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 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.

Figure 1

Depurinated tRNA^Phe increases ARP reactivity. A. Fluorescence derived from the wybutosine residue remaining in yeast tRNA^Phe was measured, after tRNA^Phe was incubated in 0.1 M ammonium formate (pH2.9) at 37 °C for ◆: 0h, ◇: 0.2h, ■: 0.5h, □: 1.5h, ●: 5.5h, or ○: 10h. B. After acid treatment tRNA^Phe was reacted with ARP followed by incubation with streptavidin-HRP on a positively charged membrane. The chemiluminescence derived from HRP was quantified. The inset shows the correlation between the remaining wybutosine and ARP reactivity. Data represent mean ±S.D. (n=4).

Figure 1

Depurinated tRNA^Phe increases ARP reactivity. A. Fluorescence derived from the wybutosine residue remaining in yeast tRNA^Phe was measured, after tRNA^Phe was incubated in 0.1 M ammonium formate (pH2.9) at 37 °C for ◆: 0h, ◇: 0.2h, ■: 0.5h, □: 1.5h, ●: 5.5h, or ○: 10h. B. After acid treatment tRNA^Phe was reacted with ARP followed by incubation with streptavidin-HRP on a positively charged membrane. The chemiluminescence derived from HRP was quantified. The inset shows the correlation between the remaining wybutosine and ARP reactivity. Data represent mean ±S.D. (n=4).

Figure 2

Abasic RNA reacts with ARP. A chemically synthesized oligo RNA with abasic sites was deprotected by visible light irradiation, followed by elongation with poly(A) polymerase. The schematic structure of the final abasic RNA is shown in panel A. Abasic sites and 6-carboxyfluorescein are designated as X and 6-FAM, respectively. A. Six hundred (600) ng of RNA with abasic sites (lane 1) or control RNA (lane 2) was loaded onto agarose gels for electrophoresis, followed by staining with SYBR green. The results indicate that elongated RNA reached approximately 2 to 3 kb. The original oligo RNA with (lane 3) or without (lane 4) abasic sites was visualized using 6-FAM rather than SYBR green. B. Elongated RNAs were examined for ARP reactivity. Abasic sites in RNA in the left lane were not deprotected by light, whereas RNAs in the right side were deprotected. Absc or ctrl denotes the RNA with or without abasic sites, respectively. C. ARP derivatized abasic RNA (2.5 fmoles to 100 fmole) was spotted in the presence of 1 nmole of ribonucleotides. The concentration of abasic sites in the RNA was measured by the fluorescence derived from 6-FAM.

Figure 2

Abasic RNA reacts with ARP. A chemically synthesized oligo RNA with abasic sites was deprotected by visible light irradiation, followed by elongation with poly(A) polymerase. The schematic structure of the final abasic RNA is shown in panel A. Abasic sites and 6-carboxyfluorescein are designated as X and 6-FAM, respectively. A. Six hundred (600) ng of RNA with abasic sites (lane 1) or control RNA (lane 2) was loaded onto agarose gels for electrophoresis, followed by staining with SYBR green. The results indicate that elongated RNA reached approximately 2 to 3 kb. The original oligo RNA with (lane 3) or without (lane 4) abasic sites was visualized using 6-FAM rather than SYBR green. B. Elongated RNAs were examined for ARP reactivity. Abasic sites in RNA in the left lane were not deprotected by light, whereas RNAs in the right side were deprotected. Absc or ctrl denotes the RNA with or without abasic sites, respectively. C. ARP derivatized abasic RNA (2.5 fmoles to 100 fmole) was spotted in the presence of 1 nmole of ribonucleotides. The concentration of abasic sites in the RNA was measured by the fluorescence derived from 6-FAM.

Figure 3

Characterization of ARP reactivity to abasic RNA. One hundred (100) pg of synthetic abasic RNA (closed circles) or control RNA (open circles) was reacted with ARP at different temperatures (A), incubation times (B), pH (C), or concentrations of ARP (D). The basic reaction conditions were carried out in the presence of 2 mM of ARP at 37 °C for 1h at pH7.5. Data represent mean ±S.D. (n=4).

Figure 4

Gamma irradiation increased ARP reactivity. Two hundred fifty (250) μg/ml of in vitro synthesized RNA was irradiated at the doses indicated, then directly incubated with ARP at a final concentration 2 mM for 1h. The derivatized RNA was analyzed by streptavidin-HRP as described in Materials and Methods. Data represent mean ±S.D. (n=4).

Figure 5

RNA oxidation using the Fenton reaction increased ARP reactivity. A. In vitro synthesized RNA was subjected to oxidation in the presence of H₂O₂ and Fe(II)-ascorbate at the indicated concentrations at 37 °C for 30 min, then precipitated with ethanol to eliminate unreacted oxidants. The oxidized RNA was examined by ARP reactivity as described in Materials and Methods. B. The oxidized RNA which was derivatized with ARP was loaded on an agarose gel and transferred to a positive charged membrane. The membrane was incubated with streptavidin-HRP, and developed with chemiluminescence reagent (upper panel). Then, the membrane was stained with methylene blue to visualize RNA (lower panel).

Figure 6

Abasic formation in oxidized RNA. RNA oxidized by Fe(II)/ascorbate/H₂O₂ was derivatized with ARP, then hydrolyzed enzymatically. The hydrolysate was subjected to LC/MS as described in Materials and Methods. Panel A shows the extracted ion chromatograms with 464.2 ±0.3 m/z. Ribose-ARP complex and formaldehyde-ARP complex were analyzed as positive and negative controls, respectively. Panel B shows the mass spectra of the oxidized RNA hydrolysate eluted at 6 min.

Figure 6

Abasic formation in oxidized RNA. RNA oxidized by Fe(II)/ascorbate/H₂O₂ was derivatized with ARP, then hydrolyzed enzymatically. The hydrolysate was subjected to LC/MS as described in Materials and Methods. Panel A shows the extracted ion chromatograms with 464.2 ±0.3 m/z. Ribose-ARP complex and formaldehyde-ARP complex were analyzed as positive and negative controls, respectively. Panel B shows the mass spectra of the oxidized RNA hydrolysate eluted at 6 min.

Figure 7

RNA oxidation induced by peroxynitrite increased ARP reactivity. Two hundred fifty (250) μg/ml of in vitro synthesized RNA was oxidized by peroxynitrite with the indicated concentrations in 50 mM potassium phosphate buffer (pH7.4) for 30 min at room temperature. After the reaction, oxidized RNA was analyzed using the ARP assay. Data represent mean ±S.D. (n=4).

Figure 8

ARP reactivity of total cellular RNA was increased after oxidative stress. HeLa cells were treated with H₂O₂ for 1h (A) or peroxynitrite for 15 min (B) using the indicated concentrations. Total RNA was isolated from the cells and abasic sites analyzed with the ARP assay. Results indicate a dose related rise in abasic sites in response to hydrogenperoxide or peroxynitrite. Data represent mean ±S.D. (n=4).