Reporter-Based BRET Sensors for Measuring Biological Functions In Vivo

Abstract

Genetic reporter systems provide a good alternative to monitor cellular functions in vitro and in vivo and are contributing immensely in experimental research. Reporters like fluorescence and bioluminescence genes, which support optical measurements, provide exquisite sensitivity to the assay systems. In recent years several activatable strategies have been developed, which can relay specialized molecular functions from inside the cells. The application of bioluminescence resonance energy transfer (BRET) is one such strategy that has been proved to be extremely valuable as an in vitro or in vivo assay to measure dynamic events such as protein-protein interactions (PPIs).The BRET assay using RLuc-YFP was introduced in biological research in the late 1990s and demonstrated the interaction of two proteins involved in circadian rhythm. Since then, BRET has become a popular genetic reporter-based assay for PPI studies due to several inherent attributes that facilitate high-throughput assay development such as rapid and fairly sensitive ratio-metric measurement, the assessment of PPI irrespective of protein location in cellular compartment and cost effectiveness. In BRET-based screening, within a defined proximity range of 10-100 Å, the excited energy state of the luminescent molecule excites the acceptor fluorophore in the form of resonance energy transfer, causing it to emit at its characteristic emission wavelength. Based on this principle, several such donor-acceptor pairs, using Renilla luciferase or its mutants as donor and either GFP2, YFP, mOrange, TagRFP or TurboFP as acceptor, have been reported for use.In recent years, the applicability of BRET has been greatly enhanced by the adaptation of the assay to multiple detection devices such as a luminescence plate reader, a bioluminescence microscope and a small animal optical imaging platform. Apart from quantitative measurement studies of PPIs and protein dimerization, molecular spectral imaging has expanded the scope for fast screening of pharmacological compounds that modulate PPIs by unifying in vitro, live cell and in vivo animal/plant measurement, all using one assay. Using examples from the literature, we will describe methods to perform in vitro and in vivo BRET imaging experiments and some of its applications.

Development of Non-invasive ABPP-BRET Platform Technology Enable Real-Time Detection of Post-Translational Modification and Target Engagement with Absolute Specificity in Living Systems

“…The RLuc 8.6 substrate coelenterazine h was added to the cells, and the average radiance was recorded in the donor emission channel and the acceptor emission channel. The BRET measurements were calculated using the mBRET values for the STS treated versus untreated samples 26 . The calculated mBRET value for the STS treated sample is 60 ± 0.4, which is significantly higher than the untreated control, 11 ± 9.2 (Figure 4a).…”

“…The acceptor/donor channel emission (A/D) ratio was calculated for the donor (D) only, and the donor + acceptor (D+A) sample. A/D channel emission ratio of the D only sample was subtracted from the D+A sample to obtain the corrected BRET ratio (cBRET) 26 . Subsequently, mBRET values were calculated by multiplying the values of the corrected BRET ratio to 1000.…”

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

“…The bioluminescence resonance energy transfer (BRET) assay ( Figure 5 ) involving the use of RLuc has become popular since the late 1990s for measurements of protein–protein interactions and conformational rearrangements in live cells [ 138 , 139 , 140 ], for non-invasive bioimaging [ 141 ], and as probes for biosensing [ 123 , 142 ]. The sensitivity of BRET assays has recently been improved by introducing new BRET components: RLuc2 and RLuc8 with improved quantum yields, stability, and brightness [ 143 ] as well as a great variety of acceptors (GFP2, YFP, Venus, mOrange, TagRFP, TurboFP, semiconductor quantum dots or carbon-dots) [ 144 , 145 , 146 ]. However, the main application of RLuc is luciferase genetic reporter assay that has become an invaluable and routine tool for molecular biology research, including identification and characterization of protein functional variants [ 147 , 148 ], investigations of gene expression [ 148 , 149 , 150 ], transcription factors [ 151 , 152 , 153 , 154 , 155 ], receptor activity [ 156 ], miRNA expression [ 157 , 158 , 159 ], monitoring mRNA splicing events in cells [ 160 , 161 ], virus investigations [ 162 , 163 , 164 ], drug discovery [ 165 , 166 ], and identification and evaluation of virus inhibitors [ 167 , 168 ].…”

A Genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor

“…In addition to the above, Bioluminescence Resonance Energy Transfer (BRET) has been used in developing a range of genetically encoded, live cell sensors [31][32][33][34] . BRET relies on the non-radiative resonance energy transfer from a light emitting luciferase protein (donor) upon oxidation of its substrate to a fluorescent protein (acceptor) with an excitation spectrum overlapping with the luciferase emission spectra.…”