RNAs are involved in interaction networks with other biomolecules and are crucial for proper cell function. Yet their biochemical analysis remains challenging. For Förster Resonance Energy Transfer (FRET), a common tool to study such interaction networks, two interacting molecules have to be fluorescently labeled. “Spinach” is a genetically encodable RNA aptamer that starts to fluoresce upon binding of an organic molecule. Therefore, it is a biological fluorophore tag for RNAs. However, spinach has never been used in a FRET assembly before. Here, we describe how spinach is quenched when close to acceptors. We used RNA–DNA hybridization to bring quenchers or red organic dyes in close proximity to spinach. Furthermore, we investigate RNA–protein interactions quantitatively on the example of Pseudomonas aeruginosa phage coat protein 7 (PP7) and its interacting pp7-RNA. We utilize spinach quenching as a fully genetically encodable system even under lysate conditions. Therefore, this work represents a direct method to analyze RNA–protein interactions by quenching the spinach aptamer.

中文翻译：

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使用特定的RNA-蛋白质相互作用来淬灭荧光RNA菠菜

RNA参与与其他生物分子的相互作用网络，对于正常的细胞功能至关重要。然而，他们的生化分析仍然具有挑战性。对于研究此类相互作用网络的常用工具福斯特共振能量转移（FRET），必须对两个相互作用的分子进行荧光标记。“菠菜”是一种可遗传编码的RNA适体，在结合有机分子后便开始发出荧光。因此，它是RNA的生物荧光标记。但是，菠菜从未在FRET组件中使用过。在这里，我们描述了菠菜在接近受体时如何被淬灭。我们使用RNA-DNA杂交使淬灭剂或红色有机染料紧邻菠菜。此外，我们以铜绿假单胞菌为例，定量研究RNA-蛋白质相互作用噬菌体外壳蛋白7（PP7）及其相互作用的pp7-RNA。即使在裂解液条件下，我们也利用菠菜淬灭作为完全可遗传编码的系统。因此，这项工作代表了一种通过淬灭菠菜适体来分析RNA与蛋白质相互作用的直接方法。