The function and fate of cellular RNAs are often governed by the phosphorylation state at the 5' end or the identity of whatever cap may be present there. Here we describe methods for examining these important 5'-terminal features on any cellular or synthetic RNA of interest that can be detected by Northern blotting. One such method, PABLO, is a splinted ligation assay that makes it possible to accurately quantify the percentage of 5' ends that are monophosphorylated. Another, PACO, is a capping assay that reveals the percentage of 5' ends that are diphosphorylated. A third, boronate gel electrophoresis in conjunction with deoxyribozyme-mediated cleavage, enables different types of caps (e.g., m7Gppp caps versus NAD caps) to be distinguished from one another and the percentage of each to be determined. After completing all three tests, the percentage of 5' ends that are triphosphorylated can be deduced by process of elimination. Together, this battery of assays allows the 5' terminus of an RNA to be profiled in unprecedented detail.

中文翻译：

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RNA 5' 末端的分析：磷酸盐计数和帽表征

细胞 RNA 的功能和命运通常由 5' 端的磷酸化状态或那里可能存在的任何帽的身份控制。在这里，我们描述了在任何细胞或合成 RNA 上检查这些重要的 5'-末端特征的方法，这些特征可以通过北方印迹检测到。一种这样的方法，PABLO，是一种夹板连接分析，它可以准确地量化 5' 端被单磷酸化的百分比。另一个，PACO，是一种加帽分析，它揭示了 5' 末端被二磷酸化的百分比。第三种，硼酸盐凝胶电泳与脱氧核酶介导的切割相结合，可以区分不同类型的帽（例如 m7Gppp 帽与 NAD 帽）并确定每种帽的百分比。完成所有三个测试后，可以通过消除过程推断出三磷酸化的 5' 端的百分比。总之，这一系列检测允许以前所未有的细节分析 RNA 的 5' 末端。