Department of Chemistry, University of Colorado Denver, Denver, Colorado, United States of America.
PLoS One. 2020 Aug 28;15(8):e0235102. doi: 10.1371/journal.pone.0235102. eCollection 2020.
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, 18-mers, 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.
肌苷在许多生物过程中无处不在且必不可少，包括 RNA 编辑。此外，由于其在疾病的发展/进展中的潜在作用，RNA 上的氧化应激已成为越来越受关注的话题。在这项工作中，我们研究了三种逆转录酶 (RT) 在含有肌苷 (I)、8-氧代-7,8-二氢肌苷 (8oxo-I)、鸟苷 (G) 或 8-氧代-7,8-二氢鸟苷 (8-oxoG) 的 RNA 模板存在下催化 cDNA 合成的能力，并探讨了这些嘌呤衍生物作为功能的位置的影响。为此，我们使用了包含修饰物的 29 个核苷酸的 RNA (作为模板)，并使用 17 个核苷酸、18 个核苷酸或 19 个核苷酸 (作为引物) 进行逆转录 DNA。一般来说，病毒 RT，AMV / HIV / MMLV，对包含 G 或 I 的模板以及包含 8-oxoG 或 8-oxoI 的模板的 cDNA 合成反应性相似。值得注意的差异是：1）使用 18 个核苷酸的 DNA（探索 cDNA 合成越过病变/修饰）会导致在存在 G:dA 摆动对的情况下抑制 DNA 延伸，而 I、8-氧代 I 或 8-氧代 G 导致相应 cDNA 的完全合成，后两者显示出更有效的过程；2）HIV RT 对 cDNA 合成附近的修饰碱基对更敏感；3）转录起始位置相差两个位置的修饰对整体过程有不利影响。使用 AMV RT 建立了稳态动力学，以确定对典型 dNTP（N = G、C、T、A）的底物特异性。总的来说，我们有证据表明，含有肌苷的 RNA 模板可能会掺入 dC > dT > > dA，其中在存在 dA 的情况下的反应性依赖于 pH（在 pH 7.3 时被破坏）；并且缺乏 C2-外环胺，如含有 8-氧代 I 的模板所示，导致对 dA 的掺入选择性增加而不是 dC。该数据将有助于评估肌苷和/或氧化产生的损伤的存在对病毒过程的影响，并补充了以前的报告，其中 I 专门编码为 G。在比较 AMV 和 MMLV RT 时获得了类似的结果。
