Effects of 8-hydroxy-GTP and 2-hydroxy-ATP on in vitro transcription

Oxidation of RNA precursors may disturb genetic information. In this study, the effects of oxidized RNA precursors on in vitro transcription were examined. Two oxidized ribonucleoside triphosphates, 8-hydroxyguanosine 5′-triphosphate (8-OH-GTP) and 2-hydroxyadenosine 5′-triphosphate (2-OH-ATP), were added to in vitro transcription reactions. The addition of 8-OH-GTP and 2-OH-ATP reduced the amount of RNA synthesized in vitro. Moreover, to examine qualitative alteration of the mRNA, it was converted to cDNA by reverse transcriptase, and the cDNA was then amplified by PCR. The PCR product was subsequently cloned into plasmid DNA, and the DNA sequence was analyzed for each bacterial colony. The two oxidized ribonucleotides induced mutations in cDNA, suggesting the disturbance of genetic information during transcription and/or reverse transcription. 8-OH-GTP induced T → G plus T → C mutations, and 2-OH-ATP caused T → C mutations. These results indicate that the formation of these oxidized RNA precursors in cells affects transcription quantitatively and qualitatively.