An assay for RNA oxidation induced abasic sites using the Aldehyde Reactive Probe

There have been several reports describing elevation of oxidized RNA in ageing or age-related diseases, however RNA oxidation has been assessed solely based on 8-hydroxy-guanosine levels. In this study, Aldehyde Reactive Probe (ARP), which was originally developed to detect DNA abasic sites, was used to assess RNA oxidation. It was found that ARP reacted with depurinated tRNA(Phe) or chemically synthesized RNA containing abasic sites quantitatively to as little as 10 fmoles, indicating that abasic RNA is recognized by ARP. RNA oxidized by Fenton-type reactions, γ-irradiation or peroxynitrite increased ARP reactivity dose-dependently, indicating that ARP is capable of monitoring oxidized RNA mediated by reactive oxygen species or reactive nitrogen species. Furthermore, oxidative stress increased levels of ARP reactive RNA in cultured cells. These results indicate the versatility of the assay method for biologically relevant oxidation of RNA. Thus, this study developed a sensitive assay for analysis of oxidized RNA.     

Improved measurement of dibenzo[a,l]pyrene-induced abasic sites by the aldehyde-reactive probe assay

Dibenzo[a,l]pyrene (DB[a,l]P) induces abundant amounts of depurinating adducts that spontaneously dissociate to form abasic sites in DNA. However, several previous studies that used the aldehyde-reactive probe (ARP) assay, could not verify abasic site formation by DB[a,l]P. Therefore, we examined whether a modification of the ARP assay would allow greater quantification of abasic sites. A previous study indicated that the abasic site quantification is improved by letting ARP trap the nascent abasic sites in cells, before extracting DNA for the assay. To test whether the addition of ARP to the DB[a,l]P–DNA adduct-forming reaction would improve abasic site quantification, we treated calf thymus DNA (0.625 mg/mL) with DB[a,l]P (80 μM) and 3-methylcholanthrene-treated rat liver microsomes with or without ARP (3 mM). The inclusion of ARP in the adduct-forming reaction resulted in significantly greater detection of abasic sites (62 lesions/10⁶ bp versus 3.7 lesions/10⁶ bp). DB[a,l]P also induces DNA strand breaks. The strand breaks may occur at abasic sites and by other mechanisms, such as oxidative damage. ARP/O-methoxyamine-abasic site conjugates are refractory to strand breakage, however, ARP or O-methoxyamine (3–10 mM) could only partially protect DB[a,l]P-induced DNA degradation, presumably by protecting the abasic sites, but not the other strand breaks. These results suggest that if DNA strand breakages occur at the abasic sites or at bases flanking them, and the fragments are lost during DNA extraction, abasic site estimation could be compromised. To obtain an independent line of evidence for abasic site formation in DB[a,l]P-treated cells, mouse Mβ16 fibroblasts were treated with DB[a,l]P and O-methoxyamine. O-Methoxyamine is known to potentiate cytotoxicity of abasic site-inducing chemicals by forming abasic site conjugates, which partially inhibits their repair. O-Methoxyamine was found to increase DB[a,l]P cytotoxicity in these cells, supporting the idea that DB[a,l]P formed abasic sites. In summary, the inclusion of ARP in the DB[a,l]P–DNA adduct-forming reaction traps and protects the nascent abasic sites, allowing an improved quantification of abasic sites.     

Improved measurement of dibenzo[a,l]pyrene-induced abasic sites by the aldehyde-reactive probe assay

Dibenzo[a,l]pyrene (DB[a,l]P) induces abundant amounts of depurinating adducts that spontaneously dissociate to form abasic sites in DNA. However, several previous studies that used the aldehyde-reactive probe (ARP) assay, could not verify abasic site formation by DB[a,l]P. Therefore, we examined whether a modification of the ARP assay would allow greater quantification of abasic sites. A previous study indicated that the abasic site quantification is improved by letting ARP trap the nascent abasic sites in cells, before extracting DNA for the assay. To test whether the addition of ARP to the DB[a,l]P-DNA adduct-forming reaction would improve abasic site quantification, we treated calf thymus DNA (0.625 mg/mL) with DB[a,l]P (80 microM) and 3-methylcholanthrene-treated rat liver microsomes with or without ARP (3 mM). The inclusion of ARP in the adduct-forming reaction resulted in significantly greater detection of abasic sites (62 lesions/10(6) bp versus 3.7 lesions/10(6) bp). DB[a,l]P also induces DNA strand breaks. The strand breaks may occur at abasic sites and by other mechanisms, such as oxidative damage. ARP/O-methoxyamine-abasic site conjugates are refractory to strand breakage, however, ARP or O-methoxyamine (3-10 mM) could only partially protect DB[a,l]P-induced DNA degradation, presumably by protecting the abasic sites, but not the other strand breaks. These results suggest that if DNA strand breakages occur at the abasic sites or at bases flanking them, and the fragments are lost during DNA extraction, abasic site estimation could be compromised. To obtain an independent line of evidence for abasic site formation in DB[a,l]P-treated cells, mouse Mbeta16 fibroblasts were treated with DB[a,l]P and O-methoxyamine. O-Methoxyamine is known to potentiate cytotoxicity of abasic site-inducing chemicals by forming abasic site conjugates, which partially inhibits their repair. O-Methoxyamine was found to increase DB[a,l]P cytotoxicity in these cells, supporting the idea that DB[a,l]P formed abasic sites. In summary, the inclusion of ARP in the DB[a,l]P-DNA adduct-forming reaction traps and protects the nascent abasic sites, allowing an improved quantification of abasic sites.     

Detection of abasic sites and oxidative DNA base damage using an ELISA-like assay

Reactive oxygen species produce a wide spectrum of DNA damage, including oxidative base damage and abasic (AP) sites. Many procedures are available for the quantification and detection of base damage and AP sites. However, either these procedures are laborious or the starting materials are difficult to obtain. A biotinylated aldehyde-specific reagent, ARP, has been shown to react specifically with the aldehyde group present in AP sites, resulting in biotin-tagged AP sites in DNA. The biotin-tagged AP sites can then be determined colorimetrically with an ELISA-like assay, using avidin/biotin-conjugated horseradish peroxidase as the indicator enzyme. The ARP assay is thus a simple, rapid, and sensitive method for the detection of AP sites in DNA. Furthermore, removal of damaged base by DNA N-glycosylases generates AP sites that can be measured by the ARP reagent. By coupling the ARP assay with either endonuclease III from Escherichia coli or 8-oxoguanine N-glycosylase (OGG1) from yeast, investigators can rapidly determine the amount of oxidative pyrimidine damage (endonuclease III-sensitive sites) or purine damage (OGG1-sensitive sites) in cellular DNA, respectively. An increased level of oxidative damage has been implicated in several age-related human diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease, as well as the aging process. The sensitivity and simplicity of the ARP assay thus make it a valuable method for investigators who are interested in estimating the level of oxidative DNA damage in cells and tissues derived from patients with various age-related diseases or cancers.     

A novel, sensitive, and specific assay for abasic sites, the most commonly produced DNA lesion.

Free radicals produce a wide spectrum of damages; among these are DNA base damages and abasic (AP) sites. Although several methods have been used to detect and quantify AP sites, they either are relatively laborious or require the use of radioactivity. A novel reagent for detecting abasic sites in DNA was prepared by reacting O-(carboxymethyl)hydroxylamine with biotin hydrazide in the presence of carbodiimide. This reagent, called Aldehyde Reactive Probe (ARP), specifically tagged AP sites in DNA with biotin residues. The number of biotin-tagged AP sites was then determined colorimetrically by an ELISA-like assay using avidin/biotin complex conjugated to horseradish peroxidase as the indicator enzyme. With heat/acid-depurinated calf thymus or bacteriophage f1 DNA, ARP detected femtomoles of AP sites in DNA. Using this assay, DNA damages generated in calf thymus, phi X174 RF, and f1 single-stranded DNA, X-irradiated in phosphate buffer, were easily detectable at 10 rad (0.1 Gy). Furthermore, ARP sites were detectable in DNA isolated from heat-inactivated X-irradiated (10 Gy) and methyl methanesulfonate (MMS)-treated (5 microM) Escherichia coli cells. The rate of production of ARP sites was proportional to the X-ray dose as well as to the concentration of MMS. Thus, the sensitivity and simplicity of the ARP assay should provide a potentially powerful method for the quantitation of AP sites or other DNA lesions containing an aldehyde group.     

Copper generated reactive oxygen leads to formation of lysine-DNA adducts

This work describes the addition of a lysine derivative to guanine base in a nucleoside, an oligonucleotide, and to a large DNA that occurs via oxidation by copper generated reactive oxygen species. Nucleophiles present during oxidation leads to the formation of adducts. In this work, 2′-deoxyguanosine is oxidized by copper generated reactive oxygen species in the presence of a lysine derivative, Nα-acetyl-lysine methyl ester. Under these conditions the guanidinohydantoin-lysine adduct is observed in a relative yield of 27% when compared to other guanine oxidation products. MS² strongly supports that lysine is added to the 5-position during the formation of guanidinohydantoin-lysine. A fourteen-nucleotide DNA duplex was oxidized under similar conditions. Digestion showed formation of the same guanidinohydantoin-lysine nucleoside. The reaction was then examined on a 392-nucleotide DNA substrate. Oxidation in the presence of the lysine ester showed adduct formation as stops in a primer extension assay. Adducts predominately formed at a 5′-GGG at position 415. Six of the seven sites that showed reaction greater than 3-fold above background were guanine sites. We conclude from this study that copper can catalyze the formation of DNA-protein adducts and may form in cells with elevated copper and oxidative stress.     

Sensitivity of human type II topoisomerases to DNA damage: stimulation of enzyme-mediated DNA cleavage by abasic, oxidized and alkylated lesions

Type II topoisomerases are essential enzymes that are also the primary cellular targets for a number of important anticancer drugs. These drugs act by increasing levels of topoisomerase II-mediated DNA cleavage. Recent studies indicate that endogenous forms of DNA damage, such as abasic sites and base mismatches, also stimulate the DNA scission activity of the enzyme. To extend our understanding of how type II topoisomerases react to DNA damage, the effects of abasic sites, and oxidized and alkylated bases on DNA cleavage mediated by human topo-isomerase IIα and β were determined. Based on experiments that incorporated random abasic sites into plasmid DNA, human type II enzymes can locate lesions even within a background of several thousand undamaged base pairs. As determined by experiments that utilized site-specific forms of DNA lesions, oxidized or monoalkylated purines that allow base pairing and induce little distortion in the double helix have modest effects on topoisomerase II-mediated DNA cleavage. In contrast, 1,N⁶-ethenoadenine, a bulky lesion that disrupts base pairing, enhanced DNA cleavage ~10-fold. 1,N⁶-Ethenoadenine is the first lesion found to rival the stimulatory effects of apurinic sites on the DNA scission activity of eukaryotic type II topoisomerases.     

Endogenous DNA damage clusters in human hematopoietic stem and progenitor cells

Clustered DNA damages-multiple oxidized bases, abasic sites, or strand breaks within a few helical turns-are potentially mutagenic and lethal alterations induced by ionizing radiation. Endogenous clusters are found at low frequencies in unirradiated normal human cells and tissues. Radiation-sensitive hematopoietic cells with low glycosylase levels (TK6 and WI-L2-NS) accumulate oxidized base clusters but not abasic clusters, indicating that cellular repair genotype affects endogenous cluster levels. We asked whether other factors, i.e., in the cellular microenvironment, affect endogenous cluster levels and composition in hematopoietic cells. TK6 and WI-L2-NS cells were grown in standard medium (RPMI 1640) alone or supplemented with folate and/or selenium; oxidized base cluster levels were highest in RPMI 1640 and reduced in selenium-supplemented medium. Abasic clusters were low under all conditions. In primary hematopoietic stem and progenitor cells from four non-tobacco-using donors, cluster levels were low. However, in cells from tobacco users, we observed high oxidized base clusters and also abasic clusters, previously observed only in irradiated cells. Protein levels and activity of the abasic endonuclease Ape1 were similar in the tobacco users and nonusers. These data suggest that in highly damaging environments, even normal DNA repair capacity can be overwhelmed, leaving highly repair-resistant clustered damages.     

Determination of hydroperoxides by the ferric-xylenol orange method

Abstract

Organic hydroperoxides are some of the first semi-stable products of the interaction between free radicals (and other reactive oxygen species) with biological systems, so that they are potential indicators of the formation and effects of these reactive molecules. Many assays have been developed for the detection and measurement of hydroperoxides, but all have significant drawbacks. For example, while the acid iodometric technique is the only one giving indisputably quantitative results,¹ it requires anaerobic conditions, making it difficult to use in routine assays. A promising alternative method, with similar sensitivity but unaffected by oxygen, was developed for a wide range of hydroperoxides. It is based on the reaction between the hydroperoxide and ferrous iron in acid medium and the measurement of the ferric iron produced by formation of a complex with xylenol orange. The assay was initially used to measure H₂O₂, but later also in the measurement of a wide range of organic hydroperoxides, including some present in biological fluids.² In attempting to apply the assay to solutions of oxidized proteins, we found that accurate hydroperoxide concentrations could not be obtained by the recommended protocols. Therefore, we examined the variables affecting the assay and validated a new protocol able to provide more reliable results.     

Quantification of DNA strand breaks and abasic sites by oxime derivatization and accelerator mass spectrometry: application to gamma-radiation and peroxynitrite

We report a highly sensitive method to quantify abasic sites and deoxyribose oxidation products arising in damaged DNA. The method exploits the reaction of aldehyde- and ketone-containing deoxyribose oxidation products and abasic sites with [(14)C]methoxyamine to form stable oxime derivatives, as originally described by Talpaert-Borle and Liuzzi [Reaction of apurinic/apyrimidinic sites with [(14)C]methoxyamine. A method for the quantitative assay of AP sites in DNA, Biochim. Biophys. Acta 740 (1983) 410-416]. The sensitivity of the method was dramatically improved by the application of accelerator mass spectrometry to quantify the (14)C, with a limit of detection of 1 lesion in 10(6) nucleotides in 1 microg of DNA. The method was validated using DNA containing a defined quantity of abasic sites, with a >0.95 correlation between the quantities of abasic sites and those of methoxyamine labels. The original applications of this and similar oxyamine derivatization methods have assumed that abasic sites are the only aldehyde-containing DNA damage products. However, deoxyribose oxidation produces strand breaks and abasic sites containing a variety of degradation products with aldehyde and ketone moieties. To assess the utility of methoxyamine labeling for quantifying strand breaks and abasic sites, the method was applied to plasmid DNA treated with gamma-radiation and peroxynitrite. For gamma-radiation, there was a 0.99 correlation between the quantity of methoxyamine labels and the quantity of strand breaks and abasic sites determined by a plasmid nicking assay; the abasic sites comprised less than 10% of the radiation-induced DNA damage. Studies with peroxynitrite demonstrate that the method, in conjunction with DNA repair enzymes that remove damaged bases to produce aldehydic sugar residues or abasic sites, is also applicable to quantifying nucleobase lesions in addition to strand break products. Compared to other abasic site quantification techniques, the modified method offers the advantage of providing a straightforward and direct measurement of aldehyde- and ketone-containing strand breaks and abasic sites, with the potential for direct labeling in cells prior to DNA isolation.     

Nuclear and mitochondrial DNA oxidation in Alzheimer's disease

Abstract

The study of Alzheimer's disease neuropathology has been intimately associated with the field of oxidative stress for nearly 20 years. Indeed, increased markers of oxidative stress have been associated with this neurodegenerative condition, resulting from oxidation of lipids, proteins and nucleic acids. Increased nuclear and mitochondrial DNA oxidation are observed in Alzheimer's disease, stemming from increased reactive oxygen species attack to DNA bases and from the impairment of DNA repair mechanisms. Moreover, mitochondrial DNA is found to be more extensively oxidized than nuclear DNA. This review is intended to summarizes the most important cellular reactive oxygen species producers and how mitochondrial dysfunction, redox-active metals dyshomeostasis and NADPH oxidases contribute to increased oxidative stress in Alzheimer's disease. A summary of the antioxidant system malfunction will also be provided. Moreover, we will highlight the mechanisms of DNA oxidation and repair. Importantly, we will discuss evidence relating the DNA repair machinery and accumulated DNA oxidation with Alzheimer's disease.     

Assays for urinary biomarkers of oxidatively damaged nucleic acids

Abstract

The analysis of oxidized nucleic acid metabolites can be performed by a variety of methodologies: liquid chromatography coupled with electrochemical or mass-spectrometry detection, gas chromatography coupled with mass spectrometry, capillary electrophoresis and ELISA (Enzyme-linked immunosorbent assay). The major analytical challenge is specificity. The best combination of selectivity and speed of analysis can be obtained by liquid chromatography coupled with tandem mass spectrometric detection. This, however, is also the most demanding technique with regard to price, complexity and skills requirement. The available ELISA methods present considerable specificity problems and cannot be recommended at present. The oxidized nucleic acid metabolites in urine are assumed to originate from the DNA and RNA. However, direct evidence is not available. A possible contribution from the nucleotide pools is most probably minimal, if existing. Recent investigation on RNA oxidation has shown conditions where RNA oxidation but not DNA oxidation is prominent, and while investigation on DNA is of huge interest, RNA oxidation may be overlooked. The methods for analyzing oxidized deoxynucleosides can easily be expanded to analyze the oxidized ribonucleosides. The urinary measurement of oxidized nucleic acid metabolites provides a non-invasive measurement of oxidative stress to DNA and RNA.     

Mutagenic effects of abasic and oxidized abasic lesions in Saccharomyces cerevisiae

2-Deoxyribonolactone (L) and 2-deoxyribose (AP) are abasic sites that are produced by ionizing radiation, reactive oxygen species and a variety of DNA damaging agents. The biological processing of the AP site has been examined in the yeast Saccharomyces cerevisiae. However, nothing is known about how L is processed in this organism. We determined the bypass and mutagenic specificity of DNA containing an abasic site (AP and L) or the AP analog tetrahydrofuran (F) using an oligonucleotide transformation assay. The tetrahydrofuran analog and L were bypassed at 10-fold higher frequencies than the AP lesions. Bypass frequencies of lesions were greatly reduced in the absence of Rev1 or Polζ (rev3 mutant), but were only marginally reduced in the absence of Polη (rad30 mutant). Deoxycytidine was the preferred nucleotide inserted opposite an AP site whereas dA and dC were inserted at equal frequencies opposite F and L sites. In the rev1 and rev3 strains, dA was the predominant nucleotide inserted opposite these lesions. Overall, we conclude that both Rev1 and Polζ are required for the efficient bypass of abasic sites in yeast.     

Elimination and Utilization of Oxidized Guanine Nucleotides in the Synthesis of RNA and Its Precursors

Reactive oxygen species are produced as side products of oxygen utilization and can lead to the oxidation of nucleic acids and their precursor nucleotides. Among the various oxidized bases, 8-oxo-7,8-dihydroguanine seems to be the most critical during the transfer of genetic information because it can pair with both cytosine and adenine. During the de novo synthesis of guanine nucleotides, GMP is formed first, and it is converted to GDP by guanylate kinase. This enzyme hardly acts on an oxidized form of GMP (8-oxo-GMP) formed by the oxidation of GMP or by the cleavage of 8-oxo-GDP and 8-oxo-GTP by MutT protein. Although the formation of 8-oxo-GDP from 8-oxo-GMP is thus prevented, 8-oxo-GDP itself may be produced by the oxidation of GDP by reactive oxygen species. The 8-oxo-GDP thus formed can be converted to 8-oxo-GTP because nucleoside-diphosphate kinase and adenylate kinase, both of which catalyze the conversion of GDP to GTP, do not discriminate 8-oxo-GDP from normal GDP. The 8-oxo-GTP produced in this way and by the oxidation of GTP can be used for RNA synthesis. This misincorporation is prevented by MutT protein, which has the potential to cleave 8-oxo-GTP as well as 8-oxo-GDP to 8-oxo-GMP. When ¹⁴C-labeled 8-oxo-GTP was applied to CaCl₂-permeabilized cells of a mutT⁻ mutant strain, it could be incorporated into RNA at 4% of the rate for GTP. Escherichia coli cells appear to possess mechanisms to prevent misincorporation of 8-oxo-7,8-dihydroguanine into RNA.     

Effects of oolonghomobisflavan A on oxidation of low-density lipoprotein

Oxidation of low-density lipoprotein (LDL) by reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been suggested to be involved in the onset of atherosclerosis. Oolong tea contains unique polyphenols including oolonghomobisflavan A (OFA). In this study, the effects of OFA on LDL oxidation by ROS and RNS were investigated in vitro. OFA suppressed formation of cholesterol ester hydroperoxides in LDL oxidized by peroxyl radical and peroxynitrite, and formation of thiobarbituric acid reactive substances in LDL oxidized by Cu²⁺. In addition, OFA inhibited fragmentation, carbonylation, and nitration of apolipoprotein B-100 (apo B-100) in the oxidized LDL, in which heparin-binding activity of apo B-100 was protected by OFA. Our results suggest that OFA exhibits antioxidant activity against both lipid peroxidation and oxidative modification of apo B-100 in LDL oxidized by ROS and RNS. Polyphenols in oolong tea may prevent atherosclerosis by reducing oxidative stress.     

Lowered Nudix type 5 expression leads to cellular senescence in IMR-90 fibroblast cells

Abstract

The molecule 8-oxo-7,8-dihydroguanine (8-oxoGua), an oxidized form of guanine, can pair with adenine or cytosine during nucleic acid synthesis. RNA sequences that contain 8-oxoGua cause translational errors that lead to the synthesis of abnormal proteins. Human Nudix type 5 (NUDT5), a MutT-related protein, catalyzes the hydrolysis of 8-oxoGDP to 8-oxoGMP, thereby preventing the misincorporation of 8-oxoGua into RNA. To investigate the biological roles of NUDT5 in human fibroblast cells, we established cell lines with decreased levels of NUDT5 expression. In NUDT5 knockdown cells, the RNA oxidation levels were significantly higher, the rates of cellular senescence and cell apoptosis were significantly increased, and the cell viability was significantly decreased in comparison with control cells. These results suggested that the NUDT5 protein could play significant roles in the prevention of RNA oxidation and survival in human fibroblast cells.     

Processing of abasic DNA clusters in hApeI-silenced primary fibroblasts exposed to low doses of X-irradiation

Clustered damage in DNA includes two or more closely spaced oxidized bases, strand breaks or abasic sites that are induced by high- or low-linear-energy-transfer (LET) radiation, and these have been found to be repair-resistant and potentially mutagenic. In the present study we found that abasic clustered damages are also induced in primary human fibroblast cells by low-LET X-rays even at very low doses. In response to the induction of the abasic sites, primary fibroblasts irradiated by low doses of X-rays in the range 10–100 cGy showed dose-dependent up-regulation of the DNA repair enzyme, ApeI. We found that the abasic clusters in primary fibroblasts were more lethal to cells when hApeI enzyme expression was down-regulated by transfecting primary fibroblasts with hApeI siRNA as determined by clonogenic survival assay. Endonuclease activity of hApeI was found to be directly proportional to hApeI gene-silencing efficiency. The DNA repair profile showed that processing of abasic clusters was delayed in hApeI-siRNA-silenced fibroblasts, which challenges the survival of the cells even at very low doses of X-rays. Thus, the present study is the first to attempt to understand the induction of cluster DNA damage at very low doses of low-LET radiation in primary human fibroblasts and their processing by DNA repair enzyme ApeI and their relation with the survival of the cells.     

Detection and interpretation of 8-oxodG and 8-oxoGua in urine,plasma and cerebrospinal fluid

Background

DNA and RNA oxidations have been linked to diseases such as cancer, arteriosclerosis, neurodegeneration and diabetes. The prototype base modification studied is the 8-hydroxylation of guanine. DNA integrity is maintained by elaborate repair systems and RNA integrity is less studied but relies mainly on degradation.

Scope of review

DNA and RNA oxidations are measured by very similar techniques. The scope of this review is to highlight the preferred methods of measurement of oxidized nucleic acid metabolites, to highlight novel findings particularly in RNA oxidation, and to present the interpretation of the measurements.

Major conclusions

Tissue levels are snap-shots of the level in a specific organ or cell system and reflect the balance between formation rate and elimination rate (repair), and must be interpreted as such. Urinary excretion is a global measure of oxidative stress in an organism and is therefore best suited for situations or diseases where large parts or the entire organism is stressed by oxidation. It represents the body average rate by which either RNA or DNA is oxidized and is interpreted as oxidative stress. Oxidations of RNA and DNA precursors have been demonstrated and the quantitative importance is debated.

General significance

Careful experimental designs and appropriate choice of methodology are paramount for correct testing of hypotheses related to oxidative stress, and pitfalls are plentiful. There is accumulating evidence that DNA oxidation is associated with disease, particularly cancer, and recent evidence points at an association between RNA oxidation and neurodegenerative diseases and diabetes. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.