Department of Chemistry, Wayne State University, Detroit, Michigan, USA.
Biophys J. 2012 Mar 7;102(5):1097-107. doi: 10.1016/j.bpj.2011.12.052. Epub 2012 Mar 6.
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.
发夹环相互作用形成于两个发夹的环残基具有 Watson-Crick 互补性时。在单分子环境中，发夹环相互作用对于三级折叠和假结形成很重要，而在双分子环境中，它们为分子识别提供了基础。在某些情况下，发夹环复合物可以作为链位移反应的前奏，其中两个发夹解析形成稳定的扩展分子间双链。发夹环复合物形成及其随后的链位移反应的动力学和热力学仍知之甚少。在这里，包括等温滴定量热法、表面等离子体共振和单分子荧光在内的生物物理技术已被用于研究控制发夹环复合物稳定性及其随后结构重排的因素。我们表明，对 RNA 双链形成的普遍理解可以扩展到发夹环复合物，但与相同序列的简单双链相比，发夹环复合物显示出异常水平的稳定性。这些相互作用在室温下多次形成和断裂，然后才缓慢、不可逆地向前转变为链位移形式。此外，我们使用 smFRET 表明，稳定和不稳定发夹环复合物之间的主要区别几乎完全基于它们的离解速率。在误差范围内，稳定和不稳定的复合物以相同的速率形成，但不太稳定的物质迅速解离，这使我们能够了解这些复合物如何帮助在折叠途径或基因调控事件中产生特异性。
