A General LC-MS-Based Method for Direct and De Novo Sequencing of RNA Mixtures Containing both Canonical and Modified Nucleotides.
A General LC-MS-Based Method for Direct and De Novo Sequencing of RNA Mixtures Containing both Canonical and Modified Nucleotides.
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一种基于 LC-MS 的通用方法，用于对含有规范和修饰核苷酸的 RNA 混合物进行直接和从头测序。
DOI：
10.1007/978-1-0716-1374-0_17
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Zhang,Shenglong
中科院分区：
文献类型：
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作者：
Zhang,Ning;Shi,Shundi;Yuan,Xiaohong;Ni,Wenhao;Wang,Xuanting;Yoo,Barney;Jia,TonyZ;Li,Wenjia;Zhang,Shenglong
与基于dna的RNA测序方法相比，基于质谱（MS）的测序在直接测序RNA方面具有优势，因为它完全不依赖于酶和样品制备过程中的碱基互补误差。此外，它允许在一项研究中对不同的RNA修饰进行测序，而不是每次研究只对一种特定的修饰类型进行测序。然而，在RNA的重新MS测序中仍然存在许多技术挑战，使得难以对混合RNA进行MS测序或区分异构体修饰，如假尿嘧啶（Ψ）和尿嘧啶(U)。我们最近的研究将二维疏水末端标记策略整合到MS-based测序（2D-HELS MS Seq）中，以系统地解决当前RNA的MS测序挑战，使其能够直接和从头测序纯化的单个RNA和含有典型核苷酸和修饰核苷酸的混合RNA。在这里，我们描述了使用2D-HELS MS Seq对代表性单rna和混合rna寡核苷酸进行测序的方法，每种核苷酸具有不同的序列和/或含有修饰的核苷酸，如Ψ和5-甲基胞嘧啶（m5C）。
Mass spectrometry (MS)-based sequencing has advantages in direct sequencing of RNA, compared to cDNA-based RNA sequencing methods, as it is completely independent of enzymes and base complementarity errors in sample preparation. In addition, it allows for sequencing of different RNA modifications in a single study, rather than just one specific modification type per study. However, many technical challenges remain in de novo MS sequencing of RNA, making it difficult to MS sequence mixed RNAs or to differentiate isomeric modifications such as pseudouridine (Ψ) from uridine (U). Our recent study incorporates a two-dimensional hydrophobic end labeling strategy into MS-based sequencing (2D-HELS MS Seq) to systematically address the current challenges in MS sequencing of RNA, making it possible to directly and de novo sequence purified single RNA and mixed RNA containing both canonical and modified nucleotides. Here, we describe the method to sequence representative single-RNA and mixed-RNA oligonucleotides, each with a different sequence and/or containing modified nucleotides such as Ψ and 5-methylcytosine (m5C), using 2D-HELS MS Seq.