Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, 812-8582, Japan.
BMC Biol. 2021 Oct 14;19(1):225. doi: 10.1186/s12915-021-01160-8.
Cell-free DNA (cfDNA), which is extracellular DNA present in the circulating plasma and other body fluids, is currently investigated as a minimally invasive, highly informative biomarker. While nucleosome-sized cfDNA fragments have been investigated intensively, shorter DNA fragments in the plasma have not been studied due to several technical limitations.
We aimed to investigate the existence of shorter cfDNA fragments in the blood. Using an improved cfDNA purification protocol and a 3'-end-labeling method, we found DNA fragments of approximately 50 nucleotides in length in the human plasma, present at a molar concentration comparable to that of nucleosome-sized fragments. Unfortunately, these short fragments cannot be recovered by widely used cfDNA isolation methods. In addition, they are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. Therefore, we established a library-preparation protocol based on our unique ssDNA ligation technique and applied it to the isolated cfDNA. Deep sequencing of these libraries revealed that the short fragments are derived from hundreds of thousands of genomic sites in open chromatin regions and enriched with transcription factor-binding sites. Remarkably, antisense strands of putative G-quadruplex motifs occupy as much as one-third of the peaks by these short fragments.
We propose a new class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.
无细胞 DNA（cfDNA）是存在于循环血浆和其他体液中的细胞外 DNA，目前正作为一种微创、高信息量的生物标志物进行研究。虽然核小体大小的 cfDNA 片段已得到深入研究，但由于存在若干技术限制，血浆中的较短 DNA 片段尚未得到研究。
我们旨在研究血液中是否存在较短的 cfDNA 片段。使用改良的 cfDNA 纯化方案和 3'-末端标记方法，我们在人血浆中发现了约 50 个核苷酸长度的 DNA 片段，其摩尔浓度与核小体大小片段相当。不幸的是，这些短片段不能通过广泛使用的 cfDNA 分离方法回收。此外，它们由单链 DNA（ssDNA）组成，因此在之前的研究中未被检测到。因此，我们建立了一种基于我们独特的 ssDNA 连接技术的文库制备方案，并将其应用于分离的 cfDNA。对这些文库进行深度测序表明，这些短片段源自开放染色质区域的数十万基因组位点，并富含转录因子结合位点。值得注意的是，这些短片段的峰中有三分之一左右由潜在 G-四链体模体的反义链占据。
我们提出了一类新的血浆 cfDNA，由短的单链片段组成，这些片段可能形成非典型的 DNA 结构。
