Automated FRET Two‐Hybrid Analysis

ABSTRACT The fluorescence resonance energy transfer (FRET) two‐hybrid assay enables live‐cell detection of biomolecular complexes but faces high‐throughput screening (HTS) limitations due to laborious image analysis. We developed an automated platform using the Luminance‐Uniformity‐based Region of I...

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Veröffentlicht in:Journal of biophotonics Jg. 18; H. 9; S. e70033 - n/a
Hauptverfasser: Wei, Zhiqiang, Xu, Yanling, Wang, Jingzhen, Sun, Beini, Huang, Qialing, Zhuang, Zhengfei, Chen, Tongsheng, Hu, Min
Format: Journal Article
Sprache:Englisch
Veröffentlicht: Weinheim WILEY‐VCH Verlag GmbH & Co. KGaA 01.09.2025
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Schlagworte:
Algorithms
automated data processing
Automation
Energy transfer
Fluorescence resonance energy transfer
Fluorescence Resonance Energy Transfer - methods
FRET quantitative analysis
FRET two‐hybrid assay
Humans
Image analysis
Image processing
Image Processing, Computer-Assisted
Stoichiometry
Two-Hybrid System Techniques
FRET two‐hybrid assay
image processing
FRET quantitative analysis
stoichiometry
automated data processing
ISSN:1864-063X, 1864-0648, 1864-0648
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Zusammenfassung:ABSTRACT The fluorescence resonance energy transfer (FRET) two‐hybrid assay enables live‐cell detection of biomolecular complexes but faces high‐throughput screening (HTS) limitations due to laborious image analysis. We developed an automated platform using the Luminance‐Uniformity‐based Region of Interest Selection (LURS) algorithm, accelerating processing 12‐fold (6 h  → 30 min) for three‐channel FRET imaging. Validation with FRET standards (C32V: E D C 32 V = 0.30 ± 0.01 , S C 32 V = 1.06 ± 0.14 ; CVC: E D CVC = 0.40 ± 0.02 , S CVC = 1.90 ± 0.11 ) matched reference values. Applied to Bcl‐xL/Bak interactions under A1331852 treatment, LURS revealed dose‐dependent stoichiometry reduction ( 1.87 → 1.12 ). The method achieved precise signal extraction while preserving native cellular conditions, overcoming throughput constraints in dynamic protein interaction studies. We developed an automated FRET two‐hybrid platform using the LURS algorithm, accelerating image analysis 12‐fold (6 h → 30 min). Validated with C32V/CVC constructs, it accurately measured FRET efficiency and stoichiometry ( n D / n A = 1.06 ± 0.14/C32V; 1.90 ± 0.11/CVC), matching reported values. Applied to Bcl‐xL/Bak interaction under A1331852, it revealed stoichiometry reduction (1.87 → 1.12), enabling HTS‐compatible drug discovery.
Bibliographie:This work was supported by grants from the Key‐Area Research and Development Program of Guangdong Province (Grant No. 2022B0303040003), National Natural Science Foundation of China (NSFC) (Grant No. 62135003 and 62475077), Guangdong Basic and Applied Basic Research Foundation (Grant No. 2024A1515010586), and Graduate Research Innovation Program of South China Normal University (Grant No. 2024KYLX074).
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ISSN:1864-063X
1864-0648
1864-0648
DOI:10.1002/jbio.70033