ABSTRACT
DNA damage causes genomic instability underlying many diseases, with traditional analytical approaches providing minimal insight into the spectrum of DNA lesions in vivo. Here we used untargeted chromatography-coupled tandem mass spectrometry-based adductomics (LC-MS/MS) to begin to define the landscape of DNA modifications in rat and human tissues. A basis set of 114 putative DNA adducts was identified in heart, liver, brain, and kidney in 1-26-month-old rats and 111 in human heart and brain by 'stepped MRM' LC-MS/MS. Subsequent targeted analysis of these species revealed species-, tissue-, age- and sex-biases. Structural characterization of 10 selected adductomic signals as known DNA modifications validated the method and established confidence in the DNA origins of the signals. Along with strong tissue biases, we observed significant age-dependence for 36 adducts, including N2-CMdG, 5-HMdC and 8-Oxo-dG in rats and 1,N6-ϵdA in human heart, as well as sex biases for 67 adducts in rat tissues. These results demonstrate the potential of adductomics for discovering the true spectrum of disease-driving DNA adducts. Our dataset of 114 putative adducts serves as a resource for characterizing dozens of new forms of DNA damage, defining mechanisms of their formation and repair, and developing them as biomarkers of aging and disease.
Subject(s)
DNA Adducts , DNA , Animals , Female , Humans , Male , Rats , Chromatography, Liquid/methods , DNA/chemistry , DNA Adducts/genetics , Rodentia , Tandem Mass Spectrometry/methodsABSTRACT
The protocol describes the preparation and purification of interstrand DNA-DNA cross-links derived from the reaction of an N(4) -aminocytidine residue with an abasic site in duplex DNA. The procedures employ inexpensive, commercially available chemicals and enzymes to carry out post-synthetic modification of commercially available oligodeoxynucleotides. The yield of cross-linked duplex is typically better than 90%. If purification is required, the cross-linked duplex can be readily separated from single-stranded DNA starting materials by denaturing gel electrophoresis. The resulting covalent hydrazone-based cross-links are stable under physiologically relevant conditions and may be useful for biophysical studies, structural analyses, DNA repair studies, and materials science applications. © 2016 by John Wiley & Sons, Inc.
Subject(s)
Cytidine/chemistry , Nucleic Acid Heteroduplexes/chemistry , Oligodeoxyribonucleotides/chemistry , Chemistry Techniques, Synthetic , Cross-Linking Reagents/chemistry , DNA/chemistry , Native Polyacrylamide Gel Electrophoresis , Oligodeoxyribonucleotides/chemical synthesis , Phosphorus Radioisotopes/chemistryABSTRACT
Interstrand DNA-DNA cross-links are highly toxic to cells because these lesions block the extraction of information from the genetic material. The pathways by which cells repair cross-links are important, but not well understood. The preparation of chemically well-defined cross-linked DNA substrates represents a significant challenge in the study of cross-link repair. Here a simple method is reported that employs "post-synthetic" modifications of commercially available 2'-deoxyoligonucleotides to install a single cross-link in high yield at a specified location within a DNA duplex. The cross-linking process exploits the formation of a hydrazone between a non-natural N(4) -amino-2'-deoxycytidine nucleobase and the aldehyde residue of an abasic site in duplex DNA. The resulting cross-link is stable under physiological conditions, but can be readily dissociated and re-formed through heating-cooling cycles.
