Polymorphism is inherent for G-quadruplexes (G4s), and the different structural forms are important for the participation in different biological functions of telomeres. A lot of progress has been made in the exploration of G4 polymorphism. However, quick separation and reliable assessment methods for different conformations of G4 are still very few. In this work, the polymorphism of three sequences d[(G₃T)₃G₃], d[(G₃C)₃G₃] and d[(G₃T)₄] annealed in six different solutions were investigated by means of reversed-phase high performance liquid chromatography (RP-HPLC), liquid chromatography–mass spectrometry (LC–MS), fluorescence spectroscopy, circular dichroism spectroscopy, together with native-polyacrylamide gel electrophoresis. Different G4 conformations of these three sequences can be separated clearly by RP-HPLC, and further characterized by on-line LC–MS analysis. It is revealed that high-order structures other than intramolecular quadruplexes were favored for d[(G₃T)₃G₃] and d[(G₃C)₃G₃] under the annealing conditions. However, flanking loop impeded d[(G₃T)₄] to form higher-order structures than dimer. In addition, the nature and concentration of cation, as well as the annealing solution component, all have decent influence on the stability and relative ratios of various G4 building blocks. Based on the above findings, RP-HPLC and LC–MS combined with spectroscopic techniques can be used as a facile and powerful tool for quick separation and identification of G4s in solutions, and for effective assessment of DNA sequences and annealing environments on G4 polymorphism. The established protocol provides a novel strategy for evaluating G4 polymorphism, which will facilitate studies on quadruplex structures and their biophysical properties.

多态性是G-四链体（G4）固有的，并且不同的结构形式对于参与端粒的不同生物学功能很重要。在探索G4多态性方面已经取得了许多进展。然而，针对G4不同构型的快速分离和可靠的评估方法仍然很少。在这项工作中，三个序列d [（G ₃ T）₃ G ₃ ]，d [（G ₃ C）₃ G ₃ ]和d [（G ₃ T）₄的多态性通过反相高效液相色谱（RP-HPLC），液相色谱-质谱（LC-MS），荧光光谱，圆二色谱，天然聚丙烯酰胺凝胶电泳等方法研究了在6种不同溶液中退火的]。可以通过RP-HPLC清楚地分离出这三个序列的不同G4构象，并通过在线LC-MS分析进一步表征。结果表明，在退火条件下，d [（G ₃ T）₃ G ₃ ]和d [（G ₃ C）₃ G ₃ ]优于分子内四链体。但是，侧翼环阻碍了d [（G ₃ T）₄]形成比二聚体更高的结构。另外，阳离子的性质和浓度，以及退火溶液的成分，都对各种G4结构单元的稳定性和相对比率具有相当大的影响。基于以上发现，RP-HPLC和LC-MS结合光谱技术可作为一种简便而强大的工具，用于快速分离和鉴定溶液中的G4，并有效评估G4多态性的DNA序列和退火环境。建立的协议为评估G4多态性提供了一种新颖的策略，这将有助于研究四链体结构及其生物物理特性。