Site-specific fluorescent labeling of RNA is crucial for obtaining the structural and dynamic information of RNAs by fluorescence techniques. Post-synthetic modification of RNA based on N-hydroxysuccinimide (NHS) coupling reaction is an economic, efficient and simple strategy to introduce fluorophore to samples. However, this strategy are not that frequently used in RNA molecules, and the reported reaction conditions and yields varied among different systems. This study results mainly focused on screening the reaction conditions (reactants concentrations, dimethylsulfoxide concentration, solution conditions, pH and reaction time) between NHS-linked fluorophore and aminoallyl-RNA (aa-RNA) to optimize the yield of fluorescent RNA up to 55%, doubled the initial yield. What's more, as low as one tenth of fluorescent reagent was used in our protocol compared with the reported protocols, greatly reducing the experimental cost. The protocol can be applied as a general guide potentially for RNA labeling by NHS-ester coupling reaction.

中文翻译：

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N-羟基琥珀酰亚胺酯与氨基烯丙基修饰的 RNA 偶联用于荧光标记的优化。

RNA 的位点特异性荧光标记对于通过荧光技术获得 RNA 的结构和动态信息至关重要。基于 N-羟基琥珀酰亚胺 (NHS) 偶联反应的 RNA 合成后修饰是一种经济、高效且简单的将荧光团引入样品的策略。然而，这种策略在 RNA 分子中并不常用，并且报告的反应条件和产量因系统而异。本研究结果主要集中在筛选NHS连接的荧光团与氨基烯丙基-RNA（aa-RNA）之间的反应条件（反应物浓度、二甲亚砜浓度、溶液条件、pH和反应时间），以优化荧光RNA的产率高达55% ，使初始收益率翻了一番。更重要的是，与报道的协议相比，我们的协议中使用了低至十分之一的荧光试剂，大大降低了实验成本。该协议可用作 NHS-酯偶联反应进行 RNA 标记的一般指南。