Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions

Abstract

Summary Lanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1–92) and HDM2 (1–128). 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. We observed much larger signal changes (>2,500%) and Z′-factors of 0.5 or more when we grew cells in 96- or 384-well plates and analyzed PPI changes using a TGL plate reader. The modular design and exceptional dynamic range of lanthanide-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.

In Situ Rolling Circle Amplification Förster Resonance Energy Transfer (RCA-FRET) for Washing-Free Real-Time Single-Protein Imaging

“…Surprisingly, first experiments using the exact same sample preparation conditions as for CW FRET did not result in any Tb PL. Because Tb is very stable in various buffers (including TBS) and has been applied to many in-solution assays and for live cell and in vivo imaging without significant PL quenching, 20,[35][36][37][38] we assumed that the fixation procedure combined with incubation of the fixed cells with an aqueous buffer (i.e., the TBS buffer washed out some of the PFA fixative) caused the Tb PL quenching. We found that removal of TBS from the cells (i.e., without addition of fresh TBS after the three usual washing steps) and direct imaging resulted in bright PL signals ( ) demonstrated that the detection channels were specific to their probes and that FRET could only be detected when both probes were present.…”

“…The improved signal-tobackground ratios of TG and TG-FRET imaging (compared to CW imaging) underline the significant benefits that lanthanide complexes can bring to advanced high-contrast PL imaging. 9,34,37,[41][42][43][44][45][46] Notably, lanthanide-based TG PL imaging of fixed cells or tissues is not common and considering that the well-established solution-phase TG-FRET assays have only recently been implemented into advanced live cell and in vivo imaging, we can anticipate that the same performance can be expected for carefully optimized TG imaging on fixed cells. Similar to immunohistochemistry, the performance of in situ PLA depends on the fixation and pretreatment conditions, the applied antibody-DNA probes, and the careful adaption of assay components and their concentrations, which are important optimization steps that are beyond our in situ RCA-FRET proof-of-concept study.…”

E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

“…This major problem can be addressed by lanthanide-based resonance energy transfer (LRET), where a time-gate delay of 10–100 μs is employed between probe excitation and signal acquisition to eliminate ns-long background fluorescence signal. Indeed, rapid development of this emerging method has been observed in recent years, as luminescent complexes of lanthanide cations (Tb 3+ and Eu 3+ , in particular) have been employed as donor chromophores to fluorescent proteins [ 35 , 36 , 37 ]. In light of this, the binding of Tb 3+ could be, in principle, employed to identify SPL2 ubiquitination substrates and E2s in vivo.…”

Förster resonance energy transfer biosensors for fluorescence and time-gated luminescence analysis of rac1 activity

“…In this study, we used Rac1 as a model system to elaborate on our recently reported biosensor design that incorporates rigid, helical linker domains and luminescent terbium (Tb(III)) complexes as energy transfer donors 20 , 21 . Our objectives were two-fold: (i) to develop LRET-type biosensors with ER/K linkers for multi-well plate TGL assays of Rac1 inhibition; and (ii) to evaluate the effects of ER/K linkers on the dynamic range of FRET-type Rac1 biosensors.…”