The replicative and translational machinery utilizes the unique geometry of canonical G·C and A·T/U Watson-Crick base pairs to discriminate against DNA and RNA mismatches in order to ensure high fidelity replication, transcription, and translation. There is growing evidence that spontaneous errors occur when mismatches adopt a Watson-Crick-like geometry through tautomerization and/or ionization of the bases. Studies employing NMR relaxation dispersion recently showed that wobble dG·dT and rG·rU mismatches in DNA and RNA duplexes transiently form tautomeric and anionic species with probabilities (≈0.01-0.40%) that are in concordance with replicative and translational errors. Although computational studies indicate that these exceptionally short-lived and low-abundance species form Watson-Crick-like base pairs, their conformation could not be directly deduced from the experimental data, and alternative pairing geometries could not be ruled out. Here, we report direct NMR evidence that the transient tautomeric and anionic species form hydrogen-bonded Watson-Crick-like base pairs. A guanine-to-inosine substitution, which selectively knocks out a Watson-Crick-type (G)N2H2···O2(T) hydrogen bond, significantly destabilized the transient tautomeric and anionic species, as assessed by lack of any detectable chemical exchange by imino nitrogen rotating frame spin relaxation (R1ρ) experiments. An 15N R1ρ NMR experiment targeting the amino nitrogen of guanine (dG-N2) provides direct evidence for Watson-Crick (G)N2H2···O2(T) hydrogen bonding in the transient tautomeric state. The strategy presented in this work can be generally applied to examine hydrogen-bonding patterns in nucleic acid transient states including in other tautomeric and anionic species that are postulated to play roles in replication and translational errors.

中文翻译：

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dG·dT 中瞬态互变异构和阴离子状态形成 Watson-Crick 样碱基对的直接 NMR 证据

复制和翻译机制利用规范 G·C 和 A·T/U Watson-Crick 碱基对的独特几何结构来区分 DNA 和 RNA 错配，以确保高保真复制、转录和翻译。越来越多的证据表明，当不匹配通过碱基的互变异构化和/或电离采用类​​似 Watson-Crick 的几何结构时，会发生自发错误。最近使用 NMR 弛豫分散的研究表明，DNA 和 RNA 双链体中的摆动 dG·dT 和 rG·rU 错配瞬时形成互变异构和阴离子物质，其概率 (≈0.01-0.40%) 与复制和翻译错误一致。尽管计算研究表明这些特别短命和低丰度的物种形成了类似沃森-克里克的碱基对，它们的构象不能直接从实验数据中推导出来，也不能排除其他配对几何形状。在这里，我们报告了直接 NMR 证据表明瞬态互变异构和阴离子物质形成氢键合的 Watson-Crick 样碱基对。鸟嘌呤到肌苷的取代，选择性地敲除 Watson-Crick 型 (G)N2H2...O2(T) 氢键，显着破坏了瞬态互变异构和阴离子物种的稳定性，正如缺乏任何可检测的化学交换所评估的那样通过亚氨基氮旋转框架自旋弛豫（R1ρ）实验。以鸟嘌呤的氨基氮 (dG-N2) 为目标的 15N R1ρ NMR 实验为 Watson-Crick (G)N2H2…O2(T) 氢键在瞬态互变异构状态下提供了直接证据。