Sarfraz Nadia, Shafik Luke K, Stickelman Zachary R, Shankar Uma, Moscoso Emilia, Braselmann Esther
Department of Chemistry, Georgetown University Washington District of Columbia USA
RSC Chem Biol. 2024 Jan 8;5(2):109-116. doi: 10.1039/d3cb00197k. eCollection 2024 Feb 7.
We recently developed Riboglow-FLIM, where we genetically tag and track RNA molecules in live cells through measuring the fluorescence lifetime of a small molecule probe that binds the RNA tag. Here, we systematically and quantitatively evaluated key elements of Riboglow-FLIM that may serve as the foundation for Riboglow-FLIM applications and further tool development efforts. Our investigation focused on measuring changes in fluorescence lifetime of representative Riboglow-FLIM probes with different linkers and fluorophores in different environments. measurements revealed distinct lifetime differences among the probe variants as a result of different linker designs and fluorophore selections. To expand on the platform's versatility, probes in a wide variety of mammalian cell types were examined using fluorescence lifetime imaging microscopy (FLIM), and possible effects on cell physiology were evaluated by metabolomics. The results demonstrated that variations in lifetime were dependent on both probe and cell type. Interestingly, distinct differences in lifetime values were observed between cell lines, while no overall change in cell health was measured. These findings underscore the importance of probe selection and cellular environment when employing Riboglow-FLIM for RNA detection, serving as a foundation for future tool development and applications across diverse fields and biological systems.
我们最近开发了Riboglow-FLIM，通过测量与RNA标签结合的小分子探针的荧光寿命，对活细胞中的RNA分子进行基因标记和追踪。在此，我们系统地、定量地评估了Riboglow-FLIM的关键要素，这些要素可能为Riboglow-FLIM的应用以及进一步的工具开发奠定基础。我们的研究重点是测量具有不同连接子和荧光团的代表性Riboglow-FLIM探针在不同环境下荧光寿命的变化。测量结果显示，由于连接子设计和荧光团选择的不同，探针变体之间存在明显的寿命差异。为了扩展该平台的通用性，我们使用荧光寿命成像显微镜（FLIM）对多种哺乳动物细胞类型中的探针进行了检测，并通过代谢组学评估了其对细胞生理学的可能影响。结果表明，寿命变化既取决于探针，也取决于细胞类型。有趣的是，不同细胞系之间观察到了寿命值的明显差异，但未检测到细胞健康状况的总体变化。这些发现强调了在使用Riboglow-FLIM进行RNA检测时探针选择和细胞环境的重要性，为未来跨不同领域和生物系统的工具开发和应用奠定了基础。
