Synthetic Phosphorylation Networks with Fluorescence and Luminescence Expansion.

Abstract

Synthetic receptors have emerged as powerful tools for precisely modulating cellular function. However, existing synthetic receptor platforms rely mainly on transcription-mediated reporting processes that are incompatible with the rapid and real-time dynamics of cellular signaling events. To address this limitation, we present SPN-FLUX (synthetic phosphorylation networks with fluorescence and luminescence expansion), a fully post-translational platform that integrates synthetic phosphorylation networks with split fluorescent or luminescent proteins, enabling rapid and tunable reporting of cellular processes. SPN-FLUX is responsive to extracellular stimuli within 1 h, providing a robust alternative to transcription-based approaches. Using mammalian cells as a model, we showcase SPN-FLUX's versatility by designing a membrane-bound receptor that activates upon ligand-induced dimerization, as well as a constitutively active intracellular biosensor. We further validate SPN-FLUX's biosensing capabilities by examining its responsiveness to hypoxic conditions, showcasing the ability to detect environmental changes dynamically. The modularity and programmability of SPN-FLUX establish it as a powerful platform for advancing synthetic biology and biosensing, with broad applications in both biomedical research and environmental monitoring.