核糖核酸
胞苷脱氨酶
RNA编辑
脱氨基
免疫沉淀
细胞生物学
计算生物学
抗体
生物
化学
分子生物学
生物化学
遗传学
基因
酶
出处
期刊:Nature Methods
[Springer Nature]
日期:2019-09-23
卷期号:16 (12): 1275-1280
被引量:276
标识
DOI:10.1038/s41592-019-0570-0
摘要
N6-methyladenosine (m6A) is a widespread RNA modification that influences nearly every aspect of the messenger RNA lifecycle. Our understanding of m6A has been facilitated by the development of global m6A 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 m6A sites. In DART-seq, the cytidine deaminase APOBEC1 is fused to the m6A-binding YTH domain. APOBEC1-YTH expression in cells induces C-to-U deamination at sites adjacent to m6A residues, which are detected using standard RNA-seq. DART-seq identifies thousands of m6A sites in cells from as little as 10 ng of total RNA and can detect m6A accumulation in cells over time. Additionally, we use long-read DART-seq to gain insights into m6A distribution along the length of individual transcripts.
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