Translesion synthesis by AMV, HIV, and MMLVreverse transcriptases using RNA templates containing inosine, guanosine, and their 8-oxo-7,8-dihydropurine derivatives

Abstract

Inosine is ubiquitous and essential in many biological processes, including RNA-editing. In addition, oxidative stress on RNA has been a topic of increasing interest due, in part, to its potential role in the development/progression of disease. In this work we probed the ability of three reverse transcriptases (RTs) to catalyze the synthesis of cDNA in the presence of RNA templates containing inosine (I), 8-oxo-7,8-dihydroinosine (8oxo-I), guanosine (G), or 8-oxo-7,8-dihydroguanosine (8-oxoG), and explored the impact that these purine derivatives have as a function of position. To this end, we used 29-mers of RNA (as template) containing the modifications at position-18 and reverse transcribed DNA using 17-mers, 18mers, or 19-mers (as primers). Generally reactivity of the viral RTs, AMV / HIV / MMLV, towards cDNA synthesis was similar for templates containing G or I as well as for those with 8-oxoG or 8-oxoI. Notable differences are: 1) the use of 18-mers of DNA (to explore cDNA synthesis past the lesion/modification) led to inhibition of DNA elongation in cases where a G:dA wobble pair was present, while the presence of I, 8-oxoI, or 8-oxoG led to full synthesis of the corresponding cDNA, with the latter two displaying a more efficient process; 2) HIV RT is more sensitive to modified base pairs in the vicinity of cDNA synthesis; and 3) the presence of a modification two positions away from transcription initiation has an adverse impact on the overall process. Steady-state kinetics were established using AMV RT to determine substrate specificities towards canonical dNTPs (N = G, C, T, A). Overall we found evidence that RNA templates containing inosine are likely to incorporate dC > dT > > dA, where reactivity in the presence of dA was found to be pH dependent (process abolished at pH 7.3); and that the absence of the C2-exocyclic amine, as displayed with templates containing 8-oxoI, leads to increased selectivity towards incorporation of dA over dC. The data will be useful in assessing the impact that the presence of inosine and/or oxidatively generated lesions have on viral processes and adds to previous reports where I codes exclusively like G. Similar results were obtained upon comparison of AMV and MMLV RTs.

Experimental and theoretical rationalization for the base pairing abilities of inosine, guanosine, adenosine, and their corresponding 8‐oxo‐7,8‐dihydropurine, and 8‐bromopurine analogues within A‐form duplexes of RNA

“…+4 °C. This result is in agreement with favorable interactions that occur in biochemical processes such as reverse transcription, [ 32 ] where RNA templates containing inosine are able to incorporate dA on their corresponding DNA primer and allow cDNA synthesis, albeit at lower efficiency rates and where the G-containing RNA analogue does not display this behavior. This result may be interpreted as a lack of H-bonding interactions from the excocyclic amine in inosine, or forced conformational changes to fit a favorable H-bonding pattern (vide infra).…”

“…The only base pair that exhibited increase thermal stability upon destitution of the exocyclic amine (G-to-I exchange) was in the case where base pairing occurred with A ( Figure 7C ). Besides the importance of G:A base pairs in various biological contexts [ 52 ] our laboratory recently reported on a case where reverse transcription allowed for the incorporation of dA opposite I, but not opposite G, [ 32 ] this case was of particular interest to us. The modeling provided useful data in this regard, where the amine group destabilized a G:A base pair in conformations that were more plausible ( Figure 5 , entries 22 and 34) and inhibited the formation of a planar base pair.…”

“…The synthesis for the phosphoramidite of 8-oxoG, [ 19 ] 8-oxoA, [ 31 ] and 8-oxoI/8-BrI [ 32 ] were previously reported by us and the same methodology was used to prepare all oligonucleotides, via solid-phase synthesis. The synthesis of oligonucleotides of RNA containing 8-BrA was not possible in our hands due to its transformation to the corresponding 8-methylamine derivative (upon AMA-deprotection of the synthesized oligonucleotide, Figure S-4 ).…”

Processing of RNA Containing 8-Oxo-7,8-Dihydroguanosine (8-oxoG) by the Exoribonuclease Xrn-1

“…We then opted to probe a longer sequence, 29-nt-long ( Figure 4 ). Oligonucleotides 17 – 19 have been previously reported, and do not display the formation of secondary structure ( via CD, Figure 4 and Supplementary Figures S76–S78 ) ( Glennon et al, 2020 ). These RNA sequences were used to probe for the presence of one or two consecutive 8-oxoG lesions within the sequence.…”