluminescence resonance energy transfer (BRET) is a biophysical technique used to monitor proximity within live cells. BRET exploits the naturally occurring phenomenon of dipole-dipole energy transfer from a donor enzyme (luciferase) to an acceptor fluorophore following enzyme-mediated oxidation of a substrate. This results in production of a quantifiable signal that denotes proximity between proteins and/or molecules tagged with complementary luciferase and fluorophore partners. BRET assays have been used to observe an array of biological functions including ligand binding, intracellular signaling, receptor-receptor proximity, and receptor trafficking, however, BRET assays can theoretically be used to monitor the proximity of any protein or molecule for which appropriate fusion constructs and/or fluorophore conjugates can be produced. Over the years, new luciferases and approaches have been developed that have increased the potential applications for BRET assays. In particular, the development of the small, bright and stable Nanoluciferase (NanoLuc; Nluc) and its use in NanoBRET has vastly broadened the potential applications of BRET assays. These advances have exciting potential to produce new experimental methods to monitor protein-protein interactions (PPIs), protein-ligand interactions, and/or molecular proximity. In addition to NanoBRET, Nluc has also been exploited to produce NanoBiT technology, which further broadens the scope of BRET to monitor biological function when NanoBiT is combined with an acceptor. BRET has proved to be a powerful tool for monitoring proximity and interaction, and these recent advances further strengthen its utility for a range of applications.

AB - Bioluminescence resonance energy transfer (BRET) is a biophysical technique used to monitor proximity within live cells. BRET exploits the naturally occurring phenomenon of dipole-dipole energy transfer from a donor enzyme (luciferase) to an acceptor fluorophore following enzyme-mediated oxidation of a substrate. This results in production of a quantifiable signal that denotes proximity between proteins and/or molecules tagged with complementary luciferase and fluorophore partners. BRET assays have been used to observe an array of biological functions including ligand binding, intracellular signaling, receptor-receptor proximity, and receptor trafficking, however, BRET assays can theoretically be used to monitor the proximity of any protein or molecule for which appropriate fusion constructs and/or fluorophore conjugates can be produced. Over the years, new luciferases and approaches have been developed that have increased the potential applications for BRET assays. In particular, the development of the small, bright and stable Nanoluciferase (NanoLuc; Nluc) and its use in NanoBRET has vastly broadened the potential applications of BRET assays. These advances have exciting potential to produce new experimental methods to monitor protein-protein interactions (PPIs), protein-ligand interactions, and/or molecular proximity. In addition to NanoBRET, Nluc has also been exploited to produce NanoBiT technology, which further broadens the scope of BRET to monitor biological function when NanoBiT is combined with an acceptor. BRET has proved to be a powerful tool for monitoring proximity and interaction, and these recent advances further strengthen its utility for a range of applications.

KW - BRET

KW - NanoLuc

KW - Nluc

KW - NanoBRET

KW - fluorophore

KW - ligand binding

KW - CRISPR

KW - RESONANCE ENERGY-TRANSFER

KW - PROTEIN-PROTEIN INTERACTIONS

KW - RENILLA-RENIFORMIS LUCIFERASE

KW - BIOLUMINESCENCE-RESONANCE

KW - TRANSFER BRET

KW - COMPLEMENTATION ASSAY

KW - NANOLUC LUCIFERASE

KW - LIGAND-BINDING

KW - DRUG DISCOVERY

KW - SPLIT REPORTER

U2 - 10.3389/fbioe.2019.00056

DO - 10.3389/fbioe.2019.00056

M3 - Review article

VL - 7

JO - Frontiers in Bioengineering and Biotechnology

JF - Frontiers in Bioengineering and Biotechnology

SN - 2296-4185

IS - MAR

M1 - 56

ER -