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.
Reynolds Oklahoma Center on Aging, Department of Geriatric Medicine, University of Oklahoma Health Sciences Center, 975 NE 10th street, Stanton L. Young BRC 1305, Oklahoma City, OK 73104, USA. mikiei-tanaka@ouhsc.edu
Mikiei
M
Tanaka
Song
S
Han
Han
H
Song
Pascal A
PA
Küpfer
Christian J
CJ
Leumann
William E
WE
Sonntag
D000375:Aging; D001080:Apurinic Acid / Q000737:chemistry; D001681:Biological Assay / Q000379:methods*; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D005260:Female; D005720:Gamma Rays; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010084:Oxidation-Reduction / Q000187:drug effects / Q000528:radiation effects; D030421:Peroxynitrous Acid / Q000494:pharmacology; D011119:Polynucleotides / Q000737:chemistry; D021281:Quantitative Structure-Activity Relationship; D012313:RNA / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D026361:Reactive Nitrogen Species / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
D011119:Polynucleotides; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; C001790:apyrimidinic acid; D001080:Apurinic Acid; D006151:Guanosine; C074188:O-(biotinylcarbazoylmethyl)hydroxylamine; D030421:Peroxynitrous Acid; C046215:8-hydroxyguanosine; D012313:RNA; D001710:Biotin