Institute of Atomic and Molecular Sciences, Academia Sinica , Taipei 106, Taiwan, R.O.C.
J Am Chem Soc. 2015 Jan 14;137(1):210-8. doi: 10.1021/ja5089327. Epub 2014 Dec 24.
The role of G-quadruplexes (G4s) in biological systems has been widely studied. It is found that they have an important function in gene transcription and regulation. In this work, we have identified two topologies of hairpin and G4 structures formed by a native G-rich sequence (WT22: 5'-GGGCCACCGGGCAGGGGGCGGG-3') from the WNT1 promoter region using nuclear magnetic resonance (NMR) spectroscopy. With the help of site-specific isotope labeling, the topologies of these two structures are unambiguously characterized. Circular dichroism and NMR results are analyzed to determine the kinetics associated with the potassium ion-induced hairpin-to-G4 transition, which is very slow-on the time scale of 4800 s-compared to the previously reported folding kinetics of G4 formation. In addition, the free energies of the unfolding of these two structures are obtained using differential scanning calorimetry. Combining the kinetic and thermodynamic data, we have established the free energy landscape of this two-state folding system. Considering that similar conformational change may exist in other native G-rich sequences, this work highlights an important hairpin to G4 conformational transition which can be used in manipulation of gene regulation or ligand modulation in vivo.
G-四链体（G4s）在生物系统中的作用已经得到了广泛的研究。研究发现，它们在基因转录和调控中具有重要的功能。在这项工作中，我们使用核磁共振（NMR）光谱技术鉴定了 WNT1 启动子区域中一个天然富含 G 的序列（WT22：5'-GGGCCACCGGGCAGGGGGCGGG-3'）形成的发夹和 G4 结构的两种拓扑结构。借助于定点同位素标记，这些结构的拓扑结构得到了明确的表征。通过圆二色性和 NMR 分析来确定与钾离子诱导发夹到 G4 转变相关的动力学，与之前报道的 G4 形成折叠动力学相比，这个转变非常缓慢——在 4800 秒的时间尺度上。此外，使用差示扫描量热法获得了这两种结构的解折叠自由能。结合动力学和热力学数据，我们建立了这个两态折叠系统的自由能景观。考虑到类似的构象变化可能存在于其他天然富含 G 的序列中，这项工作突出了一个重要的发夹到 G4 构象转变，可用于体内基因调控或配体调节的操作。
