Organisms have evolved to live in a variety of complex environments, which clearly has required cellular biology to accommodate to extreme conditions of hydraulic pressure and elevated temperature. In this work, we exploit single-molecule Forster resonance energy transfer (FRET) spectroscopy to probe structural changes in DNA hairpins as a function of pressure and temperature, which allows us to extract detailed thermodynamic information on changes in free energy (ΔG°), free volume (ΔV°), enthalpy (ΔH°), and entropy (ΔS°) associated with DNA loop formation and sequence-dependent stem hybridization. Specifically, time-correlated single-photon counting experiments on freely diffusing 40A DNA hairpin FRET constructs are performed in a 50 μm × 50 μm square quartz capillary cell pressurized from ambient pressure up to 3 kbar. By pressure-dependent van't Hoff analysis of the equilibrium constants, ΔV° for hybridization of the DNA hairpin can be determined as a function of stem length (nstem = 7-10) with single base-pair resolution, which further motivates a simple linear deconstruction into additive stem (ΔV°stem = ΔV°bp x nstem) and loop (ΔV°loop) contributions. We find that increasing pressure destabilizes the DNA hairpin stem region [ΔV°bp = +1.98(16) cm3/(mol bp)], with additional positive free volume changes [ΔV°loop = +7.0(14) cm3/mol] we ascribe to bending and base stacking disruption of the 40-dA loop. From a van't Hoff temperature-dependent analysis of the DNA 40A hairpin equilibria, the data support a similar additive loop/stem deconstruction of enthalpic (ΔH° = ΔH°loop + ΔH°stem) and entropic (ΔS° = ΔS°loop + ΔS°stem) contributions, which permits insightful comparison with predictions from nearest-neighbor thermodynamic models for DNA duplex formation. In particular, the stem thermodynamics is consistent with exothermically favored (ΔH°stem < 0) and entropically penalized (ΔS°stem < 0) hydrogen bonding but with additional enthalpic (ΔH°loop > 0) and entropic (ΔS°loop > 0) contributions due to loop bending effects consistent with distortion of dA base stacking in the 40-dA linker.

中文翻译：

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极端压力下的DNA发夹杂交：单分子FRET研究。

生物已经进化为生活在各种复杂的环境中，这显然需要细胞生物学来适应液压和高温的极端条件。在这项工作中，我们利用单分子福斯特共振能量转移（FRET）光谱来探测DNA发夹结构随压力和温度的变化，这使我们能够提取有关自由​​能变化（ΔG°）的详细热力学信息，与DNA环形成和依赖序列的茎杂交相关的自由体积（ΔV°），焓（ΔH°）和熵（ΔS°）。具体而言，在从环境压力升至3 kbar的50μm×50μm方形石英毛细管中，对自由扩散的40A DNA发夹FRET构建体进行时间相关的单光子计数实验。通过对平衡常数进行压力依赖的van't Hoff分析，可以确定DNA发夹杂交的ΔV°是茎长度（nstem = 7-10）的函数，具有单个碱基对的分辨率，这进一步促进了简单的分析。线性解构成加性词干（ΔV°stem =ΔV°bp x nstem）和循环（ΔV°loop）的贡献。我们发现增加的压力会破坏DNA发夹茎区域的稳定度[ΔV°bp = +1.98（16）cm3 /（mol bp）]，而额外的正自由体积变化[ΔV°loop = +7.0（14）cm3 / mol]我们归因于40 dA环路的弯曲和基础堆叠破坏。根据DNA的40A发夹平衡的van't Hoff温度依赖性分析，数据支持类似的焓（ΔH°=ΔH°loop +ΔH°stem）和熵（ΔS°=ΔS°loop）的加法环/茎构解。 +ΔS°stem）贡献，可以与最近邻的热力学模型对DNA双链体形成的预测进行深入的比较。特别地，阀杆的热力学与放热有利的（ΔH°stem <0）和熵受罚的（ΔS°stem <0）氢键相符，但具有额外的焓（ΔH°loop> 0）和熵（ΔS°loop> 0）。由于环路弯曲效应而产生的影响与40-dA接头中dA基础堆叠的变形一致。