High Dynamic-Range Lanthanide-Based Biosensors for Live-Cell Imaging and High Throughput Screening

FRET biosensors are a powerful tool for elucidating cellular signaling mechanisms due to their ability to capture the spatiotemporal dynamics of protein-protein interactions (PPIs) in native environment. While popular, many FRET biosensors possess a low dynamic range that barely exceeds the noise of many imaging systems and cannot be used to detect cellular PPIs at medium or high throughput applications, especially in cell-based format. The main objective of this study is to create a lanthanide-based FRET (LRET) biosensor toolkit that aims to simplify and accelerate the generation of high dynamic range biosensors for live-cell imaging and cell-based multi-well plate analysis of PPIs. The kit incorporates a Tb(III) complex as the energy transfer donor, a GFP as the acceptor, and an alpha-helical linker termed ER/K linker as a middle spacer in single-chain biosensor design. Tb and Tb-to-GFP sensitized emission signals have ms-scale lifetimes that permit the use of time-gated luminescence (TGL) detections in which pulsed light is used to excite the Tb complex and sensitized GFP signals are measured after a brief delay. ER/K linkers maintain the donors and acceptor far apart when the sensor is in the open configuration. The unique features of LRET and the incorporation of ER/K linkers generate biosensors with extraordinary dynamic ranges for imaging. TGL of luminescent lanthanide probes has been long used in HTS because it can overcome many limitations of conventional FRET. Here we also explore the potential of LRET sensors in cell-based medium-to-high throughput screening of small molecule inhibitors for PPIs. Our results show that LRET biosensors offer a versatile platform technology for interrogating PPIs and their function in live cells.