Department of Biochemistry, Duke University School of Medicine, Durham, NC, USA.
Department of Chemistry, Duke University, Durham, NC, USA.
Nucleic Acids Res. 2018 Nov 16;46(20):11099-11114. doi: 10.1093/nar/gky885.
A(syn)-U/T and G(syn)-C+ Hoogsteen (HG) base pairs (bps) are energetically more disfavored relative to Watson-Crick (WC) bps in A-RNA as compared to B-DNA by >1 kcal/mol for reasons that are not fully understood. Here, we used NMR spectroscopy, optical melting experiments, molecular dynamics simulations and modified nucleotides to identify factors that contribute to this destabilization of HG bps in A-RNA. Removing the 2'-hydroxyl at single purine nucleotides in A-RNA duplexes did not stabilize HG bps relative to WC. In contrast, loosening the A-form geometry using a bulge in A-RNA reduced the energy cost of forming HG bps at the flanking sites to B-DNA levels. A structural and thermodynamic analysis of purine-purine HG mismatches reveals that compared to B-DNA, the A-form geometry disfavors syn purines by 1.5-4 kcal/mol due to sugar-backbone rearrangements needed to sterically accommodate the syn base. Based on MD simulations, an additional penalty of 3-4 kcal/mol applies for purine-pyrimidine HG bps due to the higher energetic cost associated with moving the bases to form hydrogen bonds in A-RNA versus B-DNA. These results provide insights into a fundamental difference between A-RNA and B-DNA duplexes with important implications for how they respond to damage and post-transcriptional modifications.
与 B-DNA 相比，A-RNA 中的 A(syn)-U/T 和 G(syn)-C+Hoogsteen（HG）碱基对（bps）由于尚未完全理解的原因，其能量上相对于 Watson-Crick（WC）bps 更为不利，高出>1 kcal/mol。在这里，我们使用 NMR 光谱、光学熔融实验、分子动力学模拟和修饰核苷酸来确定导致 A-RNA 中 HG bps 不稳定的因素。在 A-RNA 双链体中去除单个嘌呤核苷酸的 2'-羟基并不能相对于 WC 稳定 HG bps。相比之下，使用 A-RNA 中的凸起来放松 A 型构象可以将侧翼位点形成 HG bps 的能量成本降低到 B-DNA 水平。嘌呤-嘌呤 HG 错配的结构和热力学分析表明，与 B-DNA 相比，A 型构象由于糖骨架重排需要空间容纳顺式碱基，使顺式嘌呤的能量不利 1.5-4 kcal/mol。基于 MD 模拟，由于在 A-RNA 中与 B-DNA 相比形成氢键时碱基移动的能量成本更高，嘌呤-嘧啶 HG bps 还会额外增加 3-4 kcal/mol 的罚分。这些结果提供了 A-RNA 和 B-DNA 双链体之间基本差异的深入了解，对它们如何响应损伤和转录后修饰具有重要意义。
