NanoBRET™
The assay marks an industry standard system to measure target engagement in living cells. This system consists of a
full-length protein, which is ectopically expressed target protein fused to the NanoLuc® (NLuc) and a BRET tracer. As
the protein is expressed in a human cell line, expression of the protein-NLuc fusion construct occurs entirely within a
cellular context. Therefore, not only a full length construct (which is often impossible to isolate from expression
systems) is utilized but the expressed construct will also contain all post-translational modifications (PTMs) ensuring
a minimally artificial system, closer to the native protein behaviour. In addition, cellular trafficking and competing
metabolites and substrate proteins are present in this environment giving more accurate cellular target engagement data.
Disruption of the cell membrane (e.g. through Digitonin treatment) also enables an in vitro assay, containing the
physiological protein, giving additional in vitro data.
The assay is based on bioluminescence resonance energy transfer (BRET), which is induced by sufficient proximity of the
NLuc (donor) and the tracer molecule (acceptor). The energy generated through bioluminescence is transmitted to the BRET
acceptor, exciting the fluorophore, and in turn, leads to fluorescence emission that correlates to the binding of
tracers to the fusion protein.
For the measurement, a plasmid containing the target cDNA fused to the NLuc and the BRET acceptor tracer is required.
Plasmids for a variety of targets can be ordered
here
or cloned into
NLuc containing plasmids, deposited at Addgene (#200878 and #200879).
Tracers are listed within this database with a
number commercially available together with their respective plasmids or can be synthesized using reactive NanoBRET™590
dyes
.
After transfection of the full-length protein NLuc fusion, a tracer targeting the protein of interest can be titrated
for the determination of an apparent KD. Since the tracer contains a fluorophore, which acts as a BRET acceptor,
proximity between NLuc and the BRET acceptor leads to the emission of BRET signal at 590 nm. Addition of competing
compounds, displaces the tracer from from the NLuc-tagged protein of interest, therefore prevents proximity between BRET
acceptor and NLuc, leading to a decrease in BRET signal.
NanoBRET™ is a versatile method, used for diverse applications including:
-
Kinome-wide selectivity screening:
Robers MB, Wilkinson JM, Vasta JD, Berger LM, Berger BT, Knapp S.
Single tracer-based protocol for broad-spectrum kinase profiling in live cells with NanoBRET. STAR Protoc. 2021 Sep 15;2(4):100822.
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Direct utilization of DEL-screening hits for follow up studies:
Teske KA, et al. DELs enable the development of BRET probes for target engagement studies in cells. Cell Chem Biol. 2023 Aug 17;30(8):987-998.e24.
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Determination of cell membrane permeability of PROTACs:
Schwalm MP, Krämer A, Dölle A, Weckesser J, Yu X, Jin J, Saxena K, Knapp S.
Tracking the PROTAC degradation pathway in living cells highlights the importance of ternary complex measurement for PROTAC optimization. Cell Chem Biol. 2023 Jul 20;30(7):753-765.e8.