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

Abstract

There have been several reports describing elevation of oxidized RNA in aging 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. We 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, we have developed a sensitive assay for analysis of oxidized RNA.

Redox Biology of Aging: Focus on Novel Biomarkers

“…It was proposed that since RNA molecules are very similar to DNA, the oxidative lesions observed in DNA could also be observed on the corresponding bases in RNA [83], and these lesions may serve as marker of oxidative damage in RNA with increased accuracy. In the past years, the oxidized product of guanosine, 7,8-dihydro-8-oxo-guanosine (8-OHG), has been actively investigated [84]. 8-OHG is the most examined oxidation products of the RNA due to its similarity to the 8-oxoG lesion in DNA and the ability to use many of the same methodologies verified on DNA substrates for the analysis of RNA oxidation products.…”

Protein Synthesis and the Stress Response

“…In bacteria, these target macromolecules include elongation factors Tu [30][31][32][33][34], Ts (EF-Ts) [32] and G (EF-G) [8,9,22,32,35], several ribosomal proteins [31,36,37], tRNA [38][39][40][41][42] and aminoacyl-tRNA synthetases (aaRS) [31,34,36,37,43,44] (Table 1). rRNA and mRNA has also been shown to be oxidized in vivo in eukaryotes [19,25,26], but in bacteria this has not been tested.…”

“…In this context, oxidation of RNA has been found in eukaryotes during oxidative stress, senescence or some age related diseases [19,25,26]. This reaction would depend on the ability of RNA to bind Fe +2 which catalyzes its oxidation.…”

“…RNA oxidation is less well studied, although we know that ribonucleotide bases can be oxidized to 5-hydroxyuridine, 5-hydroxycytidine, 8-hydroxyadenosine and 8-hydroxyguanosine [18]. In addition to these oxidative modifications, nucleotides can also completely loss their bases during oxidative stress [19].…”

A chemical toxicity sensor based on the electrochemiluminescence quantification of apurinic/apyrimidinic sites in double-stranded DNA monolayer

“…The probe was found to penetrate the plasma membrane and react with the AP sites in DNA, which was then quantified by colorimetry after labeling with avidin-HRP [17]. Tanaka et al found that ARP could also be used to quantify the AP sites in RNA with a detection limit of 10 fmol [18]. Kojima et al improved the reaction rate of the probe by incorporating a hydrophobic and a hydrophilic residue into an aminooxy group [19].…”