Automated FRET Two-Hybrid Analysis

IF 2 3区物理与天体物理 Q3 BIOCHEMICAL RESEARCH METHODS

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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来源期刊

Journal of Biophotonics 生物-生化研究方法

CiteScore

5.70

自引率

7.10%

发文量

248

审稿时长

1 months

期刊介绍： The first international journal dedicated to publishing reviews and original articles from this exciting field, the Journal of Biophotonics covers the broad range of research on interactions between light and biological material. The journal offers a platform where the physicist communicates with the biologist and where the clinical practitioner learns about the latest tools for the diagnosis of diseases. As such, the journal is highly interdisciplinary, publishing cutting edge research in the fields of life sciences, medicine, physics, chemistry, and engineering. The coverage extends from fundamental research to specific developments, while also including the latest applications.