Xu Youzhi, Feng Xiaojun, Du Wei, Liu Xin, Luo Qingming, Liu Bi-Feng
Division of Biomedical Photonics at Wuhan National Laboratory for Optoelectronics, Department of Systems Biology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China
Anal Chem. 2008 Sep 15;80(18):6935-41. doi: 10.1021/ac801335y. Epub 2008 Aug 12.
In this paper, nonequilibrium capillary electrophoresis (NECE) was attempted for the first time to investigate a dual equilibrium system, where the intramolecular G-quadruplex folding was in competition with the intermolecular duplex formation. Samples of an equilibrium mixture of human telomeric DNA and its complementary strands were separated in capillaries under nonequilibrium conditions without K (+). Polyethylene oxide was added to the running buffer facilitating the separation of single-stranded DNA, duplex, and G-quadruplex. Thus, the folding/unfolding rate constants of the G-quadruplex and the association/dissociation constants of the duplex could be simultaneously derived from the same experiment. Results indicated that the duplex formation induced minimal influence on the G-quadruplex folding. On the basis of the kinetic characterization of the G-quadruplex at varying temperatures, the thermodynamic parameters of the G-quadruplex could also be determined. Thus, the NECE method provided a new avenue for studying the kinetics and thermodynamics of nucleic acids within dual equilibrium systems with significant advantages of extreme-low sample cost (approximately 10 (-18) mol) and high repeatability.
在本文中，首次尝试采用非平衡毛细管电泳（NECE）来研究一种双重平衡体系，其中分子内G-四链体折叠与分子间双链形成相互竞争。在无K(+)的非平衡条件下，在毛细管中分离人端粒DNA及其互补链的平衡混合物样品。向运行缓冲液中添加聚环氧乙烷，有助于单链DNA、双链和G-四链体的分离。因此，可从同一实验中同时得出G-四链体的折叠/解折叠速率常数和双链的缔合/解离常数。结果表明，双链形成对G-四链体折叠的影响最小。基于不同温度下G-四链体的动力学特征，还可确定G-四链体的热力学参数。因此，NECE方法为研究双重平衡体系中核酸的动力学和热力学提供了一条新途径，具有极低样品成本（约10(-18)mol）和高重复性的显著优势。
