Yang Yuening, Lu Yanming, Wang Yan, Wen Xianghui, Qi Changhai, Piao Weilan, Jin Hua
Laboratory of Genetics and Disorders, Key Laboratory of Molecular Medicine and Biotherapy, Aerospace Center Hospital, School of Life Science, Beijing Institute of Technology, Beijing, China.
Department of Pathology, Aerospace Center Hospital, Beijing, China.
Front Cell Dev Biol. 2024 Jul 11;12:1392159. doi: 10.3389/fcell.2024.1392159. eCollection 2024.
Various methods have been developed so far for detecting -methyladenosine (mA). The total mA level or the mA status at individual positions on mRNA can be detected and quantified through some sequencing-independent biochemical methods, such as LC/MS, SCARLET, SELECT, and mA-ELISA. However, the mA-detection techniques relying on high-throughput sequencing have more effectively advanced the understanding about biological significance of mA-containing mRNA and mA pathway at a transcriptomic level over the past decade. Various SGS-based (Second Generation Sequencing-based) methods with different detection principles have been widely employed for this purpose. These principles include mA-enrichment using antibodies, discrimination of mA from unmodified A-base by nucleases, a fusion protein strategy relying on RNA-editing enzymes, and marking mA with chemical/biochemical reactions. Recently, TGS-based (Third Generation Sequencing-based) methods have brought a new trend by direct mA-detection. This review first gives a brief introduction of current knowledge about mA biogenesis and function, and then comprehensively describes mA-profiling strategies including their principles, procedures, and features. This will guide users to pick appropriate methods according to research goals, give insights for developing novel techniques in varying areas, and continue to expand our boundary of knowledge on mA.
到目前为止，已经开发出了各种检测N6-甲基腺苷（mA）的方法。通过一些不依赖测序的生化方法，如液相色谱/质谱联用（LC/MS）、 Scarlet法、SELECT法和mA酶联免疫吸附测定（mA-ELISA），可以检测和定量mRNA上各个位置的总mA水平或mA状态。然而，在过去十年中，依赖高通量测序的mA检测技术在转录组水平上更有效地推进了人们对含mA的mRNA和mA通路生物学意义的理解。为此，各种基于第二代测序（SGS）且检测原理不同的方法已被广泛应用。这些原理包括使用抗体富集mA、通过核酸酶区分mA与未修饰的A碱基、基于RNA编辑酶的融合蛋白策略以及通过化学/生化反应标记mA。最近，基于第三代测序（TGS）的方法通过直接检测mA带来了新趋势。本综述首先简要介绍了目前关于mA生物合成和功能的知识，然后全面描述了mA分析策略，包括其原理、步骤和特点。这将指导用户根据研究目标选择合适的方法，为不同领域开发新技术提供思路，并不断拓展我们对mA的知识边界。
