Short single-stranded DNAs with putative non-canonical structures comprise a new class of plasma cell-free DNA

Abstract Background 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. Results 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. Conclusions We propose a new class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.

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Short single-stranded DNA with putative non-canonical structures comprises a novel class of plasma cell-free DNA

10.1101/2021.05.25.445702 ◽

2021 ◽

Author(s):

Osamu Hisano ◽

Takashi Ito ◽

Fumihito Miura

Keyword(s):

Binding Sites ◽

Open Chromatin ◽

Cell Free Dna ◽

Single Stranded Dna ◽

Dna Structures ◽

Free Dna ◽

G Quadruplex ◽

Labeling Method ◽

Purification Protocol ◽

Library Preparation Protocol

Cell-free DNA (cfDNA) in human blood is currently investigated as a minimally invasive, highly informative biomarker. Here, we aimed to investigate the existence of the 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 human plasma, present at a molar concentration comparable to that of the nucleosome-sized fragments. These short fragments cannot be recovered by widely used cfDNA isolation methods, and are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. 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 peaks called with these short fragments. Hence, we propose a novel class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.

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Oxidized Extracellular DNA as a Stress Signal in Human Cells

Oxidative Medicine and Cellular Longevity ◽

10.1155/2013/649747 ◽

2013 ◽

Vol 2013 ◽

pp. 1-12 ◽

Cited By ~ 58

Author(s):

Aleksei V. Ermakov ◽

Marina S. Konkova ◽

Svetlana V. Kostyuk ◽

Vera L. Izevskaya ◽

Ancha Baranova ◽

...

Keyword(s):

Oxidative Stress ◽

Nuclear Translocation ◽

Cultured Cells ◽

Oxidative Modification ◽

Extracellular Dna ◽

Related Factor ◽

Dna Fragments ◽

Cell Free Dna ◽

Free Dna ◽

Stress Signal

The term “cell-free DNA” (cfDNA) was recently coined for DNA fragments from plasma/serum, while DNA present inin vitrocell culture media is known as extracellular DNA (ecDNA). Under oxidative stress conditions, the levels of oxidative modification of cellular DNA and the rate of cell death increase. Dying cells release their damaged DNA, thus, contributing oxidized DNA fragments to the pool of cfDNA/ecDNA. Oxidized cell-free DNA could serve as a stress signal that promotes irradiation-induced bystander effect. Evidence points to TLR9 as a possible candidate for oxidized DNA sensor. An exposure to oxidized ecDNA stimulates a synthesis of reactive oxygen species (ROS) that evokes an adaptive response that includes transposition of the homologous loci within the nucleus, polymerization and the formation of the stress fibers of the actin, as well as activation of the ribosomal gene expression, and nuclear translocation of NF-E2 related factor-2 (NRF2) that, in turn, mediates induction of phase II detoxifying and antioxidant enzymes. In conclusion, the oxidized DNA is a stress signal released in response to oxidative stress in the cultured cells and, possibly, in the human body; in particular, it might contribute to systemic abscopal effects of localized irradiation treatments.

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Enrichment of short mutant cell-free DNA fragments enhanced detection of pancreatic cancer

EBioMedicine ◽

10.1016/j.ebiom.2019.02.010 ◽

2019 ◽

Vol 41 ◽

pp. 345-356 ◽

Cited By ~ 15

Author(s):

Xiaoyu Liu ◽

Lingxiao Liu ◽

Yuan Ji ◽

Changyu Li ◽

Tao Wei ◽

...

Keyword(s):

Pancreatic Cancer ◽

Mutant Cell ◽

Dna Fragments ◽

Cell Free Dna ◽

Free Dna

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Neutrophil-Extracellular Traps, Cell-Free DNA, and Immunothrombosis in Companion Animals: A Review

Veterinary Pathology ◽

10.1177/0300985819861721 ◽

2019 ◽

Vol 57 (1) ◽

pp. 6-23 ◽

Cited By ~ 4

Author(s):

Robert Goggs ◽

Unity Jeffery ◽

Dana N. LeVine ◽

Ronald H. L. Li

Keyword(s):

Companion Animals ◽

High Mobility ◽

Extracellular Dna ◽

Death Process ◽

Cell Free Dna ◽

Pathogen Invasion ◽

Extracellular Traps ◽

Trap Formation ◽

Free Dna ◽

Extracellular Trap

Immunothrombosis is a potentially beneficial physiological process that aids innate immunity and host defense against pathogen invasion. However, this process can also be damaging when it occurs to excess or in critical blood vessels. Formation of extracellular traps by leukocytes, particularly neutrophils, is central to our understanding of immunothrombosis. In addition to degranulation and phagocytosis, extracellular traps are the third mechanism by which neutrophils combat potential pathogens. These traps consist of extracellular DNA decorated with bactericidal cellular proteins, including elastase, myeloperoxidase, and cathepsins. Neutrophils can release these structures as part of a controlled cell-death process or via a process termed vital NETosis that enables the cells to extrude DNA but remain viable. There is accumulating evidence that NETosis occurs in companion animals, including dogs, horses, and cats, and that it actively contributes to pathogenesis. Numerous studies have been published detailing various methods for identification and quantification of extracellular trap formation, including cell-free DNA, measurements of histones and proteins such as high-mobility group box–1, and techniques involving microscopy and flow cytometry. Here, we outline the present understanding of these phenomena and the mechanisms of extracellular trap formation. We critically review the data regarding measurement of NETosis in companion animals, summarize the existing literature on NETosis in veterinary species, and speculate on what therapeutic options these insights might present to clinicians in the future.

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Sequencing data of cell-free DNA fragments in living-related liver transplantation for inborn errors of metabolism

Data in Brief ◽

10.1016/j.dib.2020.105183 ◽

2020 ◽

Vol 29 ◽

pp. 105183

Author(s):

Xiaofan Zhu ◽

Hoi Ioi Ng ◽

Liming Xuan ◽

Yan Long ◽

Yan Mao ◽

...

Keyword(s):

Liver Transplantation ◽

Inborn Errors Of Metabolism ◽

Dna Fragments ◽

Sequencing Data ◽

Inborn Errors ◽

Cell Free Dna ◽

Free Dna ◽

Living Related Liver Transplantation ◽

Living Related

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Quantification of plasma cell-free DNA levels in dogs with various tumors

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638719880245 ◽

2019 ◽

Vol 31 (6) ◽

pp. 836-843 ◽

Cited By ~ 7

Author(s):

Michihito Tagawa ◽

Genya Shimbo ◽

Hisashi Inokuma ◽

Kazuro Miyahara

Keyword(s):

Disease Progression ◽

Distant Metastasis ◽

Malignant Tumors ◽

Clinical Stage ◽

Extracellular Dna ◽

Dna Integrity ◽

Healthy Controls ◽

Cell Free Dna ◽

Free Dna ◽

Integrity Index

Circulating cell-free DNA (cfDNA) is extracellular DNA released into the bloodstream by apoptotic or necrotic tumor cells, with cfDNA determination proposed as a noninvasive, sensitive marker for the diagnosis of human cancer. We evaluated cfDNA quantification as a diagnostic and prognostic tool in dogs with various tumors. We quantified plasma cfDNA concentration by absolute real-time PCR of long interspersed nuclear elements in 50 dogs with malignant tumors, 13 dogs with benign tumors or nodules, and 11 healthy controls. Six patients with malignant tumors were followed-up, and plasma cfDNA was quantified throughout disease progression. We found that plasma cfDNA concentrations were significantly elevated in dogs with malignant tumors compared with dogs with benign nodules or healthy controls. The DNA integrity index (the ratio between long and short cfDNA fragments) was significantly lower in dogs with malignant tumors compared to healthy controls. Significantly higher cfDNA levels and a lower DNA integrity index were observed in dogs with lymphoma or leukemia, hemangiosarcoma, and distant metastasis; cfDNA levels correlated well with clinical stage and tended to increase during or before periods of disease progression, suggesting potential efficacy of cfDNA for the detection of distant metastasis and to monitor the clinical stage of neoplasia.

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Plasma cell-free DNA and fraction of circulating KRAS mutations as prognostic in patients with metastatic colorectal cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2014.32.3_suppl.490 ◽

2014 ◽

Vol 32 (3_suppl) ◽

pp. 490-490 ◽

Cited By ~ 2

Author(s):

David Sefrioui ◽

Nasrin Vasseur ◽

Richard Sesboüé ◽

France Blanchard ◽

Alice Oden-Gangloff ◽

...

Keyword(s):

Colorectal Cancer ◽

Metastatic Colorectal Cancer ◽

Plasma Cell ◽

Circulating Tumor Dna ◽

Dna Fragments ◽

Wild Type ◽

Kras Mutations ◽

Cell Free Dna ◽

Free Dna ◽

Tumor Dna

490 Background: It has been suggested that detection of circulating tumor DNA may be relevant in patients with metastatic colorectal cancer (mCRC). The main objective of the present study was to evaluate a method based on the TaqMan Mutation Detection Assay (TMDA) for the detection of circulating KRAS mutations in mCRC patients. Moreover, we also investigated the prognostic impact of the plasma cell-free DNA and the fraction of circulating KRAS mutations. Methods: The study was conducted from April to July 2013 and plasma samples were prospectively collected in a series of 35 mCRC patients treated with chemotherapy (CT). QIAamp Circulating Nucleic Acid kit was used for DNA extraction and Quant-iT High Sensitivity dsDNA Assay for cf-DNA quantification. Detection of circulating tumor DNA was based on the KRAS mutations detected in tumour and was performed in plasma by the castPCR Technology TMDA. Response to CT was assessed according to RECIST criteria. The results of plasma cf-DNA and level of mutant DNA fragments were correlated with response and 3-months survival. Results: We isolated and quantified plasma cf-DNA in all patients with a mean concentration of 106 ng/mL. Among them, 18 were wild-type and 17 mutated for KRAS in the tumour. Detection of circulating KRAS mutations was performed with TMDA in 23 patients (10 KRAS wild-type and 13 KRAS mutated). The sensitivity was 62% (8/13) and specificity 100% (0/10) with a level of circulating mutant DNA fragments ranging from 0 to 29%. Plasma cf-DNA and level of circulating mutant DNA were both significantly correlated with the 3-months survival (mean 36 versus 524 ng/mL, p=0.0015 and 2% versus 29%, p<0.0001). There was a non significant trend for response to CT (respectively p=0.14 and p=0.12). Conclusions: TMDA method is a simple, accurate and non-invasive tool for the detection of circulating tumor DNA. Our preliminary results also suggest that plasma cf-DNA and fraction of mutant DNA fragments could be prognostic markers in mCRC patients.

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Cell-free DNA fragments increase transcription in human mesenchymal stem cells, activate tlr-dependent signal pathway and supress apoptosis

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20125806673 ◽

2012 ◽

Vol 58 (6) ◽

pp. 673-683 ◽

Cited By ~ 5

Author(s):

S.V. Kostyuk ◽

E.M. Malinovskaya ◽

A.V. Ermakov ◽

T.D. Smirnova ◽

L.V. Kameneva ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Functional Activity ◽

Human Mesenchymal Stem Cells ◽

Signal Pathway ◽

Signal Pathways ◽

Dna Fragments ◽

Cell Free Dna ◽

Free Dna ◽

Dependent Signal

Human mesenchymal stem cells (MSCs) are now widely adopted in regenerative medicine. However, many questions on the role of different signaling pathways in the regulation of stem cell (SC) functional activity within the organism remain unaswered. In damaged regions the level of cell death increases and DNA fragments from dead cells (cell-free DNA, cfDNA) are accumulated in blood. We showed that in adipose-derived MSCs exposed in vitro to cfDNA fragments the transcription level increased (the total amount of cellular RNA and the rRNA amount rose). GC-rich CfDNA fragments (GC-DNA) activated the TLR9-dependent signal pathway: the expression of TLR9 and of TLR9-signaling pathway adapter - MyD88 - was up-regulated. AT-rich DNA fragments did not increase the TLR9 expression, though, the MyD88 expression level rose. So we suggest that AT-DNA acts via some other receptors that nevertheless activate MyD88-dependent signalling in MSCs. We also showed that cfDNA fragments decreased the activity of caspase, an apoptotic enzyme. So, cfDNA can significantly influence the functional activity of MSC by activating TLR9- and MyD88-dependent signal pathways and lowering the apoptosis level.

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Properties and Application of Cell-Free DNA as a Clinical Biomarker

International Journal of Molecular Sciences ◽

10.3390/ijms22179110 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9110

Author(s):

Felipe Silva de Miranda ◽

Valério Garrone Barauna ◽

Leandro dos Santos ◽

Gustavo Costa ◽

Paula Frizera Vassallo ◽

...

Keyword(s):

Clinical Practice ◽

Venous Blood ◽

Endothelial Damage ◽

Extracellular Dna ◽

Release Mechanism ◽

Blood Collection ◽

Cell Free Dna ◽

Molecular Features ◽

Free Dna ◽

Clinical Biomarker

Biomarkers are valuable tools in clinical practice. In 2001, the National Institutes of Health (NIH) standardized the definition of a biomarker as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. A biomarker has clinical relevance when it presents precision, standardization and reproducibility, suitability to the patient, straightforward interpretation by clinicians, and high sensitivity and/or specificity by the parameter it proposes to identify. Thus, serum biomarkers should have advantages related to the simplicity of the procedures and to the fact that venous blood collection is commonplace in clinical practice. We described the potentiality of cfDNA as a general clinical biomarker and focused on endothelial dysfunction. Circulating cell-free DNA (cfDNA) refers to extracellular DNA present in body fluid that may be derived from both normal and diseased cells. An increasing number of studies demonstrate the potential use of cfDNA as a noninvasive biomarker to determine physiologic and pathologic conditions. However, although still scarce, increasing evidence has been reported regarding using cfDNA in cardiovascular diseases. Here, we have reviewed the history of cfDNA, its source, molecular features, and release mechanism. We also show recent studies that have investigated cfDNA as a possible marker of endothelial damage in clinical settings. In the cardiovascular system, the studies are quite new, and although interesting, stronger evidence is still needed. However, some drawbacks in cfDNA methodologies should be overcome before its recommendation as a biomarker in the clinical setting.

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