Lanthanide-based resonance energy transfer biosensors for live-cell applications

Lanthanide-based, Förster resonance energy transfer (LRET) biosensors enable sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living mammalian cells. LRET biosensors are polypeptides that consist of an alpha-helical linker sequence sandwiched between a lanthanide complex-binding domain and a fluorescent protein (FP) with two interacting domains residing at each terminus. Interaction between the terminal affinity domains brings the lanthanide complex and FP in close proximity such that lanthanide-to-FP, LRET-sensitized emission is increased. A recent proof-of-concept study examined model biosensors that incorporated the affinity partners FKBP12 and the rapamycin-binding domain of m-Tor (FRB) as well as p53 (1-92) and HDM2 (1-128). The sensors contained an Escherichia coli dihydrofolate reductase (eDHFR) domain that binds with high selectivity and affinity to Tb(III) complexes coupled to the ligand trimethoprim (TMP). When cell lines that stably expressed the sensors were treated with TMP-Tb(III), TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. Much larger signal changes (>2500%) and Z'-factors of 0.5 or more were observed when cells were grown in 96-well or 384-well plates and analyzed using a TGL plate reader. In this chapter, we elaborate on the design and performance of LRET biosensors and provide detailed protocols to guide their use for live-cell microscopic imaging studies and high-throughput library screening.