Abstract
Simultaneously monitoring 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 non-emissive acceptors (ShadowY, ShadowR). Using fluorescence lifetime 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 (3) protein misfolding, as exemplified through simultaneous monitoring of alpha-synuclein oligomerization and misfolding. MDF provides a scalable framework for real-time, live-cell biosensing across high-throughput, target-specific, and tissue-level applications in complex biological systems.