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      Detecting DNA Depurination with Solid-State Nanopores

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          Abstract

          Among the different types of DNA damage that occur endogenously in the cell, depurination is especially prevalent. These lesions can initiate mutagenesis and have been implicated in a variety of diseases, including cancer. Here, we demonstrate a new approach for the detection of depurination at the single-molecule scale using solid-state nanopores. We induce depurination in short duplex DNA using acidic conditions and observe that the presence of apurinic sites results in significantly slower dynamics during electrokinetic translocation. This procedure may be valuable as a diagnostic for in situ quantification of DNA depurination.

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          Solid-state nanopores.

          Cees Dekker (2007)
          The passage of individual molecules through nanosized pores in membranes is central to many processes in biology. Previously, experiments have been restricted to naturally occurring nanopores, but advances in technology now allow artificial solid-state nanopores to be fabricated in insulating membranes. By monitoring ion currents and forces as molecules pass through a solid-state nanopore, it is possible to investigate a wide range of phenomena involving DNA, RNA and proteins. The solid-state nanopore proves to be a surprisingly versatile new single-molecule tool for biophysics and biotechnology.
          Article 0 comments Cited 431 times – based on 0 reviews     Review now
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            The molecular theory of polyelectrolyte solutions with applications to the electrostatic properties of polynucleotides.

            G Manning (1978)
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              Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress.

              F. M. Yakes, B. Van Houten (1997)
              A significant amount of reactive oxygen species (ROS) is generated during mitochondrial oxidative phosphorylation. Several studies have suggested that mtDNA may accumulate more oxidative DNA damage relative to nuclear DNA. This study used quantitative PCR to examine the formation and repair of hydrogen peroxide-induced DNA damage in a 16.2-kb mitochondrial fragment and a 17.7-kb fragment flanking the beta-globin gene. Simian virus 40-transformed fibroblasts treated with 200 microM hydrogen peroxide for 15 or 60 min exhibited 3-fold more damage to the mitochondrial genome compared with the nuclear fragment. Following a 60-min treatment, damage to the nuclear fragment was completely repaired within 1.5 hr, whereas no DNA repair in the mitochondrion was observed. Mitochondrial function, as assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction, also showed a sharp decline. These cells displayed arrested-cell growth, large increases in p21 protein levels, and morphological changes consistent with apoptosis. In contrast, when hydrogen peroxide treatments were limited to 15 min, mtDNA damage was repaired with similar kinetics as the nuclear fragment, mitochondrial function was restored, and cells resumed division within 12 hr. These results indicate that mtDNA is a critical cellular target for ROS. A model is presented in which chronic ROS exposure, found in several degenerative diseases associated with aging, leads to decreased mitochondrial function, increased mitochondrial-generated ROS, and persistent mitochondrial DNA damage. Thus persistent mitochondrial DNA damage may serve as a useful biomarker for ROS-associated diseases.
              Article 0 comments Cited 217 times – based on 0 reviews     Review now
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                Author and article information

                Contributors
                Jerome Mathe: Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (iso-abbrev): PLoS ONE
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, USA )
                ISSN (Electronic): 1932-6203
                Publication date Collection: 2014
                Publication date (Electronic): 2 July 2014
                Volume: 9
                Issue: 7
                Electronic Location Identifier: e101632
                Affiliations
                [1 ]Joint School of Nanoscience and Nanoengineering, University of North Carolina at Greensboro, Greensboro, North Carolina, United States of America
                [2 ]Department of Biomedical Engineering, Wake Forest University School of Medicine, Winston-Salem, North Carolina, United States of America
                [3 ]Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston-Salem, North Carolina, United States of America
                Université d'Evry val d'Essonne, France
                Author notes

                Competing Interests: The authors confirm that Adam Hall is an Editorial Board member at PLoS ONE. This does not alter the authors' adherence to PLoS ONE Editorial policies and criteria.

                Conceived and designed the experiments: MMM ARH. Performed the experiments: MMM. Analyzed the data: MMM. Contributed reagents/materials/analysis tools: JR EWT. Contributed to the writing of the manuscript: MMM ARH.

                Article
                Publisher ID: PONE-D-14-14124
                DOI: 10.1371/journal.pone.0101632
                PMC ID: 4079296
                PubMed ID: 24988437
                SO-VID: b966398a-c5cd-4a9a-acc4-4bd6cc91e65d
                Copyright statement: Copyright @ 2014
                License:

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                Date received : 29 March 2014
                Date accepted : 10 June 2014
                Page count
                Pages: 6
                Funding
                ARH acknowledges start-up funding from the Wake Forest University School of Medicine. JR and EWT acknowledge funding from the Dr. Arthur and Bonnie Ennis Foundation, Decatur, IL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Subject: Research Article
                Subject: Biology and life sciences
                Subject: Biochemistry
                Subject: DNA
                Subject: DNA damage
                Subject: Biophysics
                Subject: Bionanotechnology
                Subject: Biotechnology
                Subject: Small Molecules
                Subject: Engineering and Technology
                Subject: Nanotechnology
                Custom metadata
                Data Availability The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

                ScienceOpen disciplines: Uncategorized
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                ScienceOpen disciplines: Uncategorized

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