Assays for urinary biomarkers of oxidatively damaged nucleic acids

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.     

Immunochemical detection of oxidatively damaged DNA

Abstract

Oxidatively damaged DNA is implicated in various diseases, including neurodegenerative disorders, cancer, diabetes, cardiovascular and inflammatory diseases as well as aging. Several methods have been developed to detect oxidatively damaged DNA. They include chromatographic techniques, the Comet assay, ³²P-postlabelling and immunochemical methods that use antibodies to detect oxidized lesions. In this review, we discuss the detection of 8-oxo-7,8-dihydro-29-deoxyguanosine (8-oxodG), the most abundant oxidized nucleoside. This lesion is frequently used as a marker of exposure to oxidants, including environmental pollutants, as well as a potential marker of disease progression. We concentrate on studies published between the years 2000 and 2011 that used enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry to detect 8-oxodG in humans, laboratory animals and in cell lines. Oxidative damage observed in these organisms resulted from disease, exposure to environmental pollutants or from in vitro treatment with various chemical and physical factors.     

Enzymatic labeling of nucleic acids

Enzymatic labeling of nucleic acids is a fundamental tool in molecular biology with virtually every aspect of nucleic acid hybridization technique involving the use of labeled probes. Different methods for enzymatic labeling of DNA, RNA and oligonucleotide probes are available today. In this review, we will describe both radioactive and nonradioactive labeling methods, yet the choice of system for labeling the probe depends on the application under study.     

Diastereomer characterizations of nitroxide-labeled nucleic acids

Site-directed spin labeling (SDSL) obtains structural and dynamic information of a macromolecule using a site-specifically attached stable nitroxide radical. SDSL studies of arbitrary DNA and RNA sequences can be achieved using an efficient phosphorothioate labeling scheme, where a nitroxide is attached to a phosphorothioate substituted at a target site during chemical synthesis. The chemically introduced phosphorothioate contains two diastereomers (Rp and Sp), and nitroxides attached to each diastereomer may experience different local environments. Here, we report work on using anion-exchange HPLC to separate and characterize diastereomers in three DNA oligonucleotides and one RNA oligonucleotide. In all oligonucleotides studied, the Rp diastereomer was found to elute earlier than the Sp in the unlabeled oligonucleotides, while a reversal in the elution order (Sp earlier than Rp) was observed for nitroxide-labeled oligonucleotides. The results enable a one-step purification procedure for preparing diastereomerically pure nitroxide-labeled oligonucleotides. This expands the score of nucleic acids SDSL.     

On the use of ESI‐QqTOF‐MS/MS for the comparative sequencing of nucleic acids

The usability of a quadrupole—quadrupole—time‐of‐flight (QqTOF) instrument for the tandem mass spectrometric sequencing of oligodeoxynuleotides was investigated. The sample set consisted of 21 synthetic oligodeoxynucleotides ranging in length from 5 to 42 nucleotides. The sequences were randomly selected. For the majority of tested oligonucleotides, two or three different charge states were selected as precursor ions. Each precursor ion was fragmented applying several different collision voltages. Overall 282 fragment ion mass spectra were acquired. Computer‐aided interpretation of fragment ion mass spectra was accomplished with a recently introduced comparative sequencing algorithm (COMPAS). The applied version of COMPAS was specifically optimized for the interpretation of information‐rich spectra obtained on the QqTOF. Sequences of oligodeoxynucleotides as large as 26‐mers were correctly verified in >94% of cases (182 of 192 spectra acquired). Fragment ion mass spectra of larger oligonucleotides were not specific enough for sequencing. Because of the occurrence of extensive internal fragmentation causing low sequence coverage paired with a high probability of assigning fragment ions to wrong sequences, tandem mass spectra obtained from oligonucleotides consisting of 30 and more nucleotides could not be used for sequence verification neither manually nor with COMPAS. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 401–409, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Fractionation with ethanol of nucleic acids from viroid-infected plants

A combination of high salt and low ethanol concentration allowed the fractionation of nucleic acids extracted from viroid-infected leaves. By adding 0.4-0.5 vol of ethanol to 1 vol of a solution in 2 M LiCl of nucleic acids (containing mainly DNA, 4S, 5S, 7S, and viroid RNAs), 85% of the DNA and 75% of the 4S RNA remained in solution, from where they could be recovered by increasing the ethanol concentration, whereas almost all 5S, 7S, and viroid RNAs precipitated. When this process was repeated three times a 95% elimination of the initial DNA and 4S RNA was achieved. The method can be of special interest in viroid purification considering that DNA and 4S RNA are the most abundant contaminants in the starting solution of nucleic acids. It is suggested that the highly ordered secondary structure of viroid RNA may be responsible for its particular behavior in the ethanol fractionation of nucleic acids.     

Towards a universally adaptable method for quantitative extraction of high-purity nucleic acids from soil

A universally adaptable protocol for quantitative extraction of high-purity nucleic acids from soil is presented. A major problem regarding the extraction of nucleic acids from soil is the presence of humic substances, which interfere with the extraction process itself and in subsequent analytical manipulations. By the approach described here, the humic compounds are precipitated prior to cell lysis with Al(2)(SO(4))(3), and thus eliminated prior to the nucleic acid extraction. The protocol allows for removing of a considerable content and range of humic acids and should therefore be applicable for a wide spectrum of soil types. Accordingly, reproducible results in analyses of different soil types are made possible, inclusively for quantitative comparisons.     

Synthesis of nucleic acids and localization of atrial natriuretic peptide in crayfish haemocytes

Three types of cells circulate in the haemolymph of the crayfish Astacus astacus, i.e., agranular haemocytes (HCs I), small-granule haemocytes (HCs II), and large-granule haemocytes (HCs III). Their proliferation, differentiation, and function remain poorly understood. Using light and electron microscopic autoradiography with [³H]-thymidine, we found that only HCs I are capable of DNA synthesis and mitosis whereas HCs II and HCs III are replicatively inactive. To verify whether HCs I are proliferating progenitor cells for granular HCs, we have analyzed autographs of the HC population 1, 2, 7, and 21 days after a single [3H]-thymidine administration. Contrary to our expectations, we have failed to find labeled HCs II and HCs III. These findings have raised doubts as to the capacity of HCs I to differentiate into two other types of HCs. With the use of ³H-uridine autoradiography, it was found that RNA synthesis was the most active in HCs I and 2 and 4 times lower in HCs II and HCs III, respectively. ANP-like immunoreactivity was revealed in large granules of the HCs III by electron microscopic immunocytochemistry. We assume that the presence of ANP in secretory granules extends the possible functions of crayfish HCs and suggests their participation in the regulation of the watersalt balance and immune response.     

Thermodynamic study of hybridization properties of heterochiral nucleic acids

Heterochiral DNA and RNA heptamers, which contained an unnatural L-nucleotide, were synthesized, and thermodynamic analyses of their hybridization properties with complementary DNA and RNA strands were systematically conducted by UV melting experiments. The results clearly demonstrated that the incorporation of an L-ribonucleotide into the RNA strand leads to more significant destabilization of the duplexes than that of an L-deoxyribonucleotide into the DNA strand, regardless of whether the complementary strand is DNA or RNA. The destabilization of the duplexes by the substitution of D-thymidine with L-thymidine in the DNA strand is entropically driven, whereas that by the substitution of D-uridine with L-uridine in the RNA strand is enthalpically driven. The thermodynamic characteristic that the stability of homochiral duplex is far superior to that of heterochiral duplex is much more remarkable in RNA than in DNA. Thus, RNA might have been a self-replicating system superior to DNA to exclude the chiral antipode.     

Mass spectrometry of nucleic acids in molecular medicine

A stable streamlining trend in the field of medical diagnostics by practical adoption of high-tech and knowledge-intensive analytical systems providing for molecular level studies has appeared during the last few decades. An illustrative example of such technologies is mass spectrometry methods for analyzing biomolecules. This review is intended to brief the potential of the state-of-the-art inventory of spectrometry equipment and illustrate the application of mass spectrometry of nucleic acids (DNA and RNA) for solving practical problems related to the analysis of human genomic DNA and clinically significant microorganisms of bacterial and viral natures.     

Binding ability of a thymine-functionalized oligolysine towards nucleic acids

In this Letter, we investigated the binding properties towards nucleic acids of a thymine-functionalized oligolysine, composed of nucleobase-bearing amino acid moieties and underivatized l-lysine residues alternate in the backbone. The basic nucleopeptide proved to be well soluble in water and able to interact with both DNA and RNA, as suggested by circular dichroism, UV and surface plasmon resonance studies performed on the thymine-containing oligomer with both adenine-containing DNA (dA₁₂) and RNA (rA₁₂ and poly rA) molecules. In both cases the thymine-functionalized oligolysine was proven to form complexes characterized by a 1:1 T/A stoichiometric ratio, as evidenced by CD titration. UV melting experiments revealed that the complex formed between the homothymine oligolysine and rA₁₂ RNA was more stable than the complex with dA₁₂ DNA probably due to the additional H-bonding of the 2′-OH groups in RNA, that reinforces the overall interaction with the nucleopeptide. Finally, human serum stability assays were conducted on the thymine-bearing nucleopeptide which showed a half-life of 45 min.     

Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry

Oxidation of methionine residues in biopharmaceuticals is a common and often unwanted modification that frequently occurs during their manufacture and storage. It often results in a lack of stability and biological function of the product, necessitating continuous testing for the modification throughout the product shelf life. A major class of biopharmaceutical products are monoclonal antibodies (mAbs), however, techniques for their detailed structural analysis have until recently been limited. Hydrogen/deuterium exchange mass spectrometry (HXMS) has recently been successfully applied to the analysis of mAbs. Here we used HXMS to identify and localise the structural changes that occurred in a mAb (IgG1) after accelerated oxidative stress. Structural alterations in a number of segments of the Fc region were observed and these related to oxidation of methionine residues. These included a large change in the hydrogen exchange profile of residues 247–253 of the heavy chain, while smaller changes in hydrogen exchange profile were identified for peptides that contained residues in the interface of the C_H2 and C_H3 domains.     

Biochemistry of selenium-derivatized naturally occurring and unnatural nucleic acids

Selenium (Se) can provide unique biochemical and biological functions, and properties to macromolecules, including protein and RNA. Although Se has not yet been found in DNA, identification of the presence of Se in natural tRNAs has led to discovery of the naturally occurring 2-selenouridine and 5-[(methylamino)methyl]-2-selenouridine (mnm(5)se(2)U). The Se-atoms at C(2) of the modified uridines are introduced by 2-selenouridine synthase via displacement of the S-atoms in the corresponding 2-thiouridine nucleotides of the tRNAs, and selenophosphate is used as the Se donor. The research indicated that mnm(5)se(2)U is located at the first or wobble position of the anticodons in several bacterial tRNAs, including tRNA(Lys), tRNA(Glu), and tRNA(Gln). The 2-seleno functionality on this modified nucleotide probably improves the translation accuracy and/or efficiency. These observations in vivo suggest that the presence of Se can provide natural RNAs with useful properties to better function and survival. To further investigate the biochemical and structural properties of Se-derivatized nucleic acids (SeNA), we have pioneered chemical and enzymatic synthesis of Se-derivatized nucleic acids, and introduced Se into both RNA and DNA at a variety of positions by atom-specific replacement of oxygen. This review outlines the recent advancements in chemical and biochemical syntheses, and studies of SeNAs, and their potential applications in structural and functional investigation of nucleic acids and their protein complexes.     

Multitasking with molecular dynamics Typhoon: quantifying nucleic acids and autoradiographs

With increased sensitivity and specificity, fluorescent assays are rapidly becoming the method of choice for nucleic acid quantification. The utility of the Typhoon scanner has now been extended to accurately measure low levels of DNA and RNA (5 ng ml^–1) with PicoGreen and RiboGreen dyes. In addition, with a few simple modifications, autoradiographic film images can be scanned and quantified with the Typhoon series of scanners.     

Innate immune sensing of nucleic acids from pathogens

The innate immune system is important as the first line of defense to sense invading pathogens. Nucleic acids represent critical pathogen signatures that trigger a host proinflammatory immune response. Much progress has been made in understanding how DNA and RNA trigger host defense countermeasures, however, several aspects of how cytosolic nucleic acids are sensed remain unclear. This special issue reviews how the host innate immune system senses nucleic acids from Brucella abortus, Mycobacterium sp and Legionella pneumophila, viral DNA, the role of STING in DNA sensing and inflammatory diseases and the mechanism of viral RNA recognition by the small interfering RNA pathway in Drosophila melanogaster.     

高效液相色谱-串联质谱检测基因组DNA甲基化方法的建立

目的:建立高效液相色谱-串联质谱(HPLC-ESI-MS/MS)检测基因组DNA甲基化水平的方法。方法:以5-mdC和dG为标准品,采用全自动高效液相色谱系统进行分离,串联电喷雾质谱检测,选择多反应监测模式(MRM)测定标准品,绘制标准工作曲线。结果:在MRM模式下选取5-mdC(m/z 241.9→126.3)和dG(m/z 268.1→152.3)分别作为定量检测的母子离子对,各化合物能实现良好的基线分离;5-mdC和dG碰撞能均为15 eV,去簇电压分别为40和45 V,最低定量限分别为1.65和2.47 fmol;标准品的响应值比为90%~110%;5-mdC含量的天内相对标准偏差和天间相对标准偏差均小于8%。结论:HPLC-ESI-MS/MS是能应用于检测基因组DNA甲基化的一种高通量、高准确率、高分辨率、高灵敏度且重复性好的方法。     

Electrophoretic elution of nucleic acids from acrylamide and agarose gels

A simple method for electrophoretic elution of nucleic acids from gel slices is described. The procedure utilizes a standard tube gel system and can be completed in as little as one hour. Nucleic acids are recovered in a small volume with almost 100% efficiency. The procedure is applicable equally to acrylamide and agarose gels, and small as well as large RNA and DNA molecules. The eluted nucleic acids are essentially undegraded and are suitable for a variety of structural and biological analyses.     

Studies on the retention of plasmid DNA and Escherichia coli nucleic acids by hydrophobic interaction chromatography

This work presents studies on the interactions of supercoiled plasmid DNA and Escherichia coli genomic DNA (gDNA) and RNA, with an hydrophobic interaction chromatography (HIC) gel, obtained by derivatisation of Sepharose CL-6B with 1,4-butanediol diglycidyl ether. Nucleic acids purified from E. coli were injected separately in the above HIC column and eluted with 1.5 M (NH₄)₂SO₄ in the buffer. The column was able to separate single-stranded from double-stranded nucleic acids. RNA and denatured gDNA were retarded in a different way due to the interactions of the exposed hydrophobic bases with the ligands. Supercoiled plasmid DNA, on the contrary, eluted in the flowthrough. This revised version was published online in July 2006 with corrections to the Cover Date.