Direct comparison of bioluminescence-based resonance energy transfer methods for monitoring of proteolytic cleavage

Dacres, Helen; Dumancic, Mira M.; Horne, Irene; Trowell, Stephen

doi:10.1016/j.ab.2008.10.040

Cited by 35 publications

(28 citation statements)

References 67 publications

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“…Of these constructs, Venus-CaM-M13-RLuc8 had a 2-fold higher dynamic range than RLuc8-CaM-M13-Venus and RLuc8-CaM-M13-cp157Venus while other constructs showed a lower dynamic range ( Figure 2). This result was consistent with previous results showing that orientation in which the donor luciferase was located at the C-terminus of the fusion generated a higher BRET signal compared to the reverse orientation [1,13]. Overall, Venus-CaM-M13-RLuc8, which we designated as BRAC (BRet-based Autoluminescent Ca 2+ indicator), had the largest dynamic range (60%) among the constructs that we tested (Table 1).…”

Section: +supporting

confidence: 80%

Auto-luminescent genetically encoded ratiometric indicator for real-time Ca²⁺imaging at the single cell level

2011

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Section: +supporting

confidence: 80%

Auto-luminescent genetically encoded ratiometric indicator for real-time Ca²⁺imaging at the single cell level

2011

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“…Unlike fluorescence resonance energy transfer (FRET), BRET does not involve an external light source, and is therefore largely free from autofluorescence, light scattering, photobleaching and/or photoisomerization of the donor moiety [5], all of which can limit the sensitivity of FRET. Absence of these effects results in a low background and very low limits of detection for BRET assays [6]. Due to these characteristics, BRET has been used for a variety of applications including RNA detection [7], investigating protein-protein interactions [8]–[10], drug screening [5], [9], [11], imaging [8], [12], [13], and general biosensing [14], [15].…”

Section: Introductionmentioning

confidence: 99%

“…We selected a thrombin bioprobe [6], [23] for this work because it is well characterised, highly specific and clinically relevant.…”

Section: Introductionmentioning

confidence: 99%

“…In earlier work we used the BRET 2 system because of its long Förster distance [24], sensitivity and its potential application to measuring intramolecular rearrangements [6], [25] as well as dissociations. Unfortunately, however, the BRET 2 system was not bright enough to be detected in the microfluidic system used here, due to its well-documented low quantum yield (e.g., Pfleger et al [9], [26]) and the small volume (2.6 nL) sampled optically.…”

Section: Introductionmentioning

confidence: 99%

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Comparison of Static and Microfluidic Protease Assays Using Modified Bioluminescence Resonance Energy Transfer Chemistry

Dacres

Anderson

et al. 2014

PLoS ONE

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BackgroundFluorescence and bioluminescence resonance energy transfer (F/BRET) are two forms of Förster resonance energy transfer, which can be used for optical transduction of biosensors. BRET has several advantages over fluorescence-based technologies because it does not require an external light source. There would be benefits in combining BRET transduction with microfluidics but the low luminance of BRET has made this challenging until now.MethodologyWe used a thrombin bioprobe based on a form of BRET (BRETH), which uses the BRET1 substrate, native coelenterazine, with the typical BRET2 donor and acceptor proteins linked by a thrombin target peptide. The microfluidic assay was carried out in a Y-shaped microfluidic network. The dependence of the BRETH ratio on the measurement location, flow rate and bioprobe concentration was quantified. Results were compared with the same bioprobe in a static microwell plate assay.Principal FindingsThe BRETH thrombin bioprobe has a lower limit of detection (LOD) than previously reported for the equivalent BRET1–based version but it is substantially brighter than the BRET2 version. The normalised BRETH ratio of the bioprobe changed 32% following complete cleavage by thrombin and 31% in the microfluidic format. The LOD for thrombin in the microfluidic format was 27 pM, compared with an LOD of 310 pM, using the same bioprobe in a static microwell assay, and two orders of magnitude lower than reported for other microfluidic chip-based protease assays.ConclusionsThese data demonstrate that BRET based microfluidic assays are feasible and that BRETH provides a useful test bed for optimising BRET-based microfluidics. This approach may be convenient for a wide range of applications requiring sensitive detection and/or quantification of chemical or biological analytes.

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“…Originally, BRET-based approaches usually used two BRET generations called BRET 1 and BRET 2 based on the use of different luciferase, coelenterazine substrates and fluorescent acceptor conferring specific and different spectral properties of the energy donor/acceptor couple [117,118] (Fig. (5)).…”

Section: 4a Ret Principlementioning

confidence: 99%

Probing Heterotrimeric G Protein Activation: Applications to Biased Ligands

Denis

Saulière

Galandrin

et al. 2012

CPD

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Cell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of "ligand-directed trafficking" has led to a conceptual revolution in pharmacological theory, thus opening new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch but rather as filters capable of selecting the activation of specific signals and thus generating texture responses to ligands, a phenomenon often referred to as ligand-biased signaling. Also, one challenging task today remains optimization of pharmacological assays with increased sensitivity so to better appreciate the inherent texture of ligands. However, considering that a single receptor has pleiotropic signaling properties and that each signal can crosstalk at different levels, biased activity remains thus difficult to evaluate. One strategy to overcome these limitations would be examining the initial steps following receptor activation. Even, if some G protein independent functions have been recently described, heterotrimeric G protein activation remains a general hallmark for all GPCRs families and the first cellular event subsequent to agonist binding to the receptor. Herein, we review the different methodologies classically used or recently developed to monitor G protein activation and discussed them in the context of G protein biased-ligands.

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Direct comparison of bioluminescence-based resonance energy transfer methods for monitoring of proteolytic cleavage

Cited by 35 publications

References 67 publications

Auto-luminescent genetically encoded ratiometric indicator for real-time Ca²⁺imaging at the single cell level

Auto-luminescent genetically encoded ratiometric indicator for real-time Ca²⁺imaging at the single cell level

Comparison of Static and Microfluidic Protease Assays Using Modified Bioluminescence Resonance Energy Transfer Chemistry

Probing Heterotrimeric G Protein Activation: Applications to Biased Ligands

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Direct comparison of bioluminescence-based resonance energy transfer methods for monitoring of proteolytic cleavage

Cited by 35 publications

References 67 publications

Auto-luminescent genetically encoded ratiometric indicator for real-time Ca2+imaging at the single cell level

Auto-luminescent genetically encoded ratiometric indicator for real-time Ca2+imaging at the single cell level

Comparison of Static and Microfluidic Protease Assays Using Modified Bioluminescence Resonance Energy Transfer Chemistry

Probing Heterotrimeric G Protein Activation: Applications to Biased Ligands

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Product

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Auto-luminescent genetically encoded ratiometric indicator for real-time Ca²⁺imaging at the single cell level

Auto-luminescent genetically encoded ratiometric indicator for real-time Ca²⁺imaging at the single cell level