DataSheet1_Hydrogen bonding in duplex DNA probed by DNP enhanced solid-state NMR N-H bond length measurements.PDF

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.

众多生物过程与机制均依赖于DNA中碱基配对与氢键作用的细节信息。X射线晶体学（X-ray crystallography）与冷冻电镜（cryo-EM）难以量化氢键作用，原因在于难以观测氢原子的位置，但溶液态与固态核磁共振波谱法（NMR spectroscopy）可实现位点特异性的氢键探测，其中固态NMR尤其适用于体积庞大、转动缓慢的DNA复合物。此前我们的研究表明，低温动态核极化（dynamic nuclear polarization，DNP）增强型固态核磁共振技术，可在类天然条件下，于多种DNA体系中有效区分胡斯坦碱基配对（Hoogsteen base pairs，bps）与经典沃森-克里克碱基配对（canonical Watson-Crick bps）。本研究以包含两段中央腺嘌呤-胸腺嘧啶（A-T）碱基配对的12聚体DNA双链为模型，分别采用沃森-克里克构象与顺式胡斯坦构象，通过实验证明可利用DNP固态核磁共振技术测量胸腺嘧啶N3-H3键长——该键长对N-H···N氢键的细节极为敏感，且可在相同DNA序列背景下，系统比较两种碱基配对构象的氢键作用。针对该DNA双链，相较于在相近实验条件下测得的N-乙酰缬氨酸（N-acetyl-valine）参考酰胺键长1.015 ± 0.010 Å，沃森-克里克A-T碱基配对与顺式胡斯坦A（syn）-T碱基配对的胸腺嘧啶N3-H3键长分别为1.055 ± 0.011 Å与1.060 ± 0.011 Å，二者几乎一致。考虑到此前考虑零点运动的量子化学计算预测，肽段N-H有效键长约为1.041 Å，这与常温下肽与蛋白质的溶液态及固态核磁共振研究结果相符；为便于与此前研究直接对比，我们可对DNA样品的胸腺嘧啶N3-H3键长进行适当校正：以1.041 Å的肽N-H参考键长为基准，沃森-克里克A-T与顺式胡斯坦A（syn）-T碱基配对的校正后键长分别为1.083 Å与1.087 Å。值得注意的是，在该模型DNA双链的背景下，上述结果表明，沃森-克里克与胡斯坦碱基配对构象之间的N-H···N A-T氢键并无显著差异。更广泛而言，基于低温DNP固态核磁共振技术的高精度N-H键长测量方法，有望为一系列DNA复合物与碱基配对环境中的氢键作用提供精细化的对比分析手段。