Zhiqiang Wei, Yanling Xu, Jingzhen Wang, Beini Sun, Qialing Huang, Zhengfei Zhuang, Tongsheng Chen, Min Hu
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{"title":"自动FRET双混合分析。","authors":"Zhiqiang Wei, Yanling Xu, Jingzhen Wang, Beini Sun, Qialing Huang, Zhengfei Zhuang, Tongsheng Chen, Min Hu","doi":"10.1002/jbio.70033","DOIUrl":null,"url":null,"abstract":"<p><p>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 <math> <semantics><mrow><mo>→</mo></mrow> <annotation>$$ \\to $$</annotation></semantics> </math> 30 min) for three-channel FRET imaging. Validation with FRET standards (C32V: <math> <semantics> <mrow><msubsup><mi>E</mi> <mi>D</mi> <mrow><mi>C</mi> <mn>32</mn> <mi>V</mi></mrow> </msubsup> <mo>=</mo> <mn>0.30</mn> <mo>±</mo> <mn>0.01</mn></mrow> <annotation>$$ {E}_D^{\\mathrm{C}32\\mathrm{V}}=0.30\\pm 0.01 $$</annotation></semantics> </math> , <math> <semantics> <mrow><msup><mi>S</mi> <mrow><mi>C</mi> <mn>32</mn> <mi>V</mi></mrow> </msup> <mo>=</mo> <mn>1.06</mn> <mo>±</mo> <mn>0.14</mn></mrow> <annotation>$$ {S}^{\\mathrm{C}32\\mathrm{V}}=1.06\\pm 0.14 $$</annotation></semantics> </math> ; CVC: <math> <semantics> <mrow><msubsup><mi>E</mi> <mi>D</mi> <mi>CVC</mi></msubsup> <mo>=</mo> <mn>0.40</mn> <mo>±</mo> <mn>0.02</mn></mrow> <annotation>$$ {E}_D^{\\mathrm{CVC}}=0.40\\pm 0.02 $$</annotation></semantics> </math> , <math> <semantics> <mrow><msup><mi>S</mi> <mi>CVC</mi></msup> <mo>=</mo> <mn>1.90</mn> <mo>±</mo> <mn>0.11</mn></mrow> <annotation>$$ {S}^{\\mathrm{CVC}}=1.90\\pm 0.11 $$</annotation></semantics> </math> ) matched reference values. Applied to Bcl-xL/Bak interactions under A1331852 treatment, LURS revealed dose-dependent stoichiometry reduction ( <math> <semantics><mrow><mn>1.87</mn> <mo>→</mo> <mn>1.12</mn></mrow> <annotation>$$ 1.87\\to 1.12 $$</annotation></semantics> </math> ). The method achieved precise signal extraction while preserving native cellular conditions, overcoming throughput constraints in dynamic protein interaction studies.</p>","PeriodicalId":94068,"journal":{"name":"Journal of biophotonics","volume":" ","pages":"e70033"},"PeriodicalIF":2.3000,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Automated FRET Two-Hybrid Analysis.\",\"authors\":\"Zhiqiang Wei, Yanling Xu, Jingzhen Wang, Beini Sun, Qialing Huang, Zhengfei Zhuang, Tongsheng Chen, Min Hu\",\"doi\":\"10.1002/jbio.70033\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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 <math> <semantics><mrow><mo>→</mo></mrow> <annotation>$$ \\\\to $$</annotation></semantics> </math> 30 min) for three-channel FRET imaging. Validation with FRET standards (C32V: <math> <semantics> <mrow><msubsup><mi>E</mi> <mi>D</mi> <mrow><mi>C</mi> <mn>32</mn> <mi>V</mi></mrow> </msubsup> <mo>=</mo> <mn>0.30</mn> <mo>±</mo> <mn>0.01</mn></mrow> <annotation>$$ {E}_D^{\\\\mathrm{C}32\\\\mathrm{V}}=0.30\\\\pm 0.01 $$</annotation></semantics> </math> , <math> <semantics> <mrow><msup><mi>S</mi> <mrow><mi>C</mi> <mn>32</mn> <mi>V</mi></mrow> </msup> <mo>=</mo> <mn>1.06</mn> <mo>±</mo> <mn>0.14</mn></mrow> <annotation>$$ {S}^{\\\\mathrm{C}32\\\\mathrm{V}}=1.06\\\\pm 0.14 $$</annotation></semantics> </math> ; CVC: <math> <semantics> <mrow><msubsup><mi>E</mi> <mi>D</mi> <mi>CVC</mi></msubsup> <mo>=</mo> <mn>0.40</mn> <mo>±</mo> <mn>0.02</mn></mrow> <annotation>$$ {E}_D^{\\\\mathrm{CVC}}=0.40\\\\pm 0.02 $$</annotation></semantics> </math> , <math> <semantics> <mrow><msup><mi>S</mi> <mi>CVC</mi></msup> <mo>=</mo> <mn>1.90</mn> <mo>±</mo> <mn>0.11</mn></mrow> <annotation>$$ {S}^{\\\\mathrm{CVC}}=1.90\\\\pm 0.11 $$</annotation></semantics> </math> ) matched reference values. Applied to Bcl-xL/Bak interactions under A1331852 treatment, LURS revealed dose-dependent stoichiometry reduction ( <math> <semantics><mrow><mn>1.87</mn> <mo>→</mo> <mn>1.12</mn></mrow> <annotation>$$ 1.87\\\\to 1.12 $$</annotation></semantics> </math> ). The method achieved precise signal extraction while preserving native cellular conditions, overcoming throughput constraints in dynamic protein interaction studies.</p>\",\"PeriodicalId\":94068,\"journal\":{\"name\":\"Journal of biophotonics\",\"volume\":\" \",\"pages\":\"e70033\"},\"PeriodicalIF\":2.3000,\"publicationDate\":\"2025-04-23\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of biophotonics\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1002/jbio.70033\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of biophotonics","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1002/jbio.70033","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
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