Reverse Transcription Past Products of Guanine Oxidation in RNA Leads to Insertion of A and C opposite 8-Oxo-7,8-dihydroguanine and A and G opposite 5-Guanidinohydantoin and Spiroiminodihydantoin Diastereomers

Abstract

Reactive oxygen species, both endogenous and exogenous, can damage nucleobases of RNA and DNA. Among the nucleobases, guanine has the lowest redox potential, making it a major target of oxidation. Although RNA is more prone to oxidation than DNA is, oxidation of guanine in RNA has been studied to a significantly lesser extent. One of the reasons for this is that many tools that were previously developed to study oxidation of DNA cannot be used on RNA. In the study presented here, the lack of a method for seeking sites of modification in RNA where oxidation occurs is addressed. For this purpose, reverse transcription of RNA containing major products of guanine oxidation was used. Extension of a DNA primer annealed to an RNA template containing 8-oxo-7,8-dihydroguanine (OG), 5-guanidinohydantoin (Gh), or the R and S diastereomers of spiroiminodihydantoin (Sp) was studied under standing start conditions. SuperScript III reverse transcriptase is capable of bypassing these lesions in RNA inserting predominantly A opposite OG, predominantly G opposite Gh, and almost an equal mixture of A and G opposite the Sp diastereomers. These data should allow RNA sequencing of guanine oxidation products by following characteristic mutation signatures formed by the reverse transcriptase during primer elongation past G oxidation sites in the template RNA strand.

Figure 1

RNA-DNA hybrid duplexes studied.

Figure 2. Nucleotide insertion profiles opposite G or OG in the templates

Insertion of A, G, C, or T in the reaction mixture with only one of the dNTPs present by SuperScript III (A) and Omniscript (B) reverse transcriptases for G-1, OG-1, G-2, and OG-2 templates. (C) Structures of OG-C, OG-A, G-C, and G-T base pairs.

Figure 3. Nucleotide insertion assays for Gh and Sp

A - Insertion of A, G, C, or T in the reaction mixture with only one of the dNTPs present by SuperScript III reverse transcriptase for Gh-1, S-Sp-1, and R-Sp-1 templates. B - Structures of the base pairs between G or A and Gh or Sp are based on previous literature reports.^,

Figure 4

Michaelis-Menten plot for insertion of A or G opposite OG.

Figure 5. Efficiency of extension past different base pairs

A–D – polyacrylamide gels showing results of primer extension in presence of one (lanes 1, 3, 5, and 6), two (lanes 2, 4, and 7), or three (lane 8) different triphosphates aimed at comparing bypass efficiencies for base pairs between OG, Gh, S-Sp, and R-Sp and A, C or G. Samples in lanes 5 and 6 marked 2N contained a doubled concentration of corresponding dNTP compared to lanes 1 and 3. E - aligned gel lane plots showing separation between cDNA strands containing different bases inserted opposite OG, Gh, S-Sp, and R-Sp.

Figure 6. Efficiency of complete primer extension

A - polyacrylamide gel showing efficiency of synthesis of full-length cDNA based on the templates containing G, OG, Gh, S-Sp, or R-Sp at two different concentrations of dNTP mixture. B - aligned gel lane plots for different templates for reactions containing 500 and 200 μM dNTP mixture.

Scheme 1

Pathways of Guanine Oxidation.