Bhai Lakshmi, Thomas Justin K, Conroy Daniel W, Xu Yu, Al-Hashimi Hashim M, Jaroniec Christopher P
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, United States.
Department of Chemistry, Duke University, Durham, NC, United States.
Front Mol Biosci. 2023 Dec 4;10:1286172. doi: 10.3389/fmolb.2023.1286172. eCollection 2023.
Numerous biological processes and mechanisms depend on details of base pairing and hydrogen bonding in DNA. Hydrogen bonds are challenging to quantify by X-ray crystallography and cryo-EM due to difficulty of visualizing hydrogen atom locations but can be probed with site specificity by NMR spectroscopy in solution and the solid state with the latter particularly suited to large, slowly tumbling DNA complexes. Recently, we showed that low-temperature dynamic nuclear polarization (DNP) enhanced solid-state NMR is a valuable tool for distinguishing Hoogsteen base pairs (bps) from canonical Watson-Crick bps in various DNA systems under native-like conditions. Here, using a model 12-mer DNA duplex containing two central adenine-thymine (A-T) bps in either Watson-Crick or Hoogsteen confirmation, we demonstrate DNP solid-state NMR measurements of thymine N3-H3 bond lengths, which are sensitive to details of N-H···N hydrogen bonding and permit hydrogen bonds for the two bp conformers to be systematically compared within the same DNA sequence context. For this DNA duplex, effectively identical TN3-H3 bond lengths of 1.055 ± 0.011 Å and 1.060 ± 0.011 Å were found for Watson-Crick A-T and Hoogsteen A (syn)-T base pairs, respectively, relative to a reference amide bond length of 1.015 ± 0.010 Å determined for N-acetyl-valine under comparable experimental conditions. Considering that prior quantum chemical calculations which account for zero-point motions predict a somewhat longer effective peptide N-H bond length of 1.041 Å, in agreement with solution and solid-state NMR studies of peptides and proteins at ambient temperature, to facilitate direct comparisons with these earlier studies TN3-H3 bond lengths for the DNA samples can be readily scaled appropriately to yield 1.083 Å and 1.087 Å for Watson-Crick A-T and Hoogsteen A (syn)-T bps, respectively, relative to the 1.041 Å reference peptide N-H bond length. Remarkably, in the context of the model DNA duplex, these results indicate that there are no significant differences in N-H···N A-T hydrogen bonds between Watson-Crick and Hoogsteen bp conformers. More generally, high precision measurements of N-H bond lengths by low-temperature DNP solid-state NMR based methods are expected to facilitate detailed comparative analysis of hydrogen bonding for a range of DNA complexes and base pairing environments.
许多生物过程和机制都依赖于DNA中碱基配对和氢键的细节。由于难以确定氢原子的位置，通过X射线晶体学和冷冻电镜对氢键进行定量具有挑战性，但在溶液和固态中，可以通过核磁共振光谱进行位点特异性探测，后者特别适用于大型、缓慢翻滚的DNA复合物。最近，我们表明低温动态核极化（DNP）增强固态核磁共振是一种有价值的工具，可在类似天然条件下区分各种DNA系统中的Hoogsteen碱基对（bps）和经典的沃森-克里克碱基对。在这里，我们使用一个包含两个处于沃森-克里克或Hoogsteen构象的中心腺嘌呤-胸腺嘧啶（A-T）碱基对的12聚体DNA双链体模型，展示了胸腺嘧啶N3-H3键长的DNP固态核磁共振测量，该键长对N-H···N氢键的细节敏感，并允许在相同的DNA序列背景下系统地比较两种碱基对构象的氢键。对于该DNA双链体，相对于在可比实验条件下为N-乙酰缬氨酸确定的1.015±0.010 Å的参考酰胺键长，沃森-克里克A-T和Hoogsteen A（反式）-T碱基对的有效TN3-H3键长分别为1.055±0.011 Å和1.060±0.011 Å。考虑到先前考虑零点运动的量子化学计算预测有效肽N-H键长略长，为1.041 Å，这与肽和蛋白质在环境温度下的溶液和固态核磁共振研究一致，为便于与这些早期研究进行直接比较，DNA样品的TN3-H3键长可以很容易地适当缩放，相对于1.041 Å的参考肽N-H键长，沃森-克里克A-T和Hoogsteen A（反式）-T碱基对的键长分别为1.083 Å和1.087 Å。值得注意的是，在模型DNA双链体的背景下，这些结果表明沃森-克里克和Hoogsteen碱基对构象之间的N-H···N A-T氢键没有显著差异。更一般地说，基于低温DNP固态核磁共振方法的N-H键长的高精度测量有望促进对一系列DNA复合物和碱基配对环境中氢键的详细比较分析。
