Thermodynamic and kinetic analysis of an RNA kissing interaction and its resolution into an extended duplex.
Thermodynamic and kinetic analysis of an RNA kissing interaction and its resolution into an extended duplex.
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RNA 亲吻相互作用的热力学和动力学分析及其解析成延伸双链体。
DOI：
10.1016/j.bpj.2011.12.052
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发表时间：
2012
影响因子：
3.4
通讯作者：
Feig,AndrewL
中科院分区：
文献类型：
--
作者：
Salim,Nilshad;Lamichhane,Rajan;Zhao,Rui;Banerjee,Tuhina;Philip,Jane;Rueda,David;Feig,AndrewL
当两个发夹的环残基具有沃森-克里克互补性时，形成了接吻发夹相互作用。在单分子背景下，接吻相互作用对于三级折叠和假结形成是重要的，而在双分子背景下，它们为分子识别提供了基础。在某些情况下，接吻复合物可以是链置换反应的前奏，其中两个发夹解析以形成稳定的延伸的分子间双链体。接吻复合物的形成及其随后的链置换反应的动力学和热力学知之甚少。在这里，生物物理技术，包括等温滴定量热法，表面等离子体共振，和单分子荧光已被用来探测的因素，管理接吻复合物的稳定性和随后的结构重排。我们表明，RNA双链体形成的一般理解可以扩展到接吻复合物，但接吻复合物相对于相同序列的简单双链体显示出不寻常的稳定性水平。这些相互作用在室温下形成和破坏多次，然后进行缓慢的、不可逆的向前转变为链置换形式。此外，使用smFRET，我们表明，稳定和不稳定的接吻复合物之间的主要差异几乎完全基于它们的关闭率。稳定和不稳定的复合物在误差范围内以相同的速率形成，但不太稳定的物种迅速解离，使我们能够理解这些复合物如何有助于沿着沿着折叠途径或在基因调控事件期间产生特异性。
Kissing hairpin interactions form when the loop residues of two hairpins have Watson-Crick complementarity. In a unimolecular context, kissing interactions are important for tertiary folding and pseudoknot formation, whereas in a bimolecular context, they provide a basis for molecular recognition. In some cases, kissing complexes can be a prelude to strand displacement reactions where the two hairpins resolve to form a stable extended intermolecular duplex. The kinetics and thermodynamics of kissing-complex formation and their subsequent strand-displacement reactions are poorly understood. Here, biophysical techniques including isothermal titration calorimetry, surface plasmon resonance, and single-molecule fluorescence have been employed to probe the factors that govern the stability of kissing complexes and their subsequent structural rearrangements. We show that the general understanding of RNA duplex formation can be extended to kissing complexes but that kissing complexes display an unusual level of stability relative to simple duplexes of the same sequence. These interactions form and break many times at room temperature before becoming committed to a slow, irreversible forward transition to the strand-displaced form. Furthermore, using smFRET we show that the primary difference between stable and labile kissing complexes is based almost completely on their off rates. Both stable and labile complexes form at the same rate within error, but less stable species dissociate rapidly, allowing us to understand how these complexes can help generate specificity along a folding pathway or during a gene regulation event.