Abstract
BackgroundCell-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.ResultsWe 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.ConclusionsWe propose a new class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.
Highlights
Cell-free DNA, which is extracellular DNA present in the circulating plasma and other body fluids, is currently investigated as a minimally invasive, highly informative biomarker
In this study, we investigated the existence of Cell-free DNA (cfDNA) fragments shorter than the well-studied nucleosomesized fragments
With an improved cfDNA purification protocol combined with a 3′-end fluorescent labeling method, we detected single-stranded DNA (ssDNA) of approximately 50 nt in length in the human plasma at a concentration comparable to that of the conventional cfDNA
Summary
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. Cell-free DNA (cfDNA) in the blood is considered a promising biomarker that can be measured with minimal invasion (i.e., liquid biopsy) [1, 2]. Detecting fetal cfDNA in the mother’s blood is a current practice that enables safe prenatal diagnosis [3]. The half-life of cfDNA in the bloodstream is generally short. Fetal cfDNA rapidly declines in the mother's blood after delivery, leading to an estimated half-life close to 1 h [8].
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