Multiplexed Dark FRET Biosensors: An Accessible Live-Cell Platform for Target- and Cell-Specific Monitoring of Protein-Protein Interactions in 2D and 3D Model Systems.

Simultaneous monitoring of multiple protein-protein interactions in live cells remains a key challenge in biology and drug discovery. While multiplexed FRET enables parallel molecular readouts, existing approaches are often constrained by spectral overlap, complex instrumentation, or incompatibility with live-cell models. To overcome these limitations and increase accessibility to the broader biological community, we present multiplexed dark FRET (MDF), a genetically encoded platform that uses spectrally distinct donors (mNeonGreen, mScarlet-I3) paired with nonemissive acceptors (ShadowY, ShadowR). We first establish that MDF fluorophores exhibit minimal background FRET under co-expression, enabling clean separation of donor lifetimes under multiplexed conditions. Using fluorescence lifetime (FLT) detection, we demonstrate MDF's versatility through three biologically and translationally relevant examples: (1) cell-type-specific biosensing in organoids, as exemplified in 3D neuro-glial spheroids; (2) target specificity for drug discovery through discrimination of TNFR1 versus TNFR2 receptor conformations and selective FLT modulation by receptor-specific small molecules; and (3) protein misfolding, as exemplified through simultaneous monitoring of alpha-synuclein oligomerization and misfolding. We further show that MDF can be applied within a single cellular environment, demonstrating the feasibility of same-cell multiplexing under optimized transient transfection conditions. MDF provides a scalable framework for real-time, live-cell biosensing across high-throughput, target-specific, and tissue-level applications in complex biological systems.