评估用于 RNA 感测的 Riboglow-FLIM 探针

IF 4.2 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Nadia Sarfraz, Luke K. Shafik, Zachary R. Stickelman, Uma Shankar, Emilia Moscoso and Esther Braselmann
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引用次数: 0

摘要

我们最近开发了 Riboglow-FLIM,通过测量与 RNA 标记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跟踪。在这里,我们系统地定量评估了 Riboglow-FLIM 的关键要素,这些要素可作为 Riboglow-FLIM 应用和进一步工具开发工作的基础。我们的研究重点是测量带有不同连接体和荧光团的代表性 Riboglow-FLIM 探针在不同环境下的荧光寿命变化。体外测量结果表明,由于连接体设计和荧光团选择的不同,探针变体之间的荧光寿命存在明显差异。为了扩展该平台的多功能性,使用荧光寿命成像显微镜(FLIM)对各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对细胞生理的可能影响。结果表明,荧光寿命的变化取决于探针和细胞类型。有趣的是,不同细胞系的探针寿命值存在明显差异,而细胞健康状况没有发生整体变化。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,为未来不同领域和生物系统的工具开发和应用奠定了基础。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Evaluating Riboglow-FLIM probes for RNA sensing†

Evaluating Riboglow-FLIM probes for RNA sensing†

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. In vitro 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.

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来源期刊
CiteScore
6.10
自引率
0.00%
发文量
128
审稿时长
10 weeks
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