添加即得：受外部提供配体调控的活细胞测量用 FRET 受体

Add and Go: FRET Acceptor for Live-Cell Measurements Modulated by Externally Provided Ligand.

机构信息

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklay St. 16/10, 117997 Moscow, Russia.

Pirogov Russian National Research Medical University, Ostrovitianov 1, 117997 Moscow, Russia.

出版信息

Int J Mol Sci. 2022 Apr 15;23(8):4396. doi: 10.3390/ijms23084396.

DOI:10.3390/ijms23084396

PMID:35457212

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9026985/

Abstract

A substantial number of genetically encoded fluorescent sensors rely on the changes in FRET efficiency between fluorescent cores, measured in ratiometric mode, with acceptor photobleaching or by changes in fluorescence lifetime. We report on a modulated FRET acceptor allowing for simplified one-channel FRET measurement based on a previously reported fluorogen-activating protein, DiB1. Upon the addition of the cell-permeable chromophore, the fluorescence of the donor-fluorescent protein mNeonGreen decreases, allowing for a simplified one-channel FRET measurement. The reported chemically modulated FRET acceptor is compatible with live-cell experiments and allows for prolonged time-lapse experiments with dynamic energy transfer evaluation.

摘要

大量的基因编码荧光传感器依赖于荧光团之间的 FRET 效率变化，以比率模式测量，通过接受体光漂白或荧光寿命变化。我们报告了一种调制的 FRET 接受体，允许基于以前报道的荧光蛋白激活蛋白 DiB1 进行简化的单通道 FRET 测量。在添加细胞通透的生色团后，供体荧光蛋白 mNeonGreen 的荧光强度降低，从而实现简化的单通道 FRET 测量。报告的化学调制 FRET 接受体与活细胞实验兼容，并允许进行延长的延时实验，以评估动态能量转移。