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, 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 current study, 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.

Translesion Synthesis by MmLV-, AMV-, and HIV-Reverse Transcriptases Using RNA Templates Containing Inosine, Guanosine, and Their 8-oxo-7,8-Dihydropurine Derivatives

“…Overall, the results obtained using 8-oxoG and AMV-or MMLV-RT are in good agreement with a previous report, 38 where A and C added efficiently opposite 8-oxoG compared to addition of C and T opposite G. In a separate report, C and T were reported to extend DNA synthesis opposite 8-oxoG using RAV2-RT and exclusively C using HIV-RT. 56 This report contrasts with the results obtained herein, possibly due to the use of different experimental conditions.…”

“…To corroborate the dependence of reverse transcription with the base pairing interactions at this position, duplexes where the last nucleotide of the RNA template is base pairing to dC (duplexes 1:7 -4:7) were probed for RTn ( Figure 3C). As expected, formation of a Watson-Crick base pair in duplex 1:7 restored the ability of the RT to carry out cDNA synthesis in the presence of a dNTP mix; where the other three duplexes also displayed cDNA synthesis under these conditions (lanes 26,32,38,44). Steady-state kinetics showed that all duplexes enabled the incorporation of dT with similar efficiencies (Figure 3C, table).…”

“…To begin our studies, templates of RNA (29-nt long) 1-4 were set for hybridization with the corresponding DNA primer, 17-mer (5) (Figure 2A). The sequence of the template was chosen based on a report by Alenko et al, 38 with the exception that the length of the RNA strand was extended by nine nucleotides to explore continuation of cDNA synthesis in more detail. All solutions were prepared in the buffer provided by the manufacturer (see experimental details).…”

“…(37) Furthermore, reverse transcription of RNA containing 8-oxoG in the presence of AMV-or MMLV-RTs showed insertion of A and C opposite this lesion. (38) In this work, we were interested in 1) probing the impact of the exocyclic amine on both G and 8-oxoG, thus we directly compared strands of RNA containing this nucleobases to RNA containing I or 8-oxoI; 2) extending the impact of reverse transcription to HIV-RT; and 3) establishing the reactivity of the reverse transcriptase within the vicinity of the reactive site containing G, I, 8-oxoG, or 8-oxoI (for which three DNA primers of varying length were used). Interestingly some unexpected reactivity was observed on RNA containing I, where experiments carried out in the presence of dTTP or dATP allowed for the enzyme to facilitate their incorporation into DNA opposite this modification.…”

Optimized photochemistry and enzymology enable efficient analysis of RNA structures and interactions in cells and virus infections

“…14). RNA damage impedes reverse transcription by trapping the enzyme in an inactive state 19,20 . We reasoned that conditions that promote enzyme conformation dynamics, or longer incubation time, may overcome such barriers.…”