Synthetic Phosphorylation Networks with Fluorescence and Luminescence Expansion

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.