Measurement of the effect of monovalent cations on RNA hairpin stability
Measurement of the effect of monovalent cations on RNA hairpin stability
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DOI：
10.1021/ja074809o
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发表时间：
2007-12-05
影响因子：
15
通讯作者：
Tinoco, Ignacio, Jr.
中科院分区：
文献类型：
--
作者：
Vieregg, Jeffrey;Cheng, Wei;Tinoco, Ignacio, Jr.
使用光镊，我们测量了单价阳离子浓度和种类对五个大（49-124 nt）RNA发夹折叠自由能的影响，包括HIV-1 TAR和近似A中心点U和G中心点C均聚物的分子。 RNA 二级结构热力学由最近邻相互作用以及加性环和凸出项组成的模型准确描述。小（< 15 bp）双链体和发夹在 1 M NaCl 中的熔化已用于确定该模型的参数，该模型现在广泛用于预测结构和折叠动力学。在其他离子条件或较大结构中进行的系统测量很少。通过施加机械力，我们测量了在室温下在 50 至 1000 mM 的阳离子浓度范围内折叠和展开单个发夹所需的功。然后使用克鲁克斯涨落定理确定自由能。我们观察到以下情况：（1）在大多数情况下，最近邻模型准确预测了 1 M NaCl 下的折叠自由能。 (2)自由能与盐浓度的对数成正比。 (3)用钾离子代替钠离子会略微降低发夹稳定性。 TAR 发夹在 KCl 中的错误折叠频率也几乎是 KCl 的两倍，表明动力学响应存在差异。 (4)单价阳离子浓度以序列依赖性方式影响RNA稳定性。 G 中心点 C 螺旋不受盐浓度变化的影响，A 中心点 U 螺旋受到适度影响，发夹环非常敏感。令人惊讶的是，发现 TAR 的 U 中心点 C 中心点 U 凸起在所有测试条件下都同样稳定。我们还报告了单链 RNA 弹性参数的新估计。
Using optical tweezers, we have measured the effect of monovalent cation concentration and species on the folding free energy of five large (49-124 nt) RNA hairpins, including HIV-1 TAR and molecules approximating A center dot U and G center dot C homopolymers. RNA secondary structure thermodynamics are accurately described by a model consisting of nearest-neighbor interactions and additive loop and bulge terms. Melting of small (< 15 bp) duplexes and hairpins in 1 M NaCl has been used to determine the parameters of this model, which is now used extensively to predict structure and folding dynamics. Few systematic measurements have been made in other ionic conditions or for larger structures. By applying mechanical force, we measured the work required to fold and unfold single hairpins at room temperature over a range of cation concentrations from 50 to 1000 mM. Free energies were then determined using the Crooks fluctuation theorem. We observed the following: (1) In most cases, the nearest-neighbor model accurately predicted the free energy of folding at 1 M NaCl. (2) Free energy was proportional to the logarithm of salt concentration. (3) Substituting potassium ions for sodium slightly decreased hairpin stability. The TAR hairpin also misfolded nearly twice as often in KCl, indicating a differential kinetic response. (4) Monovalent cation concentration affects RNA stability in a sequence-dependent manner. G center dot C helices were unaffected by changing salt concentration, A center dot U helices were modestly affected, and the hairpin loop was very sensitive. Surprisingly, the U center dot C center dot U bulge of TAR was found to be equally stable in all conditions tested. We also report a new estimate for the elastic parameters of single-stranded RNA.