5-Carboxylcytosine and Cytosine Protonation Distinctly Alter the Stability and Dehybridization Dynamics of the DNA Duplex
5-Carboxylcytosine and Cytosine Protonation Distinctly Alter the Stability and Dehybridization Dynamics of the DNA Duplex
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DOI：
10.1021/acs.jpcb.9b11510
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发表时间：
2020-01-30
影响因子：
3.3
通讯作者：
Tokmakoff, Andrei
中科院分区：
文献类型：
--
作者：
Ashwood, Brennan;Sanstead, Paul J.;Tokmakoff, Andrei
与核碱基质子化事件相关的应用基于它们对 DNA 热力学、结构和杂交动力学的根本影响。在经典核碱基中，胞嘧啶 (C) 的 N3 质子化在生物学和纳米技术中应用最广泛。天然存在的 C 衍生物可改变 N3 pKa，从而引入额外水平的可调性。表观遗传核碱基 5-羧基胞嘧啶 (caC) 是一个特别有趣的例子，因为该衍生物形成具有相似稳定性的 Watson-Crick 碱基对，并在与标准核碱基相同的范围内表现出 pH 依赖性行为。然而，caC 中的可滴定基团对应于环外羧基而不是 N3，并且这些不同的质子化事件对 DNA 杂交热力学、动力学和碱基配对动力学的影响仍然知之甚少。在这里，我们使用含有 C 和 caC 的模型寡核苷酸以及 FTIR 和跳温红外光谱来研究这些物理特性的 pH 依赖性。我们证明，C 的 N3 质子化完全破坏了双链体稳定性，导致双链体/单链平衡发生较大变化，解链协同性降低，双链体解离速率加快。相反，虽然增加含 caC 双链体中 5-羧基质子化会导致碱基对波动增加，但 DNA 双链体可以耐受大量质子化，而不会显着扰动双链体/单链平衡。然而，5-羧基质子化通过降低过渡态自由能对杂化动力学产生很大影响。我们的热力学和动力学分析提供了关于天然核碱基中两种不同质子化机制对 DNA 生物物理特性的影响的新见解。
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.