Braun Anthony, Liao Elly, Vunnam Nagamani, Murray Marguerite, Sachs Jonathan
University of Minnesota.
Res Sq. 2025 May 12:rs.3.rs-6580769. doi: 10.21203/rs.3.rs-6580769/v1.
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.
在活细胞中同时监测多种蛋白质-蛋白质相互作用仍然是生物学和药物发现中的一项关键挑战。虽然多重荧光共振能量转移（FRET）能够实现并行分子读数，但现有方法常常受到光谱重叠、复杂仪器设备或与活细胞模型不兼容的限制。为了克服这些局限性并提高广大生物学界的可及性，我们提出了多重暗态FRET（MDF），这是一个基因编码平台，它使用光谱不同的供体（mNeonGreen、mScarlet-I3）与非发光受体（ShadowY、ShadowR）配对。通过荧光寿命检测，我们通过三个生物学和转化相关的例子展示了MDF的多功能性：（1）类器官中的细胞类型特异性生物传感，如在3D神经胶质球中所示；（2）通过区分肿瘤坏死因子受体1（TNFR1）与肿瘤坏死因子受体2（TNFR2）的受体构象，实现药物发现的靶点特异性；（3）蛋白质错误折叠，如通过同时监测α-突触核蛋白的寡聚化和错误折叠所示。MDF为在复杂生物系统中跨高通量、靶点特异性和组织水平应用的实时活细胞生物传感提供了一个可扩展的框架。
