DART-seq: an antibody-free method for global m6A detection
DART-seq: an antibody-free method for global m6A detection
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DOI：
10.1038/s41592-019-0570-0
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发表时间：
2019-12-01
期刊：
影响因子：
48
通讯作者：
Meyer, Kate D.
中科院分区：
文献类型：
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作者：
Meyer, Kate D.
N-6-甲基腺苷（m（6）A）是一种广泛存在的RNA修饰，几乎影响信使RNA生命周期的各个方面。我们对m（6）A的理解已经通过全球m（6）A定位方法的发展而得到促进，该方法使用抗体来免疫沉淀甲基化RNA。然而，这些方法有几个局限性，包括高输入RNA要求和与其他RNA修饰的交叉反应性。在这里，我们提出了DART-seq（与RNA修饰靶点相邻的脱氨基作用），这是一种用于检测m（6）A位点的无抗体方法。在DART-seq中，胞苷脱氨酶APOBEC 1与m（6）A结合YTH结构域融合。细胞中的APOBEC 1-YTH表达诱导与m（6）A残基相邻的位点处的C至U脱氨基作用，其使用标准RNA-seq检测。DART-seq从低至10 ng的总RNA中识别出细胞中数千个m（6）A位点，并可以检测细胞中m（6）A随时间的积累。此外，我们使用长读DART-seq来深入了解m（6）A沿单个转录物长度的分布沿着。
N-6-methyladenosine (m(6)A) is a widespread RNA modification that influences nearly every aspect of the messenger RNA life-cycle. Our understanding of m(6)A has been facilitated by the development of global m(6)A mapping methods, which use antibodies to immunoprecipitate methylated RNA. However, these methods have several limitations, including high input RNA requirements and cross-reactivity to other RNA modifications. Here, we present DART-seq (deamination adjacent to RNA modification targets), an antibody-free method for detecting m(6)A sites. In DART-seq, the cytidine deaminase APOBEC1 is fused to the m(6)A-binding YTH domain. APOBEC1-YTH expression in cells induces C-to-U deamination at sites adjacent to m(6)A residues, which are detected using standard RNA-seq. DART-seq identifies thousands of m(6)A sites in cells from as little as 10 ng of total RNA and can detect m(6)A accumulation in cells over time. Additionally, we use long-read DART-seq to gain insights into m(6)A distribution along the length of individual transcripts.