{"title":"开发用于观察双分子活性的 mKate3/HaloTag7 (JFX650) 和 CFP/YFP 双荧光(或佛斯特)共振能量转移对","authors":"Wenjing Wang, Jie Yang","doi":"10.1021/acssensors.4c01444","DOIUrl":null,"url":null,"abstract":"Although several imaging strategies for dual fluorescence (or Förster) resonance energy transfer (FRET) biosensors have been reported, their implementation is challenging because of the limited performance of fluorescent proteins and the spectral overlap of FRET biosensors. These processes often require additional data calibration to eliminate artifacts. Many CFP/YFP FRET biosensors have been developed. In this study, we introduced the mKate3/HT7(JFX650) FRET pair, which effectively formed two pairs of FRET pairs for dual-FRET imaging when combined with the CFP/YFP FRET pair. The FRET donor mKate3 exhibited higher brightness than its predecessor mKate. The FRET acceptor, HT7(JFX650), is a HaloTag7 protein covalently conjugated with a far-red JFX650-THL ligand. The pair comprising mKate3 and HT7(JFX650) represents an excellent FRET dyad, exhibiting a high FRET efficiency ratio. To use the FRET pair for dual FRET biosensor imaging, we constructed PKA and K<sup>+</sup> biosensors based on the mKate3/HT7(JFX650) FRET pair. These biosensors can be used along with CFP/YFP biosensors to simultaneously detect the responses of intracellular PKA/Src, PKA/Ca<sup>2+</sup>, and K<sup>+</sup>/Ca<sup>2+</sup> under different stimuli. The findings revealed that dual FRET biosensors, which are based on the combination of CFP/YFP and mKate3/HT7 (JFX650), exhibit adequate compatibility and can be used to visualize multiple molecular activities in a live cell.","PeriodicalId":24,"journal":{"name":"ACS Sensors","volume":null,"pages":null},"PeriodicalIF":8.2000,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Development of mKate3/HaloTag7 (JFX650) and CFP/YFP Dual-Fluorescence (or Förster) Resonance Energy Transfer Pairs for Visualizing Dual-Molecular Activity\",\"authors\":\"Wenjing Wang, Jie Yang\",\"doi\":\"10.1021/acssensors.4c01444\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Although several imaging strategies for dual fluorescence (or Förster) resonance energy transfer (FRET) biosensors have been reported, their implementation is challenging because of the limited performance of fluorescent proteins and the spectral overlap of FRET biosensors. These processes often require additional data calibration to eliminate artifacts. Many CFP/YFP FRET biosensors have been developed. In this study, we introduced the mKate3/HT7(JFX650) FRET pair, which effectively formed two pairs of FRET pairs for dual-FRET imaging when combined with the CFP/YFP FRET pair. The FRET donor mKate3 exhibited higher brightness than its predecessor mKate. The FRET acceptor, HT7(JFX650), is a HaloTag7 protein covalently conjugated with a far-red JFX650-THL ligand. The pair comprising mKate3 and HT7(JFX650) represents an excellent FRET dyad, exhibiting a high FRET efficiency ratio. To use the FRET pair for dual FRET biosensor imaging, we constructed PKA and K<sup>+</sup> biosensors based on the mKate3/HT7(JFX650) FRET pair. These biosensors can be used along with CFP/YFP biosensors to simultaneously detect the responses of intracellular PKA/Src, PKA/Ca<sup>2+</sup>, and K<sup>+</sup>/Ca<sup>2+</sup> under different stimuli. The findings revealed that dual FRET biosensors, which are based on the combination of CFP/YFP and mKate3/HT7 (JFX650), exhibit adequate compatibility and can be used to visualize multiple molecular activities in a live cell.\",\"PeriodicalId\":24,\"journal\":{\"name\":\"ACS Sensors\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":8.2000,\"publicationDate\":\"2024-09-28\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"ACS Sensors\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://doi.org/10.1021/acssensors.4c01444\",\"RegionNum\":1,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"CHEMISTRY, ANALYTICAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Sensors","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1021/acssensors.4c01444","RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}
Development of mKate3/HaloTag7 (JFX650) and CFP/YFP Dual-Fluorescence (or Förster) Resonance Energy Transfer Pairs for Visualizing Dual-Molecular Activity
Although several imaging strategies for dual fluorescence (or Förster) resonance energy transfer (FRET) biosensors have been reported, their implementation is challenging because of the limited performance of fluorescent proteins and the spectral overlap of FRET biosensors. These processes often require additional data calibration to eliminate artifacts. Many CFP/YFP FRET biosensors have been developed. In this study, we introduced the mKate3/HT7(JFX650) FRET pair, which effectively formed two pairs of FRET pairs for dual-FRET imaging when combined with the CFP/YFP FRET pair. The FRET donor mKate3 exhibited higher brightness than its predecessor mKate. The FRET acceptor, HT7(JFX650), is a HaloTag7 protein covalently conjugated with a far-red JFX650-THL ligand. The pair comprising mKate3 and HT7(JFX650) represents an excellent FRET dyad, exhibiting a high FRET efficiency ratio. To use the FRET pair for dual FRET biosensor imaging, we constructed PKA and K+ biosensors based on the mKate3/HT7(JFX650) FRET pair. These biosensors can be used along with CFP/YFP biosensors to simultaneously detect the responses of intracellular PKA/Src, PKA/Ca2+, and K+/Ca2+ under different stimuli. The findings revealed that dual FRET biosensors, which are based on the combination of CFP/YFP and mKate3/HT7 (JFX650), exhibit adequate compatibility and can be used to visualize multiple molecular activities in a live cell.
期刊介绍:
ACS Sensors is a peer-reviewed research journal that focuses on the dissemination of new and original knowledge in the field of sensor science, particularly those that selectively sense chemical or biological species or processes. The journal covers a broad range of topics, including but not limited to biosensors, chemical sensors, gas sensors, intracellular sensors, single molecule sensors, cell chips, and microfluidic devices. It aims to publish articles that address conceptual advances in sensing technology applicable to various types of analytes or application papers that report on the use of existing sensing concepts in new ways or for new analytes.