结构化RNA中碱基对推断的mutate-and-map协议
A mutate-and-map protocol for inferring base pairs in structured RNA
摘要：
化学作图是用于核酸结构分析的广泛技术，其中分子对不同探针的反应性以单核苷酸分辨率定量并用于约束结构建模。在过去的十年中，这个实验框架被广泛地重新审视，采用了高通量读出、化学修饰和快速数据分析的新策略。最近，我们将该技术与高通量诱变相结合。碱基配对核苷酸的点突变不仅会导致该核苷酸暴露，还会导致其相互作用配偶体暴露。对整个系统进行突变和映射，给出了分子接触映射的实验近似。在这里，我们给出了基于96孔毛细管电泳的这种突变和映射策略的内部协议，并提供了解释数据以推断核酸结构的实用技巧。
英文摘要：
Chemical mapping is a widespread technique for structural analysis of nucleic acids in which a molecule's reactivity to different probes is quantified at single-nucleotide resolution and used to constrain structural modeling. This experimental framework has been extensively revisited in the past decade with new strategies for high-throughput read-outs, chemical modification, and rapid data analysis. Recently, we have coupled the technique to high-throughput mutagenesis. Point mutations of a base-paired nucleotide can lead to exposure of not only that nucleotide but also its interaction partner. Carrying out the mutation and mapping for the entire system gives an experimental approximation of the molecules contact map. Here, we give our in-house protocol for this mutate-and-map strategy, based on 96-well capillary electrophoresis, and we provide practical tips on interpreting the data to infer nucleic acid structure.
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