Automated FRET Two-Hybrid Analysis.

IF 2.3

Journal of biophotonics Pub Date : 2025-04-23 DOI:10.1002/jbio.70033

Zhiqiang Wei, Yanling Xu, Jingzhen Wang, Beini Sun, Qialing Huang, Zhengfei Zhuang, Tongsheng Chen, Min Hu

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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 $\to$ 30 min) for three-channel FRET imaging. Validation with FRET standards (C32V: $E_{D}^{C 32 V} = 0.30 \pm 0.01$ , $S^{C 32 V} = 1.06 \pm 0.14$ ; CVC: $E_{D}^{CVC} = 0.40 \pm 0.02$ , $S^{CVC} = 1.90 \pm 0.11$ ) matched reference values. Applied to Bcl-xL/Bak interactions under A1331852 treatment, LURS revealed dose-dependent stoichiometry reduction ( $1.87 \to 1.12$ ). The method achieved precise signal extraction while preserving native cellular conditions, overcoming throughput constraints in dynamic protein interaction studies.

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自动FRET双混合分析。

荧光共振能量转移（FRET）双杂交分析使活细胞检测生物分子复合物，但面临高通量筛选（HTS）的限制，由于费力的图像分析。我们开发了一个自动化平台，使用基于亮度均匀性的兴趣区域选择（LURS）算法，加速处理12倍（6小时→$$ \to $$ 30分钟）用于三通道FRET成像。用FRET标准验证(C32V: E D C32V = 0.30±0.01 $$ {E}_D^{\mathrm{C}32\mathrm{V}}=0.30\pm 0.01 $$, S C32V = 1.06±0.14 $$ {S}^{\mathrm{C}32\mathrm{V}}=1.06\pm 0.14 $$；CVC: E - D CVC = 0.40±0.02 $$ {E}_D^{\mathrm{CVC}}=0.40\pm 0.02 $$, S - CVC = 1.90±0.11 $$ {S}^{\mathrm{CVC}}=1.90\pm 0.11 $$)符合参考值。应用于A1331852处理下的Bcl-xL/Bak相互作用，LURS显示剂量依赖性化学计量减少（1.87→1.12 $$ 1.87\to 1.12 $$）。该方法在保持原生细胞条件的同时实现了精确的信号提取，克服了动态蛋白质相互作用研究中的吞吐量限制。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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Journal of biophotonics

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