Department of Human Genetics, The University of Chicago, Chicago, IL, USA; Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA.
Department of Chemistry, Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA; Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA.
Mol Cell. 2024 Feb 1;84(3):596-610.e6. doi: 10.1016/j.molcel.2023.12.021. Epub 2024 Jan 11.
Although DNA N-methyl-deoxyadenosine (6mA) is abundant in bacteria and protists, its presence and function in mammalian genomes have been less clear. We present Direct-Read 6mA sequencing (DR-6mA-seq), an antibody-independent method, to measure 6mA at base resolution. DR-6mA-seq employs a unique mutation-based strategy to reveal 6mA sites as misincorporation signatures without any chemical or enzymatic modulation of 6mA. We validated DR-6mA-seq through the successful mapping of the well-characterized G(6mA)TC motif in the E. coli DNA. As expected, when applying DR-6mA-seq to mammalian systems, we found that genomic DNA (gDNA) 6mA abundance is generally low in most mammalian tissues and cells; however, we did observe distinct gDNA 6mA sites in mouse testis and glioblastoma cells. DR-6mA-seq provides an enabling tool to detect 6mA at single-base resolution for a comprehensive understanding of DNA 6mA in eukaryotes.
尽管 DNA N-甲基脱氧腺苷（6mA）在细菌和原生生物中大量存在，但它在哺乳动物基因组中的存在和功能尚不清楚。我们提出了直接读取 6mA 测序（DR-6mA-seq），这是一种无需抗体的方法，可在碱基分辨率上测量 6mA。DR-6mA-seq 采用独特的基于突变的策略，通过错误掺入特征揭示 6mA 位点，而无需对 6mA 进行任何化学或酶修饰。我们通过成功映射大肠杆菌 DNA 中特征良好的 G(6mA)TC 基序验证了 DR-6mA-seq。正如预期的那样，当我们将 DR-6mA-seq 应用于哺乳动物系统时，我们发现大多数哺乳动物组织和细胞中的基因组 DNA（gDNA）6mA 丰度通常较低；然而，我们确实在小鼠睾丸和神经胶质瘤细胞中观察到了不同的 gDNA 6mA 位点。DR-6mA-seq 提供了一种在单碱基分辨率下检测 6mA 的工具，有助于全面了解真核生物中的 DNA 6mA。
