Applications associated with nucleobase protonation events are grounded in their fundamental impact on DNA thermodynamics, structure, and hybridization dynamics. Of the canonical nucleobases, N3 protonation of cytosine (C) is the most widely utilized in both biology and nanotechnology. Naturally occurring C derivatives that shift the N3 pKa introduce an additional level of tunability. The epigenetic nucleobase 5-carboxylcytosine (caC) presents a particularly interesting example since this derivative forms Watson-Crick base pairs of similar stability and displays pH-dependent behavior over the same range as the canonical nucleobase. However, the titratable group in caC corresponds to the exocyclic carboxyl group rather than N3, and the implications of these divergent protonation events toward DNA hybridization thermodynamics, kinetics, and base pairing dynamics remain poorly understood. Here, we study the pH dependence of these physical properties using model oligonucleotides containing C and caC with FTIR and temperature-jump IR spectroscopy. We demonstrate that N3 protonation of C completely disrupts duplex stability, leading to large shifts in the duplex/single-strand equilibrium, a reduction in the cooperativity of melting, and an acceleration in the rate of duplex dissociation. In contrast, while increasing 5-carboxyl protonation in caC-containing duplexes induces an increase in base pair fluctuations, the DNA duplex can tolerate substantial protonation without significant perturbation to the duplex/single-strand equilibrium. However, 5-carboxyl protonation has a large impact on hybridization kinetics by reducing the transition state free energy. Our thermodynamic and kinetic analysis provides new insight on the impact of two divergent protonation mechanisms in naturally occurring nucleobases on the biophysical properties of DNA.

中文翻译：

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5-羧基胞嘧啶和胞嘧啶质子化明显改变了DNA双链体的稳定性和去杂化动力学。

与核碱基质子化事件相关的应用基于其对DNA热力学，结构和杂交动力学的基本影响。在典型的核碱基中，胞嘧啶（C）的N3质子化是生物学和纳米技术中使用最广泛的。改变N3 pKa的自然存在的C衍生物会引入更高级别的可调性。表观遗传核碱基5-羧基胞嘧啶（caC）提供了一个特别有趣的示例，因为该衍生物形成了具有相似稳定性的Watson-Crick碱基对，并在与规范核碱基相同的范围内显示了pH依赖性行为。但是，caC中的可滴定基团对应于环外羧基而不是N3，并且这些发散质子化事件对DNA杂交的热力学，动力学，和碱基配对动力学仍然知之甚少。在这里，我们使用含C和caC的模型寡核苷酸，FTIR和温度跳跃IR光谱学研究了这些物理性质的pH依赖性。我们证明，C的N3质子化会完全破坏双链体的稳定性，从而导致双链体/单链平衡发生大的变化，熔化的协同性降低以及双链体解离速率的加速。相反，虽然在含caC的双链体中增加5-羧基质子化会引起碱基对波动的增加，但DNA双链体可以耐受大量的质子化而不会严重干扰双链体/单链平衡。但是，5-羧基质子化通过降低过渡态自由能对杂交动力学有很大影响。