Single-chain lanthanide luminescence biosensors for cell-based imaging and screening of protein-protein interactions

Abstract

Research tools that enable imaging or analysis of protein-protein interactions (PPIs) directly within living cells provide unique and valuable biological insights and can also aid drug discovery efforts. Here, we present lanthanide-based, Forster resonance energy transfer (lanthanide-based FRET, or LRET) biosensors for time-gated luminescence (TGL) imaging or multiwell plate analysis of PPIs. Polypeptide chains comprised of an alpha helical linker flanked by a Tb(III) complex, GFP and two binding domains exhibit large differences in long-lifetime, Tb(III)-to-GFP LRET-sensitized emission between open (unbound) and closed (bound) states. We used TGL microscopy to image ca. 500% increases in Tb(III)-to-GFP LRET following rapamycin addition to NIH 3T3 cells that expressed biosensors bearing FKBP12 and the rapamycin binding domain of m-Tor (FRB) at each terminus. Much larger signal changes, up to ca. 2500%, were observed when cells were grown in 96-well or 384-well plates and analyzed using a TGL plate reader. We also measured the interaction of p53 and HDM2 and its inhibition within intact HeLa cells grown in 96-well plates and estimated a z-factor of 0.5 for the assay. The modular design and high dynamic range of Tb(III)-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.