Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, 60637, USA.
Department of Chemistry, University of Chicago, Chicago, IL, 60637, USA.
Angew Chem Int Ed Engl. 2021 Jan 11;60(2):873-880. doi: 10.1002/anie.202007266. Epub 2020 Nov 10.
N -methyladenosine (m A) is a crucial RNA chemical mark which plays important roles in various biological processes. The development of highly multiplexed, cost-effective, and easy-to-operate methodologies for locus-specific analysis of m A is critical for advancing our understanding of the roles of this modification. Herein, we report a method which builds upon the principle of the previously reported SELECT approach by significantly improving its efficiency and coupling it to next generation sequencing technology for high-throughput validation and detection of m A modification at selected sites (LEAD-m A-seq). Through probing cDNA extension mediated by Bst DNA polymerase at and near target cellular sites by sequencing, we evaluated m A modification at these sites, and estimated differential methylation levels (0-84 %) upon in vitro demethylation by the m A demethylase FTO with high reproducibility. We envision that this strategy can be readily used for testing a greater number of sites with a broad dynamic range and modified to study other RNA modifications.
N6 -甲基腺苷（m6A）是一种关键的 RNA 化学标记，在各种生物过程中发挥重要作用。开发高度多重、经济高效且易于操作的方法，用于 m6A 特异性分析，对于深入了解这种修饰的作用至关重要。在此，我们报告了一种方法，该方法基于先前报道的 SELECT 方法的原理，显著提高了其效率，并将其与下一代测序技术相结合，用于在选定的位点（LEAD-m6A-seq）进行 m6A 修饰的高通量验证和检测。通过测序，我们在目标细胞位点及其附近，通过 Bst DNA 聚合酶介导的 cDNA 延伸，评估了这些位点的 m6A 修饰，并在体外通过 m6A 去甲基酶 FTO 进行去甲基化后，以高重复性估计了差异甲基化水平（0-84%）。我们设想，该策略可以很容易地用于测试具有更广泛动态范围的更多位点，并进行修改以研究其他 RNA 修饰。
