Template switching enables chemical probing of native RNA structures

RNAs are often studied in non-native sequence contexts to facilitate structural studies. However, seemingly innocuous changes to an RNA sequence may perturb the native structure and generate inaccurate or ambiguous structural models. To facilitate the investigation of native RNA secondary structure by selective 2' hydroxyl acylation analyzed by primer extension (SHAPE), we engineered an approach that couples minimal enzymatic steps to RNA chemical probing and mutational profiling (MaP) reverse transcription (RT) methods - a process we call template switching and mutational profiling (Switch-MaP). In Switch-MaP, RT templates and additional library sequences are added post-probing through ligation and template switching, capturing reactivities for every nucleotide. For a candidate SAM-I riboswitch, we compared RNA structure models generated by the Switch-MaP approach to those of traditional primer-based MaP, including RNAs with or without appended structure cassettes. Primer-based MaP masked reactivity data in the 5' and 3' ends of the RNA, producing ambiguous ensembles inconsistent with the conserved SAM-I riboswitch secondary structure. Structure cassettes enabled unambiguous modeling of an aptamer construct but introduced non-native interactions in the full-length riboswitch. In contrast, Switch-MaP provided reactivity data for each nucleotide in each RNA and enabled unambiguous modeling of secondary structure, consistent with the conserved SAM-I fold. Switch-MaP is an alternative approach to primer-based and cassette-based chemical probing methods that precludes primer masking and the formation of alternative secondary structures due to non-native sequence elements. Overall design: We developed a template-switching approach to mutational profiling of SHAPE chemical probes to obtain conformational flexibility measurements for all nucleotides of a candidate riboswitch. We compared structural models generated from this new approach to two alternative procedures.

核糖核酸（RNA）常被置于非天然序列环境中开展研究，以助力结构解析工作。然而，对RNA序列进行看似无伤大雅的改动，可能会扰动其天然结构，进而生成不准确或模棱两可的结构模型。为借助选择性2'-羟基酰化引物延伸分析（selective 2' hydroxyl acylation analyzed by primer extension，SHAPE）实现天然RNA二级结构的研究，我们开发了一种将极简酶促步骤与RNA化学探测及突变谱分析（mutational profiling，MaP）逆转录（reverse transcription，RT）方法相结合的策略——我们将该流程命名为模板转换-突变谱分析（template switching and mutational profiling，Switch-MaP）。在Switch-MaP流程中，探针探测完成后，通过连接反应与模板转换步骤添加逆转录模板与额外的文库序列，从而获取每个核苷酸的反应性数据。针对一款候选SAM-I核糖开关，我们将Switch-MaP策略生成的RNA结构模型与传统基于引物的MaP方法所得结果进行了对比，其中涵盖带有或未带有附加结构盒的RNA样本。基于引物的MaP方法会遮蔽RNA 5'端与3'端的反应性数据，导致生成的结构集合模棱两可，与保守的SAM-I核糖开关二级结构不符。结构盒可实现适配体构建体的明确建模，但会在全长核糖开关中引入非天然相互作用。与之形成对比的是，Switch-MaP可获取每条RNA中每个核苷酸的反应性数据，从而实现二级结构的明确建模，且该结果与保守的SAM-I折叠构象一致。Switch-MaP是一种替代基于引物及基于结构盒的化学探测方法的策略，可避免引物遮蔽以及因非天然序列元件引发的非天然二级结构形成。整体实验设计：我们开发了一种针对SHAPE化学探针的突变谱分析模板转换策略，以获取候选核糖开关所有核苷酸的构象柔性信息，并将该新方法生成的结构模型与另外两种实验流程所得结果进行了对比。