Harvard Medical School, Microbiology Department, Boston, Massachusetts.
Utha Valley University, Orem, Utah.
Curr Protoc. 2024 Nov;4(11):e70038. doi: 10.1002/cpz1.70038.
While various methods exist for examining and visualizing the structures of RNA molecules, dimethyl sulfate-mutational profiling and sequencing (DMS-MaPseq) stands out for its simplicity and versatility. This technique has been proven effective for studying RNA structures both in vitro and in complex biological settings. We present an updated protocol of DMS-MaPseq, as well as methodology that enables it to be used for detection of antisense oligonucleotides (ASOs) binding to RNA. By applying this protocol, we successfully characterized the structural ensemble of the HIV1 Rev Response Element (RRE), along with its two alternative structures. The findings align with previously published research validating the accuracy of the method. We also demonstrate the utility of the DMS-MaPseq protocol by resolving and confirming ASO binding at the complementary sites of the P21-associated noncoding RNA DNA damage-activated (PANDA) long non-coding RNA via decreased DMS reactivity. © 2024 Wiley Periodicals LLC. Basic Protocol 1: DMS-MaPseq on HIV1-RRE Basic Protocol 2: DMS-MaPseq on PANDA with ASO probing.
虽然存在多种方法可用于检查和可视化 RNA 分子的结构，但硫酸二甲酯突变分析和测序（DMS-MaPseq）以其简单性和多功能性脱颖而出。该技术已被证明可有效用于研究体外和复杂生物环境中的 RNA 结构。我们提供了 DMS-MaPseq 的更新方案，以及可用于检测与 RNA 结合的反义寡核苷酸（ASO）的方法。通过应用此方案，我们成功地描绘了 HIV1 Rev 反应元件（RRE）的结构组合及其两种替代结构。这些发现与先前发表的研究结果一致，验证了该方法的准确性。我们还通过降低 DMS 反应性来解析和确认 ASO 在 P21 相关非编码 RNA 损伤激活（PANDA）长非编码 RNA 的互补位点上的结合，证明了 DMS-MaPseq 方案的实用性。© 2024 威立出版公司。基本方案 1：DMS-MaPseq 用于 HIV1-RRE 基本方案 2：DMS-MaPseq 用于 PANDA 与 ASO 探测。
