Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, 19104, USA.
Department of Systems Pharmacology and Translational Therapeutics Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
Nat Commun. 2023 Nov 14;14(1):7346. doi: 10.1038/s41467-023-43158-6.
Genomic DNA (gDNA) undergoes structural interconversion between single- and double-stranded states during transcription, DNA repair and replication, which is critical for cellular homeostasis. We describe "CHEX-seq" which identifies the single-stranded DNA (ssDNA) in situ in individual cells. CHEX-seq uses 3'-terminal blocked, light-activatable probes to prime the copying of ssDNA into complementary DNA that is sequenced, thereby reporting the genome-wide single-stranded chromatin landscape. CHEX-seq is benchmarked in human K562 cells, and its utilities are demonstrated in cultures of mouse and human brain cells as well as immunostained spatially localized neurons in brain sections. The amount of ssDNA is dynamically regulated in response to perturbation. CHEX-seq also identifies single-stranded regions of mitochondrial DNA in single cells. Surprisingly, CHEX-seq identifies single-stranded loci in mouse and human gDNA that catalyze porphyrin metalation in vitro, suggesting a catalytic activity for genomic ssDNA. We posit that endogenous DNA enzymatic activity is a function of genomic ssDNA.
基因组 DNA（gDNA）在转录、DNA 修复和复制过程中经历单链和双链状态之间的结构转换，这对细胞内稳态至关重要。我们描述了“CHEX-seq”，它可以在单个细胞中原位识别单链 DNA（ssDNA）。CHEX-seq 使用 3'-末端封闭、光激活的探针启动 ssDNA 复制为互补 DNA，然后对其进行测序，从而报告全基因组单链染色质景观。CHEX-seq 在人类 K562 细胞中进行了基准测试，并在小鼠和人类脑细胞培养物以及脑组织切片中免疫染色的空间定位神经元中展示了其用途。ssDNA 的量会根据扰动而动态调节。CHEX-seq 还可以在单个细胞中鉴定线粒体 DNA 的单链区域。令人惊讶的是，CHEX-seq 在小鼠和人类 gDNA 中鉴定出了一些单链位点，这些位点在体外催化卟啉金属化，表明基因组 ssDNA 具有催化活性。我们假设内源性 DNA 酶活性是基因组 ssDNA 的功能。
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