Enhanced FRET-Based Biosensors Using HaloTag and Synthetic Organic Fluorophores
- HE, XINCHANG
- Advisor(s): Zhang, Jin
Abstract
Genetically encoded Förster resonance energy transfer (FRET)-based biosensors for kinase activity illuminate the spatiotemporal networks of signaling pathways. With their high specificity and ability to monitor intracellular signals in real-time, they have found widespread applications. However, the majority of FRET-based biosensors for kinase activity still suffer from limited dynamic range and sensitivity. Here, I introduce a new series of FRET-based kinase activity biosensors that use the HaloTag labeled with a synthetic organic fluorophore in combination with fluorescent proteins as FRET pairs. These novel chemigenetic FRET-based biosensors allow us to monitor diverse kinase activities, such as PKA, PKB (Akt), PKC, and ERK, as well as the concentration of the second messenger cAMP in real-time. It is worth mentioning that these enhanced FRET-based kinase activity biosensors exhibit unprecedented dynamic range. In addition, the use of this strategy for the generation of FluoSTEP biosensors successfully increased their dynamic range by more than ten-fold. Moreover, with the use of far-red synthetic organic fluorophores red-shifted biosensors were generated that are highly suitable for multiplexed imaging. In summary, these chemigenetic FRET-based biosensors represent an enhanced class of genetically encoded sensors and allow real-time monitoring of kinase activities with high sensitivity, providing robust tools for exploring spatiotemporal networks of complex kinase signaling pathways.