# 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

## TL;DR

This paper introduces a new live-cell biosensing platform called Multiplexed Dark FRET that allows researchers to monitor multiple protein interactions in real time without interference from other signals.

## Contribution

The novel Multiplexed Dark FRET platform uses non-emissive acceptors and fluorescence lifetime detection to enable specific and scalable monitoring of multiple protein interactions in live cells.

## Key findings

- MDF enables cell-type specific biosensing in 3D neuro–glial spheroids.
- MDF can distinguish TNFR1 versus TNFR2 receptor conformations for drug discovery.
- MDF allows simultaneous monitoring of alpha-synuclein oligomerization and misfolding.

## 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.