Insight into the G-Quadruplex Topologies: A Rapid Separation and Identification Method of Multiple Coexisting...

G-quadruplexes (G4s) are noncanonical secondary structures, which have been demonstrated to be prevalent at gene regulatory regions in various genomes. In solution, G4s derived from single-stranded oligonucleotides can adopt different topologies. Although considerable research over the past three decades has led to in-depth insight into these unusual structures, there is still a great demand to develop a rapid, easy, and universal strategy to separate and identify G4 topologies, especially G4s with various coexisting structures. Herein, we took advantage of capillary zone electrophoresis (CZE) to differentiate G4s with close lengths (the difference being less than 3 nt) based on the diversity of apparent ionic radii. A random sequence (RDNA) was introduced as an internal standard, and the relative migration time ratio (RMTR) was applied as the monitoring indicator. In this way, we were able to distinguish and separate parallel types in a variety of G4 mixtures with potassium ions as the background electrolyte (BGE) and antiparallel types with sodium ions, separately, in high confidence (p < 0.001). By adjusting ion concentration, three conformations could be differentiated from each other completely at a higher sodium ion concentration (30 mM) with a confidence of at least p < 0.05. Based on the developed method, we quantified the proportion of Bcl-2's multiple peaks and found that Bcl-2 tended to transfer to a hybrid structure under high K⁺ and PEG-300 due to electrostatic interactions and molecular crowding, respectively. Our method offers a new perspective to distinguish topologies of G4s by CZE and fills the gap in the rapid identification and separation of multiple coexisting conformations of G4s, displaying many advantages including low sample consumption, high efficiency, low cost, simple operation, and so on.